Fig 2: Insulin secretion and mRNA assay in MIN6 cells after infection with equivalent titer of adenovirus.(A, B) Analysis of insulin secretion after glucose stimulation assayed by radioimmunoassay (RIA). (A) MIN6 cells were treated with 0.5 µM thapsigargin (TG) or DMSO (vehicle control) for 1 h, 0.5 mM palmitate (PA) or 0.5% BSA (vehicle control) for 12 h, and the supernatants were assayed for insulin protein level (n = 3). (B) MIN6 cells were infected with a series of double dilutions of Ad-NR4A3 adenovirus (adenovirus encoding NR4A3). Additional Ad-GFP (control adenovirus expressing GFP only) was used for complementary infection in order to ensure each infection had an equal virus titer. Post-infection, levels of secreted insulin were assayed by RIA (n = 4). Western blotting showed NR4A3 protein expression gradually decreasing, and insulin secretion level increasing accordingly. (C) mRNA levels of two insulin genes (Ins1 and Ins2) were determined with reverse transcription PCR in MIN6 cells infected with Ad-NR4A3/Ad-GFP. (D) Semi-quantitative analyses of Ins1 and Ins2 mRNA levels in different adenovirus-infected MIN6 cells (normalized to beta actin) (n = 6). Data are shown as mean ± S.E. # p<0.05; **, ## p<0.01 vs. the control group or Ad-GFP infection only.

Fig 3: Possible roles of NR4A3 in modulating the expression of insulin genes in MIN6 cell lines.(A) Diagram of constructed wild-type and deletion forms of NR4A3 cDNA. (B) Verification by western blotting MIN6 cell lines stably over-expressing NR4A3 or its deletion forms. (C) mRNA levels of NR4A3 or two insulin genes (Ins1 and Ins2) detected with reverse transcription PCR. Each image is representative of at least three experiments. (D) Semi-quantitative analyses of Ins1 and Ins2 mRNA levels normalized to beta actin in various stable cell lines (n = 5). ** P<0.01 vs. the control cells. Data are representative of three clone lines. Con, cell line transfected with vector encoding GFP; N, cell line expressing the wild-type NR4A3; ?A, cell line expressing the 2–288 amino acid (aa) deletion of AF1 (activation function-1 domain); ?D, cell line expressing the 292–364 aa deletion of the DNA binding domain (DBD); ?L, cell line expressing the 398–626 aa deletion of the ligand binding domain (LBD). The C terminal of all exogenous genes was HA-tagged to facilitate identification with western blotting.

Fig 4: Tunicamycin or dithiothreitol treatment resulted in UPR activation but no NR4A3 increase in MIN6 cells.(A, B) Relative mRNA levels of NR4A3 and Chop, respectively, determined with real-time quantitative PCR in response to different doses of tunicamycin (TM) in MIN6 cells. (D, E) Relative mRNA levels of NR4A3 and Chop, respectively, determined with real-time quantitative PCR in response to different doses of dithiothreitol (DTT) in MIN6 cells. (C, F) Spliced XBP1 (sXBP1) mRNA formation in response to different (C) TM or (D) DTT doses, respectively. Two forms of XBP1 (a UPR molecule) was detected with reverse transcription PCR. Data are shown as mean ± S.E. (n = 4). * p<0.05, ** p<0.01 vs. con (vehicle control group).

Fig 5: A model for possible role of NR4A3 in releasing pancreatic beta cells ER stress.Upon expression and activation of NR4A3 induced by factors such as long-chain free fatty acids (FFA) and thapsigargin (TG), which lead to ER stress, unfolded protein response (UPR) activation, and even apoptosis, this orphan nuclear receptor decreases insulin expression, which indirectly releases the burden of ER.