Fig. 3

Extracellular lipid deprivation enhances autophagy in PDAC cells. A Average lipid droplet (LD) size of HPDEK cells stained with BODIPY 493/503. Cells were first treated with 10 μM HCQ for 4 h followed by staining with BODIPY 493/503 and confocal fluorescence microscopy analysis. Statistical analysis was performed from ~ 150 cells/sample. HCQ: hydroxychloroquine; n = 5. B and C Representative images of LC3B-mCherry and LC3B-EGFP (B) and autophagic index quantification (C) of HPDEK cells transduced either with an empty vector (pLKO-puro, shControl) or 2 different shRNAs against ACSL3. Overnight starvation was used as a positive control for autophagy induction; n = 5–8. D Representative images (top) and co-localization score plot between mCherry and EGFP signal from the imaging flow cytometer of HPDEK cells treated with 2.5 μM Triacsin C for 24 h. E Immunoblot analysis of AsPC-1 and PANC-1 cells treated with normal or delipidated serum for 72 h followed by treatment with 10 μM HCQ for 4 h, when indicated. HCQ: hydroxychloroquine. F Immunoblot analysis of PANC-1 cells transduced as indicated and treated with delipidated serum for 72 h followed by treatment with 10 μM HCQ for 4 h and/or 1 mg/ml of oleic acid for 3 h, when indicated. HCQ: hydroxychloroquine. Graphical data are shown as the mean ± s.d. Statistical analyses were done using one-way ANOVA. **** P < 0.0001; n, number of biologically independent samples