Fig 2: TRB3 is not required for development of insulin resistance in ER-stressed C2C12 cells. (A) siRNA-mediated knockdown of TRB3 at the mRNA level 48 h after transfection of C2C12 cells with 50 nM of the indicated siRNAs. TRB3 mRNA was determined by reverse transcriptase-qPCR and normalized to ACTB. Bars represent the SE from three technical replicates. p values were calculated by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (B) siRNA-mediated knockdown of TRB3 at the protein level 48 and 72 h after transfection of C2C12 cells with 50 nM of the indicated siRNAs. (C) Twenty-four hours after transfection with the indicated siRNAs, C2C12 cells were exposed to 300 µM thapsigargin, 1 µg/ml tunicamycin, or 1 µg/ml SubAB for 24 h and serum-starved during the last 18 h of exposure to ER stressors before being stimulated with 100 nM insulin for 15 min. Phosphorylation of AKT at S473 and insulin receptors were analyzed by Western blotting. (D) Quantification of the relative phosphorylation of AKT at S473 in the Western blots of panel C. Bars represent SEs (n = 5 for cells transfected with siRNAs against eGFP, n = 3 for all other samples). p values were calculated by ordinary two-way ANOVA with Dunnett’s correction for multiple comparisons. (E) Quantification of the relative abundance of a-ß proreceptors in the Western blots of panel C. Bars represent SEs (n = 6 for cells transfected with siRNAs against eGFP, n = 3 for all other samples). p values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test to compare of samples to the insulin-stimulated, unstressed sample and Tukey’s multiple comparisons test to compare different siRNAs.

Fig 3: Bypass of the ER in insulin receptor synthesis abrogates ER stress–induced insulin resistance. (A) Schematic of the WT insulin receptor, the myristoylated FV2E-insulin receptor chimera, and activation of the chimera by AP20187. Black boxes represent the signal peptide sequence and transmembrane domain of the insulin preproreceptor, striped boxes the protein tyrosine kinase domain of the insulin receptor, and checkered boxes individual FV domains. Disulfide bonds that link a to ß chains and two insulin receptor monomers are shown as gray lines. Abbreviations: M, Myr, myristoylation signal. (B) Autophosphorylation of the FV2E-insulin receptor chimera in stably transfected Flp-In T-Rex 293 cells. Expression of the chimera was induced for 27 h with 1 µg/ml tetracycline, followed by dimerization of the chimera with 100 nM AP20187 for 1 or 4 h. (C) C2C12 cells were transiently transfected with pmaxGFP or pcDNA5/FRT/TO-MyrFV2E-INSR. Twenty-four hours after transfection, ER stress was induced for 24 h with 0.1 µM thapsigargin, 0.1 µg/ml tunicamycin, or 1 µg/ml SubAB followed by dimerization of the receptor chimera with 100 nM AP20187 for 4 h. “#” indicates an unspecific band. Quantification of (D) S473 AKT phosphorylation and (E) the relative abundance of a-ß proreceptors in panel C. Bars represent SEs (n = 3-4). p values for comparison of the treated to the untreated samples were calculated by ordinary one-way ANOVA using Dunnett’s multiple comparisons test. (F) Induction of TRB3 in C2C12 cells by ER stress. C2C12 cells were treated with 300 nM thapsigargin, 1 µg/ml tunicamycin, 1 µg/ml SubAB, or 1 µg/ml SubAA272B for 24 h. TRB3 mRNA levels were determined by reverse transcriptase-qPCR and standardized to the loading control ACTB. Bars represent SEs (n = 3). p values for comparison of treated samples to the untreated sample (“–”) were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test, taking data shown in Figure 1, E and F, into account.