Fig. 3

Triglyceride synthesis and lipid droplets are upregulated in PDAC cells starved of BCAAs. a Left, imaging of neutral lipids using BODIPY™ 493/503 (green) and nuclei using Hoechst 33342 (blue) depicts build-up of lipid droplets in PaTu 8902 and MIA PaCa-2 cells. Right, the quantification of BODIPY™ 493/503 fluorescence intensity expressed as as per-cent of HOECHST 33342. N = 2. n ≥ 4. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons tests. b Flow cytometry of PaTu 8902 (up) and MIA PaCa-2 (down); BODIPY™ 493/503 mean fluorescence intensities calculated from 10⁴ events. Cells were starved of BCAA for 48 h prior experiment. Color coding: black for control, green for 3-MA, orange for CQ, corn yellow for BafA, blue for -BCAA, red for BCKA, light blue for DGAT1i, pink for DGAT2i, grey for charcoal-stripped serum. One-way ANOVA followed by Tukey’s multiple comparisons tests. N > 3, n ≥ 6. Criteria of significance: *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. c Working scheme (right) depicting inhibitors used in the experiments, potentially causative in LD formation. Carnitine palmitoyltransferase 1A inhibitor (CPT1, etomoxir (ETO), 50 µM), diacylglycerol-O-acetyl-transferase 1 inhibitor (DGAT1i, A922500, 10 µM), diacylglycerol-O-acetyl-transferase 2 inhibitor (DGAT2i, PF-06424439, 20 µM), autophagy initiation inhibitor 3-methyladenine (3-MA, 1 mM), autophagy inhibitor bafilomycin A1 (BafA, 100 nM), autophagy inhibitor chloroquine (CQ, 100 µM), charcoal-stripped FBS (CHS-FBS). Created with BioRender.com