Fig 1: Activated PSCs promoted ferroptosis resistance in pancreatic cancer in vivo. (a–g) C57BL/6 mice were injected subcutaneously with Panc02 (Panc02 groups, 2 × 105 cells/mouse) or Panc02+PSCs (Co-Panc02 groups, 1.5 × 105 Panc02 cells + 0.5 × 105 PSCs). They were divided into Panc02 groups (treated with DMSO), Co-Panc02 groups (DMSO), Panc02+H (HGF, 50 ng/i.h., every two days), Panc02+E (Piperazine Erastin, 30 mg/kg/i.h., every two days), Co-Panc02+E (Piperazine Erastin, 30 mg/kg/i.h., every two days), and Panc02+H+E (HGF, 50 ng/i.h., Piperazine Erastin, 30 mg/kg/i.h., every two days). (a) Representative photographs of isolated tumor tissues in each treatment group at day 14. (b) Tumor volume was detected every two days. (c) The body weight of mice in each treatment group was measured every two days. (d, e)D-E. The GSH (d) and MDA (e) levels in isolated tumors were assayed at day 14 after different treatments. (f) Western blot analysis of protein expression levels of ferroptosis-related indicators (SLC7A11 and GPX4) of isolated tumor tissues in each treatment group. (g) Immunohistochemistry analysis of the expression of c-MET in isolated tumor tissues. (h) The expression of c-MET in PDAC patients and normal pancreatic tissues (T: tumor; N: normal) and the correlation between the expression of c-MET and the survival of PDAC patients were analyzed with the GEPIA database. (i) Analysis of the TCGA and GEPIA databases for the correlation c-MET mRNA expression level with the ferroptosis-related indicators (NRF2, SLC7A11, and GPX4). The TCGA database result was presented by heat map: n = 183. PE: Piperazine Erastin. Experiments were repeated three times, and the data were expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2: Inhibition of c-MET sensitizes pancreatic cancer cells to ferroptosis with the presence of activated PSCs. (a, b) Panc02 cells treated with DMSO, c-MET-IN-1, Erastin, Erastin+IN-1, RSL3, and RSL3+IN-1 cultured under normal or coculture conditions. Representative images showing the induction of cell death in Panc02 cells (a) and cell viability was measured by CCK-8 kits (b). (c–f) Panc02 cells were cocultured with activated PSCs and then treated with DMSO, IN-1 (0.05 µM), Erastin (2 µM), Erastin (2 µM)+c-MET-IN-1 (0.05 µM), RSL3 (0.1 µM), and RSL3 (0.1 µM)+c-MET-IN-1 (0.05 µM). Western blot analysis of the protein expression levels of c-MET, SLC7A11, and GPX4 in Co-Panc02 (c). ß-tubulin expression was detected as a loading control. The relative GSH (d) and MDA (e) concentrations of Co-Panc02 were analyzed; lipid ROS level of Co-Panc02 was evaluated by flow cytometry (f). (g) qRT-PCR and western blot analysis of shRNA-mediated knockdown of c-MET mRNA and protein levels in Panc02 cells. ACTB mRNA expression was detected as a loading control for qRT-PCR. ß-tubulin expression was detected as a loading control for western blot. (h) Co-Panc02 cells (Ctrl shRNA, c-MET shRNA1, and c-MET shRNA2) were treated with different concentrations of Erastin (0, 0.5, and 1 µM) or RSL3 (0, 0.05, and 0.1 µM) for 72 hours. Cell viability was measured by CCK-8 kits. (i) qRT-PCR analysis of SLC7A11 and GPX4 mRNA expression in Panc02 (Ctrl shRNA) and c-MET knockdown Panc02 (c-MET shRNA1, c-MET shRNA2, and c-MET shRNA3). (j–l) Co-Panc02 cells (Ctrl shRNA, c-MET shRNA1, and c-MET shRNA2) were treated with Erastin (2 µM) or RSL3 (0.1 µM). Western blot analysis of the protein expression levels of c-MET, SLC7A11, and GPX4 in Co-Panc02. ß-tubulin expression was detected as a loading control (j). The relative GSH and MDA concentrations of Co-Panc02 were analyzed (k); lipid ROS level of Co-Panc02 was evaluated by flow cytometry (l). IN-1 represents c-MET-IN-1. Experiments were repeated three times, and data were expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 3: p53 regulated erastin-induced ferroptosis by SLC7A11/xCT in schwannoma cells. (a) Fluorescence analysis of SLC7A11/xCT between schwannoma cells and schwannoma cells transfected with p53. Relative expression of SLC7A11/xCT decreased significantly in the control and p53-overexpression cells (scale bar: 20 µM). (b) Expression of proteins between the control and overexpression-p53 schwannoma cells. The cell viability was measured with CCK-8 following 24 h erastin treatment in different schwannoma cells.
Fig 4: The activated PSCs promote pancreatic cancer cell ferroptosis resistance. (a) A proposed model illustrating the coculture system of Panc02 cells. (b) Panc02 cells were treated with different concentrations of Erastin (0, 0.5, and 1 µM) or RSL3 (0, 0.05, and 0.1 µM) for 72 hours under normal or coculture and Fer-1 conditions. Cell viability was measured by CCK-8 kits. (c) qRT-PCR analysis of the mRNA expression levels of ferroptosis indicators (NRF2, SLC7A11, GPX4, FTH1, and NCOA4) in Panc02 cells under normal and coculture conditions. ACTB mRNA expression was detected as a loading control. (d) Western blot analysis of the protein expression levels of ferroptosis indicators (SLC7A11 and GPX4) in Panc02 treated with DMSO, coculture, Erastin (2 µM), and coculture+Erastin (2 µM). ß-tubulin expression was detected as a loading control. (e–h) Panc02 cells were treated with DMSO, coculture, Erastin (2 µM)/RSL3 (0.1 µM), and coculture+Erastin (2 µM)/RSL3 (0.1 µM). Iron (Fe2+) level of Panc02 was evaluated by flow cytometry (e). The relative GSH (f) and MDA (g) concentrations of Panc02 were analyzed. Lipid ROS level of Panc02 was evaluated by flow cytometry (h). Fer-1 represents ferrostatin-1. Co-Panc02 represents Panc02 cells which were cocultured with activated PSCs. Experiments were repeated three times, and data were expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 5: Western blotting analyzed the expression of GPX4, GSS, SLC7A11, which is ferroptosis key protein. **P < 0.01 vs the control group. Data were shown as mean ± SD, n = 3.
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