Figure 2.

Palmitate does not induce CHOP protein expression or XBP1 splicing in adipocytes. (A and B) CHOP expression in in vitro differentiated (A) 3T3-F442A adipocytes and (B) 3T3-L1 adipocytes exposed to the indicated concentrations of palmitate complexed to BSA for 48 h. Relative (rel.) CHOP signals were corrected for the loading controls GAPDH or β-actin. The bar graphs show the average and standard error of three independent repeats. Differences are not statistically significant (p = 0.42 for 3T3-F442A adipocytes and p = 0.10 for 3T3-L1 adipocytes in a repeated measures ANOVA test that compares the treated samples to the untreated sample. Equal variabilities of the differences were assumed for the treated and untreated samples and Dunnett's correction for multiple comparisons^112,113 was used). 1 μM thapsigargin (Tg) was used as a positive control for induction of ER stress. Thapsigargin-treated samples were compared to untreated samples using a two-tailed, unpaired t-test. (C and D) XBP1 splicing in in vitro differentiated (C) 3T3-F442A adipocytes and (D) 3T3-L1 adipocytes incubated for 48 h with the indicated concentrations of BSA-complexed palmitate. % splicing indicates the percentage of spliced XBP1 mRNA, for which the average and standard error of three independent experiments are shown. Abbreviations: u—unspliced XBP1 mRNA, s—spliced XBP1 mRNA. (E) MTT assay on in vitro differentiated 3T3-F442A adipocytes incubated for 48 h with the indicated concentrations of BSA-complexed palmitate. A repeated measures ANOVA test was used to compare the treated samples to the untreated sample. Equal variabilities of the differences were assumed for the treated and untreated samples and Dunnett's correction for multiple comparisons^112,113 was applied. (F) XBP1 splicing in 3T3-F442A preadipocytes incubated for 12 h with the indicated concentrations of BSA-complexed palmitate. Abbreviations: * – P < 0.05, ** – P < 0.01, *** – P < 0.001, and **** – P < 0.0001.