Fig. 1: OA induces LD formation via DGAT1 in cultured MCECs.

MCECs were seeded overnight, and upon reaching full confluence, were treated with the indicated dose of oleic acid (OA) for the specified times. A–D Representative immunofluorescence images of Bodipy staining and plate-read fluorescence intensity quantification showing that OA increased LD formation in a dose- and time-dependent manner. E–I MCECs were pretreated with or without 5 µM DGAT1 inhibitor A-922500 (DGAT1i) or 10 µM DGAT2 inhibitor PF-06424439 (DGAT2i) overnight, followed by treatment with 500 µM OA for 6 h. Representative Bodipy-stained images (E) and plate-read fluorescence intensity quantification (F) indicated that DGAT1 inhibition blocked OA-induced LD formation. G Plin2 mRNA levels were measured, showing no significant effect of DGAT1 inhibition on transcription. H, I Representative immunoblots and quantification of PLIN2 protein levels demonstrate that DGAT1 inhibition significantly reduces OA-induced PLIN2 expression. Original uncropped WB bands for Fig. 1H were presented in Supplementary Fig. 3. J–L MCECs were transfected with 20 nM siRNA targeting Dgat1 (si-Dgat1) or control siRNA (si-Ctrl) for 24 h, followed by 500 µM OA treatment for 6 h. Bodipy staining (J) and plate-reader fluorescence intensity quantification (K) show that Dgat1 knockdown blocked OA-induced LD formation. L Dgat1 mRNA levels confirming successful knockdown. RFU, relative fluorescence unit. Images in A, C, E and J were acquired using a 40× objective and a standard 10× eyepiece (effective ×400 magnification). Scale bar =20 µm. Data in B, D, F, G, and L are shown as fold-changes relative to control (CTRL = 1), whereas I and K are normalized to the OA group (OA = 1). All datasets analyzed by the Kruskal–Wallis test with Dunn’s multiple-comparisons post hoc test (B, D, F–H, K) or by the Mann–Whitney test (L). *P < 0.05 vs. 0 (B, D), CTRL (F–H), or si-C (K, L); ^#P < 0.05 vs. OA (F, I) or si-C + OA (K). n = 4.