PMCID string | Title string | Sentences string |
|---|---|---|
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | To knock down STUB1, we transfected cells with siRNA duplex oligos in OPTI-MEM (#2185849, Gibco) overnight. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Expression plasmids encoding Myc-STUB1 (#90977–1), Flag-GPX4 (#68273–1), HA-ubiquitin (Ub, #61618–1), GPX4-K107R (#68273–2), GPX4-K126R (#68273–3), GPX4-K162R (#68273–4), GPX4-K167R (#68273–5), and GPX4-K191R (#68273–6) (GPX4 mutants with lys107, lys126, lys162, lys167 and lys191 changed into arginine) were purchased from Genechem (Genechem, Shanghai, China). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | All plasmids were transfected with Lipofectamine™ 3000 Transfection Reagent (#L3000015, ThermoFisher, Shanghai, China) according to the protocol. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | STUB1 and GPX4 expression lentiviruses were purchased from Genechem (Genechem, Shanghai, China). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Transfection was performed according to the manufacturer’s protocols. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The transfected cells were screened by puromycin (2 mg/mL) (ST551, Beyotime, Beijing, China) to establish a stably expressing cell line (Fig. S1). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Cells were resuspended in a buffer containing 4% paraformaldehyde and 1% glutaraldehyde and subsequently dehydrated through graded ethanol and propylene oxide, which was then embedded in Epon 812 and sliced into 60-nm ultrathin sections. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The mitochondrial area and structure were finally assessed by transmission electron microscopy (H-7650, Hitachinaka, Japan). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | ROS or lipid ROS levels were measured in the GIST-T1 and GIST-882 cells treated with IM (50 nM for T1 and 200 nM for 882) or Ferrostatin-1 (1 μM) for 12 h, by adding 10 µM 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA) (ab113851, ROS Detection Assay Kit, Abcam) or 10 µM C11-BODIPY (#D3861, Thermo Fisher Scientific) for 30 min at 37 °C, followed by trypsinization, washing and resuspension in phosphate buffered saline (PBS). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | ROS and lipid ROS levels were then measured by confocal microscopy (Carl Zeiss, Jena, Germany) or flow cytometry. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Flow cytometry analysis was performed using the SONY SH800 Cell Sorter with the FITC filter for oxidized C11-BODIPY (emission: 510 nm) and the PE-TexasRed filter for reduced C11-BODIPY (emission: 590 nm). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | At least 10,000 cells were collected for each condition, and this experiment was independently repeated three times. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Data analysis was carried out using the FlowJo software (version 9.4.10, Tree Star). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The results are expressed as the ratio of oxidized to reduced C11-BODIPY. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Change of intracellular Fe in GIST cells was detected with the novel fluorescent probe FerroOrange (F374, Dojindo) according to the manufacturer’s protocol. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | GIST-T1 and GIST-882 cells treated with IM were incubated with 1 µM FerroOrange at 37 °C for 30 min. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The fluorescence was then measured by flow cytometry. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | GSH and GSSG levels were determined using the GSH and GSSG Assay Kit (Beyotime, S0053) following the manufacturer’s instructions. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | To prepare the samples, GIST-T1 and GIST-882 cells were mixed with protein removal reagent M in a three-fold volume and thoroughly vortexed. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Subsequently, two freeze-thaw cycles (5 s freeze, 1 min thaw) were performed alternating between liquid nitrogen and a 37 °C water bath. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | After centrifugation at 10,000 g for 10 min at 4 °C, the resulting supernatant was collected as the sample solution. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | All samples were normalized based on their protein content. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Then, the 0.2 ml sample solution was mixed with 1 mM GSH solution of the same volume and incubated at 37 °C for 5 min; after addition of 0.1 ml reagent 1 and incubation at 37 °C for another 5 min, 2 ml reagent 2 was added. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The mixture was then centrifuged at 10,000 g for 10 min. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The supernatant (1 ml) was extracted and mixed with 1 ml reagent 3, 0.25 ml reagent 4 and 0.05 ml reagent 5, incubated for 15 min. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Supernatants from each group of samples were collected, and the quantification of total GSH (GSH + GSSG) was performed by monitoring the formation of 2‐nitro‐5‐thiobenzoic acid at 412 nm. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | To determine GSSG specifically, the supernatants were derivatized using 1‐methyl‐2‐vinylpyridium trifluoromethane sulfonate. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Standard curves were established for both GSH and GSSG, and the concentration of GSH was calculated by subtracting the GSSG concentration from the total GSH (GSH + GSSG). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The measured concentrations of GSH and GSSG were then expressed as a ratio (GSH/GSSG). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Total RNA was extracted using Trizol (Yeasen Biotechnology Shanghai, China) and reverse-transcribed into cDNA. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | quantitative real-time polymerase chain reaction was carried out using the SYBR Green kit. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The relative mRNA expression was performed using the 2 method. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | GAPDH was used as internal control and the primers used are specified in Table S1. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Protein stability was determined by using the cycloheximide (CHX) chase assay. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Cells were treated with 100 μg/mL CHX for various time intervals after overexpression of STUB1. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Then GPX4 protein lysates were resolved on SDS-PAGE and analyzed by Western blotting. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Co-immunoprecipitation (CO-IP) assay was performed using the protein binding IP kit (abs955, Absin). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Cell lysates were first incubated overnight with anti-STUB1 (Abcam), anti-GPX4 (Proteintech), or control IgG (Cell Signaling Technology) at 4 °C overnight, and then incubated with protein A/G-agarose beads at 4 °C for 4 h. The precipitates were washed five times in a wash buffer and evaluated using Western blot analysis. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Western & IP lysis buffer (P0013, Beyotime) supplemented with the protease inhibitor and phosphatase inhibitors (#524625 and #539131, Millipore) was used to lyse cells on ice for 30 min. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The PVDF membranes (ISEQ00010, Millipore) were blocked with 5% skimmed milk at room temperature for 1 h, followed by incubation with primary antibodies at 4 °C overnight. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Then the membranes were washed 5 times for 5 min with TBST (TBS with 0.1% Tween 20) and incubated with HRP-conjugated secondary antibodies (CST, MA, USA) at room temperature for 1 h. The antibodies and the concentrations used are listed in Table S2. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The membrane was then washed and visualized using enhanced chemiluminescence (ECL) solution (Millipore, Beverly, USA) and analyzed using ImageJ software (Version 1.53, National Institutes of Health, Bethesda, MD). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Tissue microarray (TMA) containing 418 paraffin-embedded primary GIST surgical samples resected at our hospital between January 2014 and January 2021 with Institutional Review Board approval were constructed. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Informed consent was obtained from all subjects. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Immunohistochemistry (IHC) staining was performed with anti-STUB1 and anti-GPX4 antibodies. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The antibodies and the concentrations used are listed in Table S2. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The staining intensity and the proportion of positively stained cells were evaluated using Image J 1.53 software. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The staining intensity was graded as follows: negative = 0, mild = 1, moderate = 2, and strong = 3. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The proportion of positively stained cells was scored as follows: 1 = 0–24% positive cells; 2 = 25–49% positive cells; 3 = 50–74% positive cells; and 4 = 75%-100% positive cells. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The IHC staining score was expressed by the intensity multiplied by the proportional score. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | A staining score of <8 was regarded as the low expression group and that ≥8 as the high expression group. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Cultured cells were fixed using 4% paraformaldehyde (Sigma-Aldrich, #30525–89–4) in PBS for 20 min. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Subsequently, they were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, #9036–19–5) in PBS for 10 min and then blocked with 10% normal goat serum (Beyotime, #C0265, Jiangsu, China) for 1 h at 37 °C. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | This was followed by overnight incubation at 4 °C with primary antibodies against STUB1 (Abcam, #ab134064, 1:250) and GPX4 (Abcam, #ab125066, 1:200). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Afterwards, the cells were washed three times with PBS containing Tween 20 and then incubated with fluorescent secondary antibodies for 1 h at 37 °C. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The secondary antibody for STUB1 was green fluorescent, and the secondary antibody for GPX4 was red fluorescent. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Subsequent to this, the cells were rinsed three times with PBS and cell nuclei were stained with 1 μg/ml DAPI (Sigma-Aldrich, #D9542) for 10 min. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Image acquisition was performed using Zeiss LSM780 Laser Scanning Confocal Microscopy (Carl Zeiss, Jena, Germany). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | To quantitatively analyze colocalization, ImageJ software (Version 1.53, National Institutes of Health, Bethesda, MD) was utilized in conjunction with the Colocalization Finder plugin for calculating Pearson’s correlation coefficients . |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | GIST cells were initially seeded in 6-well CultureSlides obtained from BD Biosciences (Mountain View, CA, USA). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The cells were then fixed using 4% paraformaldehyde for a duration of 15 min and subsequently permeabilized with a solution consisting of 0.5% Triton X-100 in PBS for 30 min. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The slides were further processed for an in situ Proximity Ligation Assay following the manufacturer’s instructions, employing the Duolink® II Detection Reagents Green, Duolink® II PLA probe anti-Rabbit Minus, and Duolink® II PLA probe anti-Mouse Plus (Olink Bioscience, Uppsala, Sweden). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Primary antibodies used were STUB1 (Abcam, #ab134064) and GPX4 (Proteintech, #67763–1-Ig). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The resulting images were captured using Zeiss LSM780 Laser Scanning Confocal Microscopy (Carl Zeiss, Jena, Germany). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Subsequent quantification was performed using ImageJ software (Version 1.53, National Institutes of Health, Bethesda, MD). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Animal studies were reviewed and approved by the Institutional Review Boards of Zhongshan Hospital. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | GIST-T1 xenografts were established in 6-week-old male BALB/c nude mice (Shanghai Jiesijie Laboratory Animal Corporation) by inoculating 5 × 10 cells mixed with Matrigel (BD Biosciences) at a 1:1 ratio (volume) into the dorsolateral side. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | When the tumor reached 50mm, the mice were assigned randomly into DMSO, IM, RSL3, and IM combined with RSL3 groups. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | IM was dissolved in dimethyl sulfoxide (DMSO, #HY-Y0320, MCE), and diluted in PBS. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | RSL3 was dissolved in DMSO, diluted in corn oil (#HY-Y1888, MCE), and then intraperitoneally injected into mice with IM (100 mg/kg) or RSL3 (10 mg/kg) every 3 days. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The dosages of IM and RSL3 were determined based on previous studies and our own preliminary experimental results. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Tumor sizes were measured every 3 days, and tumor volumes were calculated as follows: 0.5×length×width. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Each group consisted of 6 mice. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | All mice were euthanized after 18 days of treatment, and the tumors were surgically excised. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | A portion of the tumors was immediately fixed in 4% paraformaldehyde, and sectioned for histological hematoxylin–eosin (HE) (Zhongshanjinqiao, Beijing, China) and IHC staining. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Experimenters were not blind to experimental groups. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | No pre-test analyses were used to estimate sample sizes. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | No data were excluded from the final analyses. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The reagents used and their commercial sources are indicated as follows: Dimethyl sulfoxide (#HY-Y0320), Corn oil (#HY-Y1888), Imatinib Mesylate (IM) (#HY-50946), RSL3 (#HY-100218A), Ferrostatin-1 (#HY-100579), MG-132 (#HY-13259) and Cycloheximide (CHX, #HY-12320), all of which were purchased from Med-Chem-Express (MCE; Shanghai, China). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Categorical variables are expressed as percentages and compared using the χ2 test and Fisher’s exact tests when appropriate, while continuous variables were presented as means ± standard deviation (SD) and compared using a double-tailed Students’ t test. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The number of animals and experimental repeats is listed in individual legends. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Data meets the assumptions of the tests. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The sample variation within each group was estimated to ensure that the variance is similar. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | All statistical tests were two-sided and statistically significance was declared when the P-value was ≤ 0.05. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Error bar = mean ± standard deviation in all graphs. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Statistical analyses were performed using GraphPad Prism (version 8.0.2, San Diego, USA). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | First, we investigated the sensitivity of GIST-T1 and GIST-882 cells to IM. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The CCK-8 results showed that the IC50 was 80 nmol (GIST-T1) and 263 nmol (GIST-882) (Fig. 1A). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The experimental doses of Ferrostatin-1 blocked the death of GIST-T1 and GIST-882 cells treated with RSL3, indicating the existence of ferroptosis in GIST (Fig. S2). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Moreover, we examined whether Ferrostatin-1 could reverse the inhibitory effect of IM, and found that Ferrostatin-1 could partially rescue the inhibitory effect of IM on both GIST-T1 and GIST-882 cells (Fig. 1B).Fig. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | 1Imatinib (IM) induces ferroptosis in gastrointestinal stromal tumor (GIST) cells. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | A GIST-T1 and 882 cells were treated with different concentrations of IM for 24 h and the IC50 of IM was determined. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | B GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h in the absence or presence of Ferrostatin-1 (1 μM), and the cell viability was assayed. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | C Cell morphology was observed via transmission electron microscopy after the cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h. The area of mitochondria are quantitatively analyzed by using the ImageJ software. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | D, E GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) in the absence or presence of Ferr-1 (1 μM) for 12 h, the relative lipid ROS levels were measured using a C11-BODIPY lipid peroxidation sensor via confocal microscope (D) and flow cytometry (E). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | In fluorescence images, the increase in lipid ROS levels resulted in the oxidation of the polyunsaturated butadienyl portion of C11-BODIPY, causing a shift in fluorescence from red to green. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Flow cytometry analysis utilized the FITC filter for oxidized C11-BODIPY (emission: 510 nm) and the PE-TexasRed filter for reduced C11-BODIPY (emission: 590 nm). |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | The results are presented as a ratio of oxidized to reduced C11-BODIPY. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | F GIST-T1 and 882 cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h, and the relative levels of Fe were assayed. |
PMC10728200 | Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination | Scale bar, 50 μm in D. Experiments were independently repeated three times. |
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