Fisetin induces autophagy in pancreatic cancer cells via endoplasmic reticulum stress- and mitochondrial stress-dependent pathways

a Cell viability of PANC-1 cells was measured by CCK-8 assay. Cells were treated with fisetin (0, 25, 50, 100, 200, and 400？μM) for 24 and 48？h, respectively. The absorbance was measured at 450？nm. Data are presented as mean？±？SD; *P？≤？0.05, #P？≤？0.05. b Real-time cell analysis (RTCA) of pancreatic cancer PANC-1 cells. Cells were seeded in 96 well Eplates. Then cells were treated with fisetin (0–400？μM)for 72？h. Cell growth was monitored using the xCELLigence RTCA DP Instrument (Roche). Impedance was recorded every 15？min using an xCELLigence system. The times on the x-axis indicate the times after treatment. The Cell Index on the y-axis reflects a consistent, logarithmic relationship to cell number. Each point represents the mean value from three replicates with SDs. c A total of 1？×？106 PANC-1-Luciferase cells were injected into the shoulders via a left subcostal incision. Control group mice were treated with DMSO via intraperitoneal injection, and treatment group mice were treated with Fisetin (300？mg/kg body weight i.p.) every other day. After 20 days treatment, mice imaged for bioluminescence. Data were expressed as average radiance (photons/second/square centimeter/steradian). d The differences in tumor volume between control and fisetin treatment groups at 20 days after treatment. Results are mean？±？SD (n？=？5), **P？≤？0.01. e The expression levels of PCNA, cleaved-caspase 3 in control and fisetin treatment groups of mice were detected by immunohistochemistry. Bar scale 50？μm. f Western blotting analyses of Ki67, PCNA, p-H3, cleaved-caspase3, and cleaved PARP. g Apoptosis of cells with treatment. Cells were treated with fisetin (200？μM) for 24 and 48？h, followed by Annexin-V/PI staining and flow cytometry analysis. Data are presented as mean？±？SD; **P？≤？0.01, ****P？≤？0.0001, NS no significanc