PMCID string | Title string | Sentences string |
|---|---|---|
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The newest data indicated an enormous potential of JNK inhibitors in NB treatment, as they have been reported to induce NB cell death via direct activation of p53, Bcl-2, and caspase-dependent pathways . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | NSC697923, an inhibitor of the ubiquitin-conjugating enzyme E2 N, promoted NB cell death by regulating both p53 and JNK pathways, apoptosis induction, and colony formation inhibition in a dose-dependent manner across multiple MYCN-amplified and MYCN-non-amplified NB cell lines . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The compound also demonstrated in vivo antineoplastic activity in NB orthotopic xenografts. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Bortezomib, a proteasome inhibitor, suppressed cell growth and angiogenesis in SH-SY5Y and CHP126 NB cell lines by modulating the JNK pathway, while its combination with all-trans retinoic acid enhanced tumour growth inhibition in human NB xenografts and a mouse model . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Recently, a highly selective in vitro JNK3 Inhibitor, FMU200, was reported to decrease cell viability in SH-SY5Y cells . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The newest data showed that another selective JNK3 inhibitor, piceatannol, was highly effective in apoptosis induction by the inhibition of Cyt-C/ Bcl-2/caspase-3-dependent pathway, protecting SH-SY5Y cells from hypoxic insult . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The other possible mechanism of activity of JNK inhibitors in NB is a disruption in cancer stemness maintained by the STAT3-JNK axis, evidenced by the remarkably lower viability of IMR5, NLF, and SK-N-AS NB cell lines, and the downregulation of the expression of the stem cell marker CXCR4 after treatment with SP600125 or JNK-IN-8 inhibitors . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Our study is the first to comprehensively investigate the anti-neoplastic effect of AS601245 (JNK inhibitor V) on the SH-SY5Y cell line via a detailed assessment of the cellular effects of the compound on cell functions. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | JNK V demonstrated a dose-dependent reduction in the proliferation of T-cell acute lymphoblastic leukaemia (T-ALL) cells by inducing apoptosis and cell cycle arrest, accompanied by a reduction in c-Myc and Bcl-2 protein levels . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Moreover, JNK V significantly diminished the adhesion and migration of multiple human colon cancer cell lines through specific gene expression modulation . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Recent gene analysis has reported that the JNK V inhibitor also shows promise for use in breast cancer treatment . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | However, the potential of JNK V has not yet been elucidated in any pre-clinical or clinical trials with regard to NB. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Importantly, the JNK V inhibitor has the most potent impact on the least-explored isoform of JNKs, JNK3. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | As NB is a neural crest-derived malignancy with a high propensity for metastasis, we selected this compound for its high specificity; strong pro-apoptotic effects, evidenced in other cancers; and its neuroprotective and anti-inflammatory properties, which may help minimise excessive toxicity to neural tissue . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | An important notion that emerges from our study is that JNK inhibition induced by the chosen inhibitor is not cytotoxic towards primary human Schwann cells (HSC)—a well-established control model for NB in vitro research. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Furthermore, JNK inhibition may also modulate the tumour microenvironment (TME) by influencing stromal development, specifically Schwannian stroma, which plays a crucial role in NB to GNB maturation . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Our results demonstrated that JNK inhibitor treatment reduced the invasion abilities of SH-SY5Y cells, so it is reasonable to hypothesise that JNK inhibition could favour the development of a more organised, Schwannian-rich stroma. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | JNK inhibitors might induce a more permissive environment for stromal differentiation and maturation by reducing tumour cell invasiveness, which may shift the NB cells towards a phenotype closer to GNB, which is associated with better prognosis . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Also of particular value in our research is the Seahorse mitochondrial metabolism analysis, which reveals a novel aspect of JNK V’s anti-neoplastic mechanism—significant disruption of both oxidative phosphorylation and glycolysis—which, together with pro-apoptotic gene regulation, ultimately contributes to its overall antineoplastic efficacy. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | A major limitation of our in vitro study is that all experimental data are generated in a single neuroblastoma cell line, SH-SY5Y, which is an MYCN-non-amplified NB cell model that represents only low- and intermediate-risk NB. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | To investigate the effect of JNK inhibition on an appropriate model of high-risk NB patients and to be able to ensure the generalizability of the results, further experiments on MYCN-amplified cell lines are mandatory. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Furthermore, future research should include not only additional NB cell lines but also primary tumour-derived cells, supportive stromal cell types, and in vivo studies to better understand tumour cell death mechanisms and more accurately reflect the TME and disease complexity. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Secondly, exclusive pharmacological JNK inhibition with only a single ATP-competitive inhibitor was tested. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Further genetic validation, including JNK silencing, knockout models, or rescue strategies, is necessary to confirm the mechanistic basis of the observed effects. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | As an exclusive reliance on a single inhibitor is limited, additional orthogonal approaches would further strengthen causal inference and also exclude off-target effects. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Thirdly, another important note is that the NB cell death observed in our cytotoxicity experiments may not be solely attributed to apoptosis, but also to alternative mechanisms such as necrosis or necroptosis, which, though not examined in this study, could also have contributed to the observed effects. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Fourthly, and moreover, the short 24 h incubation period in toxicity and mechanistic experiments may capture only the acute, short-term effects and cellular responses of JNK inhibition in NB cells, necessitating extended exposure times to access long-term cellular responses and long-term toxicity. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Further experiments with longer incubation times are necessary. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The SH-SY5Y human neuroblastoma cell line (ATCC, Manassas, VA, USA) is a thrice-cloned subline of the SK-N-SH cell line and constitutes a well-established, neuronally relevant NB in vitro model . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The cells were cultured in Eagle’s minimum essential medium (EMEM) (ATCC, Manassas, VA, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (ATCC, Manassas, VA, USA) as well as 1% penicillin–streptomycin (ScienCell, Carlsbad, CA, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | As a relevant control for NB in vitro research, primary Human Schwann Cells (HSC) (ScienCell, Carlsbad, CA, USA) isolated from the human spinal nerve were used. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | These neural crest-derived cells ensheathe and myelinate axons of peripheral nerves, and interact with cancerous cells in the tumour microenvironment (TME) . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The cells were cultured in Schwann Cell Medium (ScienCell, Carlsbad, CA, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | As an additional control, widely used in toxicology and cellular biology research, the BJ cell line (ATCC, Manassas, VA, USA)—human fibroblasts from normal foreskin—was used . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Cells were cultured in EMEM supplemented with 10% FBS, 200 mM L-glutamine (Sigma-Aldrich, Saint Louis, MO, USA), and 1% antibiotics solution. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Using those two different normal cell lines as controls allows us to evaluate both the relevance and broader safety profile of the tested compound. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | All cell lines were cultured in the same incubator at standard conditions (37 °C, 5% CO2, 95% humidity). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Every 2 to 3 days, the cell culture media were replaced. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | After the cells reached 70–80% confluence, cell passages were performed, with a brief rinse with Dulbecco’s Phosphate-Buffered Saline (DPBS) (ScienCell, Carlsbad, CA, USA), and detachment using 0.25% Trypsin-EDTA solution (ScienCell, Carlsbad, CA, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | For each experiment, the cell culture was not expanded beyond passage 15. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | To selectively inhibit the JNK pathway, we have used the commercially available JNK inhibitor AS601245 (JNK inhibitor V, JNK V)—1,3-Benzothiazol-2-yl-(2--4-pyrimidinyl) acetonitrile (Sigma-Aldrich, Saint Louis, MO, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | It is a potent, reversible, and cell-permeable ATP-competitive inhibitor of c-Jun N-terminal kinases (IC50 values: 150 nM for hJNK1, 220 nM for hJNK2, and 70 nM for hJNK3) with anti-inflammatory characteristics and a 10- to 100-fold better selectivity compared to a panel of 25 other commonly researched kinases . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The JNK V showed notable antineoplastic properties in multiple T-ALL and colon cancer in vitro studies . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Importantly, it exerts the strongest inhibitory effect on the JNK3 isoform, whose role in tumours is not fully established. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The inhibitor’s efficacy and well-established in vivo safety profile have been demonstrated in gerbils, mice, and rats following oral, intraperitoneal, and intravenous administration . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The compound was reconstituted in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Saint Louis, MO, USA) and maintained at −20 °C in darkness. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The vehicle concentration in the culture medium was not more than 0.01%. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Furthermore, cultures treated with 0.01% DMSO alone were used to rule out the potential effects of the vehicle. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The cytotoxicity assessment of the evaluated JNK V compound was conducted using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay (Thermo Scientific, Waltham, MA, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The experiment was performed on SH-SY5Y, HSC, and BJ cell lines. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Cells were seeded at a density of 5 × 10 cells/well in a 96-well plate and cultured in 100 μL of the complete growth medium for 24 h. The cells were then treated and incubated with a particular compound for a standardised time of 24 h to assess the short-term effects of the investigated compound. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | SH-SY5Y cells were exposed to a wide concentration range of JNK V (0.1–100 µM). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | HSC and BJ cells were treated with JNK V at concentrations of 0.1–25 µM, because of their substantial sensitivity to DMSO at the concentration required to dissolve higher doses of the investigated compound, specifically 50 and 100 µM JNK V, together with the reduced solubility and partial precipitation of the compound, specifically in Schwann Cell Medium. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Cells treated with 20% DMSO served as a positive control. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Cells treated with 0.01% DMSO served as the solvent control, while untreated cells constituted the negative control. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | After incubation for 24 h, 75 μL of XTT/PMS suspension was added to each well in accordance with the manufacturer’s protocol. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | After 2 h of incubation in a 5% CO2 incubator at 37 °C, the absorbance measurement was performed using the Synergy HT spectrophotometer (BioTek, Shoreline, WA, USA) at a wavelength of 450 nm. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The morphology of SH-SY5Y cells was assessed using an inverted microscope (Nikon, Tokyo, Japan), after 24 h incubation of the cells with, respectively, 0.1–100 µM JNK V in the experimental group, 0.01% DMSO in the solvent control group, or 20% DMSO in the positive control group. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | For each experimental condition, at least 10 random fields were acquired, capturing around 200 cells per replicate. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Images were captured at 10× magnification, under identical exposure settings. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | All images were saved in TIFF format. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Morphological changes, including cell shape, adhesion, neurite outgrowth, cytoplasmic integrity, and nuclear condensation, were analysed qualitatively. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | A clonogenic assay, which assesses the ability of a single cell to form a colony over 14 days, was performed on SH-SY5Y cells to evaluate their proliferation and survival abilities. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Cells treated with 0.1–25 μM JNK V constituted the experimental control, while untreated cells served as a negative control . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Firstly, cells at a density of 2 × 10 cells/well were seeded on a 12-well plate with EMEM supplemented with 10% FBS. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | After being incubated for 72 h, the JNK V inhibitor was applied. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The media was refreshed every 3 days, and cells were grown for a total of 2 weeks. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Ultimately, the cells were washed with DPBS, fixed in 10% neutral buffered formalin (NBF) (Sigma-Aldrich, Saint Louis, MO, USA) for 30 min, and visualised using 0.1% crystal violet (Sigma-Aldrich, Saint Louis, MO, USA) for 30 min. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The plate was then allowed to dry. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The colony-forming abilities of cells were assessed using an inverted microscope (Nikon, Tokyo, Japan). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | For each experimental condition, at least 10 random fields were acquired, capturing around 50 cells per replicate. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Images were captured at 400× magnification, under identical exposure settings. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Confluence was quantified based on the percentage of the cell-covered surface area within randomly selected 100 × 100 µm image fragments. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The proportion of the area covered by cells was calculated using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The wound healing (scratch) assay was performed to assess the migration abilities of SH-SY5Y cells after treatment with JNK at concentrations of 0.1–3 μM, which maintained cell viability over 80%, confirmed by a previous XTT assay . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Cells were seeded in a 24-well plate at a density of 2 × 10 cells/mL in an EMEM with 10% FBS. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | After reaching full confluence, a 200 μL sterile plastic tip was used to scrape the cell monolayer longitudinally on each well. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | All wells were then intensively rinsed three times with DPBS to mechanically eliminate non-adherent floating cells. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | To suppress the contribution of cell proliferation but allow cells to survive for the following 72 h, cells were incubated in a medium supplemented with a low serum concentration (1% FBS) together with an inhibitor at an appropriate concentration. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Untreated cells constituted a negative control. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Subsequently, the scratched area was carefully viewed, and representative images were captured at 0 h, 24 h, 48 h, and 72 h after wounding time points using an inverted phase-contrast microscope (Nikon, Tokyo, Japan). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Images were captured at 100× magnification, under identical exposure settings. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | All images were saved in TIFF format and analysed using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The percentage of wound closure was calculated by identifying the low-texture region corresponding to the acellular wound area in the Laplacian texture profile and expressing the reduction in this area relative to the initial wound width, as recommended . |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The Caspase-3 colorimetric activity assay (Abcam, Cambridge, UK) was performed to assess the effect of JNK inhibition on the apoptosis of SH-SY5Y cells. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Cells treated with 0.75–100 µM of JNK V for 24 h constituted the experimental sample, the negative control comprised untreated cells, whereas positive control cells were treated with 10 μM staurosporine for 24 h. Following incubation, the cells were dissociated with Trypsin/EDTA solution and centrifuged. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Subsequently, the samples were prepared for further protein isolation by resuspension of the cell pellet in 50 μL of cold lysis buffer, incubation for 10 min on ice, centrifugation at 10,000× g for 1 min, transfer of supernatant to new microcentrifuge tubes, and maintenance on ice. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Then, the Pierce Bicinchoninic Acid (BCA) Protein Assay (Thermo Scientific, Waltham, MA, USA), calibrated to 100 μg of protein per sample, was performed to determine the protein concentration. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Briefly, each well of the microplate was filled with 50 μL of sample per well (except for background wells), 50 μL of freshly prepared Caspase Reaction Mix using 2X Reaction Buffer with dithiothreitol (DTT), 5 μL of the substrate solution (4 mM Asp-Glu-Val-Asp-para-nitroanilide (DEVD-pNA)), and then incubated for 120 min at 37 °C. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | By monitoring the samples’ absorbance at 400 nm in the Synergy HT Microplate Reader (BioTek Instruments, Winooski, VT, USA), the quantity of p-NA was measured quantitatively. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The expression of selected genes connected with apoptosis and cellular damage control was evaluated by qRT-PCR analysis in cells treated with JNK V at 1 μM, 10 μM, and 25 μM and 0.01% DMSO as a solvent control. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Untreated cells constituted the control. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Following incubation with the investigated compound, the PureLinkTM RNA Mini Kit (Invitrogen, Waltham, MA, USA) was used to extract the total RNA, in line with the manufacturer’s guidelines. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Then, the obtained samples’ RNA levels were measured and normalised by use of the Synergy HT Microplate Reader (BioTek Instruments, Winooski, VT, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Subsequently, using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA), a final concentration of 100 ng of cDNA was produced by transcription of the isolated RNA, according to the manufacturer’s protocol. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Then, the expression profile of the MAPK10 (Hs00959268_m1), BCL2 (Hs00608023_m1), BAX (Hs00180269_m1), and NRF2 (Hs00232352_m1) was analysed using the TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | GAPDH (Hs99999905_m1) served as a reference gene. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | The overall reaction volume was 20 μL, containing 1 μL probes, 1 μL cDNA, 10 μL TaqMan Universal PCR Master Mix II (Applied Biosystems, Waltham, MA, USA), and 8 μL nuclease-free water (Invitrogen, Waltham, MA, USA). |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | Ultimately, the Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA) was used to conduct the PCR reaction in the following sequence: initial denaturation (15 min, 95 °C); cycling–denaturation (10 s, 95 °C); and annealing/extension (60 s, 60 °C), for 40 cycles each. |
PMC12732662 | Pharmacological Inhibition of JNK Signalling Exerts Anti-Neoplastic Effects on SH-SY5Y Human Neuroblastoma Cells | All collected data was quantified using 2 values. |
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