Activated Stellate Cell Paracrine HGF Exacerbated Pancreatic Cancer Cell Ferroptosis Resistance

Figure 3

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). β-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. β-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. β-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 . , , and .

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)