Fig 2: 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

Fig 3: Absence of UPR and ISR activation in the liver of mistranslating A226Y mice. (a) Western blots showing total and specific phosphorylation levels for key players in the three branches of the ER-UPR. Significance level for comparison between A226Y and WT (N = 5): p-IRE1 (Ser724)/IRE1a p = 0.43; p-eIF2a (Ser51)/eIF2a p = 0.55; GADD34 p = 0.77; ATF6 p = 0.48, BiP p = 0.64. Densitometry values were normalized to tubulin as the loading control. (b) qRT-PCR quantification of spliced XBP1 relative to un-spliced XBP1 (N = 4, p = 0.94). (c) Western blot of total ATF4 protein normalized to tubulin (N = 5, p = 0.01). (d) Western blot of total Bim protein normalized to tubulin (N = 5, p = 0.45). Plots show mean, SEM, and individual data points. * p < 0.05, calculated by unpaired two-sided Student’s t-test. N = number of independent mice in each comparison. Full gel images are provided in the Supplementary Materials.

Fig 4: Tg impacts M2(IL-4) phenotype via the UPR IRE1α pathway.A Membrane expression by FACS analysis of CD80, CD206 and Dectin-1 on M1 and M2 macrophages treated or not with Tg or Tm (n = 6–9). B Western-blot analysis of MMP-9 (n = 3) in M1 and M2 macrophages treated or not with Tg or Tm during 24 h. C Western-blot analysis of signalization proteins pSTAT1/STAT1 and pSTAT6/STAT6 (n = 5) in 24 h activated M1 and M2 macrophages treated or not with Tg or Tm. D pSTAT1/STAT1 signalization proteins in 24 h activated M2 macrophages treated or not with Tg, Tm or carbachol (cch, 1 μM) (n = 3). E Western-blot analysis of signalization proteins pSTAT1/STAT1 in 24 h activated M2 macrophages treated or not with Tg and with or without 5 μM of ceapin A7, GSK2606414 and MKC 3946, inhibitors of the ATF6, PERK and IRE1α UPR pathway respectively (n = 4). *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001).

Fig 5: PA induces endoplasmic reticulum stress in podocytes. (A) Representative images of BIP, PERK, ATF4, CHOP, ATF6 and IRE1α expression, as detected by western blot analysis after treatment with different concentrations of PA. Densitometric analysis of the results shown in (A) for (B) BIP, (C) PERK, (D) ATF4, (E) CHOP, (F) ATF6 and (G) IRE1α. Data are presented as the mean ± SEM (n=3). *P<0.05 vs. 0 µmol/l group, #P<0.05 vs. 50 µmol/l group, ΔP<0.05 vs. 150 µmol/l group. (H) BIP expression was detected by fluorescence microscopy after the cells had been treated with PA (150 µmol/l) for 24 h (magnification, ×400). The rightmost panels show the enlarged (×7) views of areas in the left panels. (I) Fluorescence intensities of five random fields per group, as shown in (H), were determined and analyzed. Data are presented as the mean ± SEM (n=3). *P<0.05 vs. control group. PA, palmitic acid; BIP, binding immunoglobulin protein; PERK, protein kinase RNA-like endoplasmic reticulum kinase; ATF, activating transcription factor; CHOP, C/EBP homologous protein; IRE1α, inositol-requiring enzyme 1α; SEM, standard error of the mean.