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. 2021 Nov 2;22(21):11906. doi: 10.3390/ijms222111906

Figure 3.

Figure 3

DIO2 knockdown results in impaired mtUPR activation, increased ROS and decreased viability and an impaired metabolic transcriptional landscape. (A) Western blot results for mtHSP70 protein levels (n = 6 independent samples; corrected for GAPDH). (B) RT-qPCR derived expression profiles of genes involved in mitochondrial UPR and ER stress and ER stress-induced apoptosis (GDF15, ATF4, CHOP, XBP1s, BiP, PDIA5 and GADD34; n = 5 independent samples; corrected for 36B4). (C) Baseline assessment of cellular viability (n = 5 independent samples). (D) Western blot analysis results showing levels of total Caspase 3 and cleaved–Caspase 3 and the calculated ratio (n = 6 independent samples). (E) Immunofluorescence staining of Ki-67 indicating proliferative activity in percentage positive cells (scale bar represents 50 µm; n = 5 independent samples). (F) Western Blot analysis results showing protein levels of (phosphorylated)-FoxO1/FoxO1, FoxO1/Tubulin, p-AMPK/AMPK, p-AKT/AKT and p-p38 MAPK/p38-MAPK (n = 6 independent samples). (G) RT-qPCR derived expression profiles of cardiac metabolic genes (PGC1α, PGC1β, PPARα, ERRα, ACACA, ACACB, ACLY, LDHA, PKM, GLUT4 and FASN; n = 4 independent samples; corrected for 36B4). (H) Cellular ROS levels in shSCR- and shDIO2-CMs under control conditions and with the addition of rotenone to induce oxidative stress (n = 4 independent samples). The dashed lines represent the relative expression levels of shSCR-CMs; * p < 0.05; Student unpaired t-test; values are compared to the mean shSCR-CM level. Data shown are expressed as means ± standard error of the mean (SEM).