Activated Stellate Cell Paracrine HGF Exacerbated Pancreatic Cancer Cell Ferroptosis Resistance

Figure 2

HGF secreted by activated PSCs mediated ferroptosis resistance in pancreatic cancer cells. (a) qRT-PCR analysis of mRNA expression levels of multiple secreted factors (CXCL-16, TNF-α, CCL7, HGF, TGF-β, PDGF, CCL2, VEGF, SDF1-A, IGF-1, IL-6, b-FGF, and CCL5) in PSCs under normal or coculture conditions. (b) ELISA-based analysis of the HGF concentration in PSCs under normal and coculture conditions. (c) Panc02 cells were treated with HGF (5 ng/ml) and different concentrations of Erastin (0, 0.2, 0.4, 0.6, 0.8, and 1 μM) or RSL3 (0, 0.02, 0.04, 0.06, 0.08, and 0.1 μM) for 72 hours. Cell viability was measured by CCK-8 kits. (d, e) qRT-PCR and western blot analysis of the mRNA and protein expression levels of ferroptosis indicators (SLC7A11 and GPX4) in Panc02 cells treated with DMSO, HGF (5 ng/ml), Erastin (2 μM), or HGF (5 ng/ml)+Erastin (2 μM). ACTB mRNA expression was detected as a loading control for qRT-PCR. β-tubulin expression was detected as a loading control for western blot. (f–h) Panc02 cells were treated with DMSO, HGF (5 ng/ml), Erastin (2 μM)/RSL3 (0.1 μM), or HGF (5 ng/ml)+Erastin (2 μM)/RSL3 (0.1 μM). The relative levels of GSH (f) and MDA (g) of Panc02 were assayed by test kits, and the relative levels of Panc02 lipid ROS were detected by flow cytometry (h). Experiments were repeated three times, and data were expressed as the . , , and .

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