Fig. 1

Palmitate induces ER stress and autophagy at early time points, but prolonged palmitate treatment impairs autophagy in a hypothalamic cell line. a and b Immunoblotting analysis (a) and quantification (b) of ER stress markers (p-PERK, PERK, p-IRE1, IRE1, ATF6, ATF4, and CHOP) in cells treated with different concentrations of palmitate (25, 50, and 100 μM) for the indicated times (n = 3 per each group). Data are mean ± SEM; p-PERK/PERK; *p = 0.0252, ***p = 0.0003 vs. control at 12 h, **p = 0.0065 vs. control at 24 h, ATF6; **p = 0.0040 vs. control at 12 h, **p = 0.0074 vs. control at 24 h, ATF4; *p = 0.0454, **p = 0.0017 vs. control at 12 h, *p = 0.00139 vs. control at 24 h, CHOP; *p = 0.0240, ****p < 0.0001 vs. control at 12 h, ***p = 0.0002 vs. control at 24 h. c and d Immunoblotting analysis (c) and quantification (d) of ER stress markers in cells treated with 0.1 mM palmitate for the indicated times (n = 3 per each group). Data are mean ± SEM; p-PERK/PERK; **p = 0.0050, ***p = 0.0007, ****p < 0.0001 vs. control, ATF6; **p = 0.0091 vs. control, ATF4; **p = 0.0033 at PA 6 h vs. control, **p = 0.0067 at PA 12 h vs. control, ***p = 0.0009 vs. control, CHOP; ****p < 0.0001 vs. control. e and f Immunoblotting analysis (e) and quantification (f) of autophagy markers (p62 and LC3-II) in cells treated with 0.1 mM palmitate for the indicated times. Baf A1 (200 nM) was added for 3 h before harvest (p62; n = 7 for control, control + Baf A1, PA 3 h, PA 3 h + Baf A1, n = 3 for PA 6 h, PA 6 h + Baf A1, PA 12 h, PA 12 h + Baf A1, LC3-II; n = 8 for control, n = 7 for control + Baf A1, PA 3 h, PA 3 h + Baf A1, n = 4 for PA 6 h, PA 6 h + Baf A1, n = 3 for PA 12 h, PA 12 h + Baf A1. Data are mean ± SEM; p62; *p = 0.0295 at PA 6 h vs. control, *p = 0.0250 at 12 h vs. control, ****p < 0.0001 vs. control, ^###p = 0.0008, LC3-II; *p = 0.0444, ***p = 0.0003, ****p < 0.0001 vs. control, ^####p < 0.0001. g and h Cells transiently expressing mRFP-GFP-LC3 were treated with 0.1 mM palmitate for the indicated times. Representative micrographs (g) and quantification (h) of the average number of mRFP-GFP-LC3 puncta per cell (n = 18 per each group). Scale bar, 10 μm. Data are mean ± SEM; Red puncta; *p = 0.0158, ****p < 0.0001 vs. control, ^####p < 0.0001, Yellow puncta; *p = 0.0257, ****p < 0.0001 vs. control, ^†p = 0.0174. n.s., no significant difference