Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Briefly, reagents were combined in a 1:1 ratio and incubated with the samples collected from LPS‐induced RAW 264.7 cells.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Nitrite levels were compared among M0 uninduced (Control), LPS‐induced M1 polarized, and IL‐4‐induced M2 polarized cells.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Absorbance was determined at 540 nm using a multi‐mode microplate reader (Synergy MX).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	RAW 264.7 cells (8 × 10 cells) were washed three times with 1× sterile PBS and cultured in serum‐free media (SFM) for 3 days in a T75 flask, as described in [34, 35].
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	After 72 h, media was collected and subjected to centrifugation for 10 min at 1000 rpm, 4°C, and 30 min at 3000 rpm, 4°C to remove cell debris.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Per the manufacturer's protocol, the remaining cell culture media was subjected to EP isolation using an ExoQuick‐TC isolation kit (Systems Biosciences, USA).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Briefly, the isolation solution was added to the collected cell culture media in a ratio of 1:5 and incubated for 24 h at 4°C.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Following this incubation, the solution was centrifuged at 1500 rpm for 30 min at 4°C to pellet the EPs and purified further according to the manufacturer's instructions.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Finally, a series of centrifugation and washing steps were followed to yield EPs.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	According to this study's purification method and characterization, we refer to our isolated vesicles by the generic term “EPs”.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Isolated EPs were further characterized using nanoparticle tracking analysis (NTA) for particle concentration and size.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Approximately 0.3 mL of isolated EPs were loaded into the sample chamber of an LM10 unit (Nanosight, Amesbury, UK), and data analysis was performed with the NTA software (Nanosight).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	In NTA, the paths of EPs act as point scatterers, undergoing Brownian motion in a chamber through which a 632 nm laser beam is passed.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Samples were analyzed using the following parameters: the shutter speed was 15 ms, with camera gains between 280 and 560.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Software settings for analysis were detection threshold: 12 multi; blur size: auto; frames per second: 23.75; measurement time: 30 ms.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	When samples contained higher numbers of particles, they were diluted before analysis, and the relative concentration was calculated according to the dilution factor.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	After purification, EPs were pelleted at 4°C for 100 min at 14000 g and resuspended in a 3.7% glutaraldehyde solution, incubated for 30 min, and re‐centrifuged at 4°C for 100 min.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The supernatant was discarded, and EP pellets were washed in 40%, 60%, and 95% ethanol for 15 min each.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	During the final step, EPs were resuspended in 95% ethanol and deposited on a silicon wafer for SEM analysis.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Samples were left overnight to dry under a laminar flow.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	MDA‐MB‐231 cells at the density of 4 × 10 per well in a 6‐well plate; M1 RAW 264.7 cells derived EPs with 2 × 10 particles/mL were added to MDA‐MB‐231 cells.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	After 24 h, the silicon wafer was dipped into 3.7% glutaraldehyde for 30 min.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Then, the extra solution was removed and transferred to 40%, 60%, and 95% ethanol.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Samples were left to dry overnight before observation under SEM.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The interaction between the EPs derived from M1 RAW 264.7 cells and MDA‐MB‐231 cells.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Samples were then observed under SEM (JEOL JSM‐IT800 Schottky FESEM).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Protein levels were detected after isolating EPs from RAW 264.7 cells and characterized by western blot analysis, based on previously published protocols [20, 30, 31].
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Briefly, total protein concentration from the samples was measured using a Qubit 4 Fluorometer (Invitrogen, Thermo Fisher).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Next, 4× Laemmli buffer was added to samples at a 1:3 ratio and heated to 75°C for 10 min.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	19.5 μL of each normalized sample was loaded onto a 12% Mini‐PROTEAN TGX Precast Protein Gel (Bio‐Rad, Hercules, CA).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Proteins were separated by electrophoresis, followed by transfer to 0.45 μm nitrocellulose membranes (ThermoFisher).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Membranes were blocked with StartingBlock T20 (TBS) blocking buffer for 30 min at 4°C, followed by washing with phosphate buffer saline‐Tween (PBST).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	RAW 264.7 cell‐derived EP proteins were assessed using anti‐Hsp70 (ab181606, Abcam).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Membranes were incubated with primary antibodies (1:1000) overnight at 4°C.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Then, membranes were washed 3× for 10 min each in PBST.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	For primary antibodies, Hsp 70 was incubated with goat anti‐rabbit IgG (A16104, ThermoFisher).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Secondary antibodies were diluted in 1:10000, and the membranes were incubated with the secondary antibodies for 30 min at 4°C.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The blot was imaged under the iBright Imaging System (Invitrogen, model #FL1500).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Each western blot was repeated three times, with the representative images shown in this manuscript.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	MBA‐MB‐231 cells were stained with BioTracker 405 Blue Mitochondria Dye (Sigma Aldrich, USA, Cat#SCT135).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Briefly, MDA‐MB‐231 cells were counted, and the mitochondria tracking dye was added to a final concentration of 40 nM. Cells were incubated at 37°C for 30 min with the dye and seeded in confocal dishes for 24 h. Cells were allowed to attach to the surface of a 4‐chamber confocal dish.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The media was replaced the next day with fresh media.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Then, EPs isolated from M0 (Control), M1, and M2 polarized RAW 264.7 cells at a concentration of 2 × 10 particles/mL were added to each well in addition to Cell Event Caspase‐3/7 Green ReadyProbes Reagent (Invitrogen, Cat#R37111).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Images were taken using an Evident FV3000 confocal microscope using a 20× objective at 0, 24, and 48 h time points.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	A cell viability assay was performed using a CyQUANT XTT (2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide) cell viability assay kit (ThermoFisher, USA).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Following the manufacturer's protocol, MDA‐MB‐231 at 5000 cells/well were seeded in a 96‐well plate for 24 h. After 24 h, M0 (control), M1, and M2 RAW 264.7 cell‐derived EPs were added at a concentration of 2 × 10 particles/mL to measure cell viability in each sample allowed to incubate.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	After 24 h incubation with EPs, 1 mL of electron coupling reagent was mixed with 6 mL of XTT reagent and incubated at 37°C for 4 h in a 5% CO2 incubator.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Absorbance was measured at 450 and 660 nm using a Synergy MX multi‐mode microplate reader.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The data analysis was performed according to the manufacturer's protocol.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Statistical analyses were performed using Microsoft Excel.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	A Student's t‐test was employed for p‐value determination across all experiments (N = 3).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	All data are expressed as the mean ± SEM (N = 3).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	All raw data readings were exported into Excel, where subsequent calculations were performed, and the resulting data were plotted.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Macrophages are innate immune cells characterized by their functional characteristics and environmental responses, known as M0 (resting/not activated), M1 (classically activated), or M2 (alternatively active).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Lipopolysaccharide (LPS) is an inducer of the M1 macrophage phenotype, while IL‐4 is an inducer of the M2 macrophage phenotype [33, 36].
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Accordingly, we used different concentrations of LPS to polarize RAW 264.7 cells and monitored their morphology via optical microscopy.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	After 24 h, round cells were observed in the non‐induced (M0 macrophages) RAW 264.7 cells, the control group (Figure 2a), while RAW 264.7 cells with LPS (M1 macrophages) looked like dendritic or star‐like shapes (Figure 2b–d), indicating a morphological change. (
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Figure 2f–h) indicate insignificant changes in shape under the influence of IL‐4 (M2 macrophages) compared to non‐induced RAW 264.7 cells (Figure 2e), the control group.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Thus, macrophage polarization was determined morphologically after 24 h under external stimuli of LPS and IL‐4 under different concentrations (0–100 ng/mL).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	We found that LPS‐induced (M1 macrophages) RAW 264.7 cells showed noticeable differences in morphology compared to non‐induced cells (M0 macrophages) in Figure 2a, while IL‐4‐induced (M2 macrophages) RAW 264.7 cells showed insignificant morphological differences compared to non‐induced (M0 macrophages) cells in Figure 2e.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	RAW 264.7 cells under the influence of different stimuli: RAW 264.7 cells were observed for M1 polarization in the presence of LPS after 24 h (a) 0, (b) 25, (c) 50, and (d) 100 ng/mL. M2 polarization was observed in the presence of IL‐4 after 24 h (e) 0, (f) 25, (g) 50, and (h) 100 ng/mL. The observation was made using a bright field microscope with a 10× objective and an external camera; images were collected using SeBaView software.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	LPS‐induced M1 macrophages have been shown to secrete markers, such as tumor necrosis factor‐α (TNF‐α), nitric oxide (NO), and interleukin‐6 (IL‐6), indicating their pro‐inflammatory state .
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	RAW 264.7 cells were challenged with LPS to induce an inflammatory or M1 polarized state.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Subsequently, TNF‐α, nitrite production, and IL‐6 levels released by cells were quantified by collecting cell culture media.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Results showed that LPS‐induced TNF‐α, nitrite, and IL‐6 concentrations increased in a dose‐dependent manner (Figure 3).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	TNF‐α and IL‐6 were quantified in cell culture media using ELISA (Figure 3a,b), and the nitrite level was assessed separately in the supernatant using the Griess reaction.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	We observed the increased nitrite production in a dose‐responsive manner (Figure 3c) compared to M0 or uninduced RAW 264.7 cells.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	These results confirmed the activated stage of RAW 264.7 cells in a pro‐inflammatory state after LPS induction.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	RAW 264.7 cell polarization using biochemical analysis: RAW 264.7 cells were cultured in 6‐well plates and incubated for 24 h before collecting cell culture media.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Collected media was subjected to enzyme‐linked immunosorbent assay (ELISA), (a) and (b) show the dose‐dependent increase in the levels of IL‐6 and TNF‐α cytokines. (
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	c) Shows the dose‐dependent increase in the levels of nitrites.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Results indicate that M0 polarized RAW 264.7 cells, the control, have low nitrites produced compared to LPS‐induced macrophages.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	N = 3 independent experiments.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Error bars = SEM; connecting bars denote the statistical comparison between groups, with p‐value < 0.05 considered statistically significant.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Next, EPs derived from three different macrophage states, M0 (uninduced), M1 (induced with LPS), and M2 (induced with IL‐4), were characterized.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The characterization was done to identify the type of particle secreted by RAW 264.7 cells under different stimuli.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	According to the MISEV guidelines [6, 7], EP size was analyzed by NTA and the mode size found among the samples was 27–76 nm, regardless of their polarization state.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	In addition to their size, it was also observed that the number of particles varies with M0, M1, and M2 polarized states (Figure 4a–c).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	A scanning electron microscope (SEM) was used to confirm their size and morphology.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The EPs had a heterogeneous size range on the order of 100 nm (Figure 4d).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Next, protein content in isolated EPs was analyzed using western blot analysis (Figure 4e).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	After confirming enrichment of heat shock protein 70 (HSP 70), isolated particles from RAW 264.7 cells were classified as EPs.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Taken together, these results indicate the successful isolation of EPs from RAW 264.7 cells in M1, M2, and M0 polarization states.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Characterization of EPs isolated from polarized RAW 264.7 cells in their M0, M1, and M2 states: (a–c) particle size distribution showing a heterogeneous population of EPs derived from RAW 264.7 macrophages.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The plots show the concentration and particle based on a 15 ms camera shutter speed and 23.75 frames per second per sample type.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The data were obtained in the NTA 2.0 analytical software and processed using Microsoft Excel. (
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	d) The SEM image of RAW 264.7 cells derived EPs.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	EP sizes were consistent with NTA analysis. (
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	e) The presence of HSP 70 was demonstrated with western blotting among all the EPs derived from M0, M1, and M2 polarized RAW 264.7 cells.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	To evaluate the potency of M0 (Control), M1, and M2 macrophage‐derived EPs on MDA‐MB‐231 cells, EPs were incubated with MDA‐MB‐231 cells for 0 to 48 h. Cells were stained with 405 mitochondrial blue dye and caspase 3/7 dye.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Figure 5a–i represents the MDA‐MB‐231 cells at the 0 h time point.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The initiation of caspase 3/7 in MDA‐MB‐231 cells was observed after 24 h, Figure 6a–i. At the 48 h time point, Figure 7e shows the dominance of green color indicative of caspase 3/7 activation in MDA‐MB‐231 cells incubated with M1‐derived EPs as compared with other EPs (Figure 7b,h).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Figure 5e, taken at 0 h, shows no activity of caspase 3 at the beginning of the experiment.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	In Figure 6e, the white arrows show the slow induction of caspase 3/7 after 24 h with M1‐derived EPs compared to EPs derived from M0 and M2 RAW 264.7 cells.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Possible cytoplasmic blebs indicated by white arrows in Figure 7e were also observed in MDA‐MB‐231 cells after 48 h of incubation with M1‐derived EPs.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Thus, these results suggest the possibility of M1‐derived EPs yielding a cytotoxic effect against MDA‐MB‐231 cells after 48 h. Evaluating caspase‐3/7 expression induced by M1 RAW 264.7 cell‐derived EPs: Confocal images show MDA‐MB‐231 cells at 0 h incubated with EPs derived from M0 macrophages (a–c), M1 macrophages (d–f), and M2 macrophages (g–i).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Cells were stained with BioTracker 405 Blue Mitochondrial Dye to visualize mitochondria (a, d, g), while the GFP channel (502 nm excitation) highlights caspase‐3/7 activation (b, e, h).
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	At the 0 h time point, no caspase‐3/7 activation is observed, confirming that the cells are healthy prior to treatment.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	The scale bar represents 20 μm.
PMC12217045	M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.	Caspase‐3/7 activation induced by M1 RAW 264.7 cell‐derived EPs at 24 h: Confocal images show MDA‐MB‐231 cells after 24 h of incubation with EPs derived from M0 macrophages (a–c), M1 macrophages (d–f), and M2 macrophages (g–i).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Briefly, reagents were combined in a 1:1 ratio and incubated with the samples collected from LPS‐induced RAW 264.7 cells.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Nitrite levels were compared among M0 uninduced (Control), LPS‐induced M1 polarized, and IL‐4‐induced M2 polarized cells.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Absorbance was determined at 540 nm using a multi‐mode microplate reader (Synergy MX).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

RAW 264.7 cells (8 × 10 cells) were washed three times with 1× sterile PBS and cultured in serum‐free media (SFM) for 3 days in a T75 flask, as described in [34, 35].

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

After 72 h, media was collected and subjected to centrifugation for 10 min at 1000 rpm, 4°C, and 30 min at 3000 rpm, 4°C to remove cell debris.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Per the manufacturer's protocol, the remaining cell culture media was subjected to EP isolation using an ExoQuick‐TC isolation kit (Systems Biosciences, USA).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Briefly, the isolation solution was added to the collected cell culture media in a ratio of 1:5 and incubated for 24 h at 4°C.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Following this incubation, the solution was centrifuged at 1500 rpm for 30 min at 4°C to pellet the EPs and purified further according to the manufacturer's instructions.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Finally, a series of centrifugation and washing steps were followed to yield EPs.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

According to this study's purification method and characterization, we refer to our isolated vesicles by the generic term “EPs”.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Isolated EPs were further characterized using nanoparticle tracking analysis (NTA) for particle concentration and size.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Approximately 0.3 mL of isolated EPs were loaded into the sample chamber of an LM10 unit (Nanosight, Amesbury, UK), and data analysis was performed with the NTA software (Nanosight).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

In NTA, the paths of EPs act as point scatterers, undergoing Brownian motion in a chamber through which a 632 nm laser beam is passed.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Samples were analyzed using the following parameters: the shutter speed was 15 ms, with camera gains between 280 and 560.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Software settings for analysis were detection threshold: 12 multi; blur size: auto; frames per second: 23.75; measurement time: 30 ms.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

When samples contained higher numbers of particles, they were diluted before analysis, and the relative concentration was calculated according to the dilution factor.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

After purification, EPs were pelleted at 4°C for 100 min at 14000 g and resuspended in a 3.7% glutaraldehyde solution, incubated for 30 min, and re‐centrifuged at 4°C for 100 min.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The supernatant was discarded, and EP pellets were washed in 40%, 60%, and 95% ethanol for 15 min each.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

During the final step, EPs were resuspended in 95% ethanol and deposited on a silicon wafer for SEM analysis.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Samples were left overnight to dry under a laminar flow.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

MDA‐MB‐231 cells at the density of 4 × 10 per well in a 6‐well plate; M1 RAW 264.7 cells derived EPs with 2 × 10 particles/mL were added to MDA‐MB‐231 cells.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

After 24 h, the silicon wafer was dipped into 3.7% glutaraldehyde for 30 min.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Then, the extra solution was removed and transferred to 40%, 60%, and 95% ethanol.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Samples were left to dry overnight before observation under SEM.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The interaction between the EPs derived from M1 RAW 264.7 cells and MDA‐MB‐231 cells.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Samples were then observed under SEM (JEOL JSM‐IT800 Schottky FESEM).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Protein levels were detected after isolating EPs from RAW 264.7 cells and characterized by western blot analysis, based on previously published protocols [20, 30, 31].

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Briefly, total protein concentration from the samples was measured using a Qubit 4 Fluorometer (Invitrogen, Thermo Fisher).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Next, 4× Laemmli buffer was added to samples at a 1:3 ratio and heated to 75°C for 10 min.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

19.5 μL of each normalized sample was loaded onto a 12% Mini‐PROTEAN TGX Precast Protein Gel (Bio‐Rad, Hercules, CA).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Proteins were separated by electrophoresis, followed by transfer to 0.45 μm nitrocellulose membranes (ThermoFisher).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Membranes were blocked with StartingBlock T20 (TBS) blocking buffer for 30 min at 4°C, followed by washing with phosphate buffer saline‐Tween (PBST).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

RAW 264.7 cell‐derived EP proteins were assessed using anti‐Hsp70 (ab181606, Abcam).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Membranes were incubated with primary antibodies (1:1000) overnight at 4°C.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Then, membranes were washed 3× for 10 min each in PBST.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

For primary antibodies, Hsp 70 was incubated with goat anti‐rabbit IgG (A16104, ThermoFisher).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Secondary antibodies were diluted in 1:10000, and the membranes were incubated with the secondary antibodies for 30 min at 4°C.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The blot was imaged under the iBright Imaging System (Invitrogen, model #FL1500).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Each western blot was repeated three times, with the representative images shown in this manuscript.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

MBA‐MB‐231 cells were stained with BioTracker 405 Blue Mitochondria Dye (Sigma Aldrich, USA, Cat#SCT135).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Briefly, MDA‐MB‐231 cells were counted, and the mitochondria tracking dye was added to a final concentration of 40 nM. Cells were incubated at 37°C for 30 min with the dye and seeded in confocal dishes for 24 h. Cells were allowed to attach to the surface of a 4‐chamber confocal dish.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The media was replaced the next day with fresh media.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Then, EPs isolated from M0 (Control), M1, and M2 polarized RAW 264.7 cells at a concentration of 2 × 10 particles/mL were added to each well in addition to Cell Event Caspase‐3/7 Green ReadyProbes Reagent (Invitrogen, Cat#R37111).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Images were taken using an Evident FV3000 confocal microscope using a 20× objective at 0, 24, and 48 h time points.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

A cell viability assay was performed using a CyQUANT XTT (2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide) cell viability assay kit (ThermoFisher, USA).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Following the manufacturer's protocol, MDA‐MB‐231 at 5000 cells/well were seeded in a 96‐well plate for 24 h. After 24 h, M0 (control), M1, and M2 RAW 264.7 cell‐derived EPs were added at a concentration of 2 × 10 particles/mL to measure cell viability in each sample allowed to incubate.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

After 24 h incubation with EPs, 1 mL of electron coupling reagent was mixed with 6 mL of XTT reagent and incubated at 37°C for 4 h in a 5% CO2 incubator.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Absorbance was measured at 450 and 660 nm using a Synergy MX multi‐mode microplate reader.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The data analysis was performed according to the manufacturer's protocol.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Statistical analyses were performed using Microsoft Excel.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

A Student's t‐test was employed for p‐value determination across all experiments (N = 3).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

All data are expressed as the mean ± SEM (N = 3).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

All raw data readings were exported into Excel, where subsequent calculations were performed, and the resulting data were plotted.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Macrophages are innate immune cells characterized by their functional characteristics and environmental responses, known as M0 (resting/not activated), M1 (classically activated), or M2 (alternatively active).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Lipopolysaccharide (LPS) is an inducer of the M1 macrophage phenotype, while IL‐4 is an inducer of the M2 macrophage phenotype [33, 36].

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Accordingly, we used different concentrations of LPS to polarize RAW 264.7 cells and monitored their morphology via optical microscopy.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

After 24 h, round cells were observed in the non‐induced (M0 macrophages) RAW 264.7 cells, the control group (Figure 2a), while RAW 264.7 cells with LPS (M1 macrophages) looked like dendritic or star‐like shapes (Figure 2b–d), indicating a morphological change. (

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Figure 2f–h) indicate insignificant changes in shape under the influence of IL‐4 (M2 macrophages) compared to non‐induced RAW 264.7 cells (Figure 2e), the control group.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Thus, macrophage polarization was determined morphologically after 24 h under external stimuli of LPS and IL‐4 under different concentrations (0–100 ng/mL).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

We found that LPS‐induced (M1 macrophages) RAW 264.7 cells showed noticeable differences in morphology compared to non‐induced cells (M0 macrophages) in Figure 2a, while IL‐4‐induced (M2 macrophages) RAW 264.7 cells showed insignificant morphological differences compared to non‐induced (M0 macrophages) cells in Figure 2e.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

RAW 264.7 cells under the influence of different stimuli: RAW 264.7 cells were observed for M1 polarization in the presence of LPS after 24 h (a) 0, (b) 25, (c) 50, and (d) 100 ng/mL. M2 polarization was observed in the presence of IL‐4 after 24 h (e) 0, (f) 25, (g) 50, and (h) 100 ng/mL. The observation was made using a bright field microscope with a 10× objective and an external camera; images were collected using SeBaView software.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

LPS‐induced M1 macrophages have been shown to secrete markers, such as tumor necrosis factor‐α (TNF‐α), nitric oxide (NO), and interleukin‐6 (IL‐6), indicating their pro‐inflammatory state .

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

RAW 264.7 cells were challenged with LPS to induce an inflammatory or M1 polarized state.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Subsequently, TNF‐α, nitrite production, and IL‐6 levels released by cells were quantified by collecting cell culture media.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Results showed that LPS‐induced TNF‐α, nitrite, and IL‐6 concentrations increased in a dose‐dependent manner (Figure 3).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

TNF‐α and IL‐6 were quantified in cell culture media using ELISA (Figure 3a,b), and the nitrite level was assessed separately in the supernatant using the Griess reaction.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

We observed the increased nitrite production in a dose‐responsive manner (Figure 3c) compared to M0 or uninduced RAW 264.7 cells.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

These results confirmed the activated stage of RAW 264.7 cells in a pro‐inflammatory state after LPS induction.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

RAW 264.7 cell polarization using biochemical analysis: RAW 264.7 cells were cultured in 6‐well plates and incubated for 24 h before collecting cell culture media.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Collected media was subjected to enzyme‐linked immunosorbent assay (ELISA), (a) and (b) show the dose‐dependent increase in the levels of IL‐6 and TNF‐α cytokines. (

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

c) Shows the dose‐dependent increase in the levels of nitrites.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Results indicate that M0 polarized RAW 264.7 cells, the control, have low nitrites produced compared to LPS‐induced macrophages.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

N = 3 independent experiments.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Error bars = SEM; connecting bars denote the statistical comparison between groups, with p‐value < 0.05 considered statistically significant.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Next, EPs derived from three different macrophage states, M0 (uninduced), M1 (induced with LPS), and M2 (induced with IL‐4), were characterized.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The characterization was done to identify the type of particle secreted by RAW 264.7 cells under different stimuli.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

According to the MISEV guidelines [6, 7], EP size was analyzed by NTA and the mode size found among the samples was 27–76 nm, regardless of their polarization state.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

In addition to their size, it was also observed that the number of particles varies with M0, M1, and M2 polarized states (Figure 4a–c).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

A scanning electron microscope (SEM) was used to confirm their size and morphology.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The EPs had a heterogeneous size range on the order of 100 nm (Figure 4d).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Next, protein content in isolated EPs was analyzed using western blot analysis (Figure 4e).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

After confirming enrichment of heat shock protein 70 (HSP 70), isolated particles from RAW 264.7 cells were classified as EPs.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Taken together, these results indicate the successful isolation of EPs from RAW 264.7 cells in M1, M2, and M0 polarization states.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Characterization of EPs isolated from polarized RAW 264.7 cells in their M0, M1, and M2 states: (a–c) particle size distribution showing a heterogeneous population of EPs derived from RAW 264.7 macrophages.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The plots show the concentration and particle based on a 15 ms camera shutter speed and 23.75 frames per second per sample type.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The data were obtained in the NTA 2.0 analytical software and processed using Microsoft Excel. (

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

d) The SEM image of RAW 264.7 cells derived EPs.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

EP sizes were consistent with NTA analysis. (

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

e) The presence of HSP 70 was demonstrated with western blotting among all the EPs derived from M0, M1, and M2 polarized RAW 264.7 cells.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

To evaluate the potency of M0 (Control), M1, and M2 macrophage‐derived EPs on MDA‐MB‐231 cells, EPs were incubated with MDA‐MB‐231 cells for 0 to 48 h. Cells were stained with 405 mitochondrial blue dye and caspase 3/7 dye.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Figure 5a–i represents the MDA‐MB‐231 cells at the 0 h time point.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The initiation of caspase 3/7 in MDA‐MB‐231 cells was observed after 24 h, Figure 6a–i. At the 48 h time point, Figure 7e shows the dominance of green color indicative of caspase 3/7 activation in MDA‐MB‐231 cells incubated with M1‐derived EPs as compared with other EPs (Figure 7b,h).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Figure 5e, taken at 0 h, shows no activity of caspase 3 at the beginning of the experiment.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

In Figure 6e, the white arrows show the slow induction of caspase 3/7 after 24 h with M1‐derived EPs compared to EPs derived from M0 and M2 RAW 264.7 cells.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Possible cytoplasmic blebs indicated by white arrows in Figure 7e were also observed in MDA‐MB‐231 cells after 48 h of incubation with M1‐derived EPs.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Thus, these results suggest the possibility of M1‐derived EPs yielding a cytotoxic effect against MDA‐MB‐231 cells after 48 h. Evaluating caspase‐3/7 expression induced by M1 RAW 264.7 cell‐derived EPs: Confocal images show MDA‐MB‐231 cells at 0 h incubated with EPs derived from M0 macrophages (a–c), M1 macrophages (d–f), and M2 macrophages (g–i).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Cells were stained with BioTracker 405 Blue Mitochondrial Dye to visualize mitochondria (a, d, g), while the GFP channel (502 nm excitation) highlights caspase‐3/7 activation (b, e, h).

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

At the 0 h time point, no caspase‐3/7 activation is observed, confirming that the cells are healthy prior to treatment.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

The scale bar represents 20 μm.

PMC12217045

M1 Macrophage-Derived Extracellular Particles Induce Cell Death in MDA-MB-231 Cells.

Caspase‐3/7 activation induced by M1 RAW 264.7 cell‐derived EPs at 24 h: Confocal images show MDA‐MB‐231 cells after 24 h of incubation with EPs derived from M0 macrophages (a–c), M1 macrophages (d–f), and M2 macrophages (g–i).