Extended Data Fig. 3: Alterations in mitochondrial protein solubility are observed in human islets following pharmacologic induction of mitochondrial rather than ER protein misfolding.
(a) TFAM and LONP1 protein levels visualized by WB (Left) and densitometry (Right) of recombinant purified human TFAM and LONP1 to assess LONP1 protease activity in the presence of 5 μM CDDO or Vehicle (DMSO). LONP1 protein levels serve as a reference/loading control. n = 3 independent experiments/group. (b) Cellular component analysis of significantly upregulated insoluble proteins (left) and cellular component analysis of significantly downregulated soluble proteins (right) from 1 μM CDDO-exposed islets compared to DMSO control islets. n = 4 independent islet donors/group. (c) Cellular component analysis of significantly upregulated insoluble proteins (left) and cellular component analysis of significantly downregulated soluble proteins (right) from 1 μg/mL tunicamycin (TUN)-exposed islets compared to DMSO control islets. n = 4 independent islet donors/group. (d) Representative WB images of selected mitochondrial proteins of human islets and (e) quantification of protein expression by densitometry (normalized to VINCULIN). n = 4 independent islet donors/group. VDAC1 serves as a soluble mitochondrial protein loading control. VINCULIN serves as a loading control for both soluble and insoluble fractions. S, soluble fraction; P, insoluble fraction. (f) Representative immunofluorescence image (n = 4/group) depicting Proteostat visualization of protein aggregates and co-localization with mtHSP70 in islets of 8-week-old C57BL/6N mice exposed to 1 μM CDDO or DMSO control for 20 h. Scale bars (Insulin, mtHSP70, Proteostat, Merge), 12.5 μm; Scale bar (zoom), 8.5 μm. Pink dashed boxes within merged image denote regions visualized at higher magnification (Zoom - far right). (g) Quantitative RT-PCR of markers of the mitochondrial unfolded protein response (UPRmt) from RNA isolated from islets of 12-week-old C57BL/6N mice exposed to 1 μM CDDO or DMSO control for 24 h. n = 3 mice/group. All data in figure are presented as mean ± SEM. Statistical analysis: 3A and 3E, *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparisons test; 3B and 3C *P < 0.05 by hypergeometric test followed by multiple hypothesis testing using FDR-corrected P values (FDR < 0.05); 3G, *P < 0.05, **P < 0.01 by unpaired two-tailed Student’s t-test.
