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: EPA, DPA, and DHA attenuate PAL-induced increases in the ER stress markers BiP and CHOP. Representative images of ER stress proteins BiP and CHOP following 16 h treatment with 500 µM PAL and/or 50 µM EPA, DPA or DHA (A) and representative image of a stain free blot used for normalization shown with molecular weight markers indicated. Quantified protein content of BiP (B) and CHOP (C). In the absence of PAL, little to no CHOP protein was detectable. All densitometry values are expressed relative to protein content determined by stain free imaging and presented in arbitrary units relative to CON. Treatments that do not share letters are considered statistically significant (p < 0.05; myoblasts were cultured and harvested independently to generate a total sample size of 5 biological replicates). Values are presented as mean ± SEM.

Fig 4: SELENOK deficiency-induced SELENOS upregulation was involved in ERS in imDCs. (a–f) The protein levels of SELENOS and the ERS markers ATF4, ATF6, BIP and CHOP in WT and KO imDCs after treatment with Tm were assayed by Western blotting, and the density values were calculated (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 were compared with DMSO- or Tm-treated WT imDCs. SELENOK, selenoprotein K; SELENOS, selenoprotein S; ERS, endoplasmic reticulum stress; imDCs, immature dendritic cells; ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; BIP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; Tm, tunicamycin; DMSO, dimethyl sulfoxide; WT, wild-type; KO, knockout.

Fig 5: MA-mediated ER stress involved caspase-3 and caspase-9 in astrocyte cell deathA-F. SVGA cells were seeded at 6 × 105 cells in a 6-well plate and transfected with various siRNA as mentioned in methods. After 48 hours, the cells were reseeded in 12-well plate and allowed to adhere overnight. Finally, these cells were treated with 500 µM MA for additional 24 hours. The effect of specific siRNA against ATF6 (A), PERK (B), ATF4 (C), IRE1a (D), p65 (E) and CHOP (F) were assessed on the levels of cleaved and pro-caspase-3 and caspase-9 using western blotting. The western blots are representative images of at least 3 independent experiments. The numbers above the blots represent mean intensity of the respective bands.