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. 2022 May 25;14(2):375–403. doi: 10.1016/j.jcmgh.2022.05.006

Figure 8.

Figure 8

SMYD5 critically regulates mitochondrial biogenesis and function. (A) RT-qPCR analysis of mRNA (presented as relative fold change) of mitochondrial biogenesis markers Tfam (n = 3) and Cox I (n = 3) in IECs isolated from Smyd5fl/fl and Smyd5ΔIEC mice. (B) RT-qPCR analysis of the abundance of mtDNA content in HCT116 cells with KO SMYD5 or control vector. The relative mtDNA content was determined using the Comparative Ct method quantification (2-ΔCt method) method. The primers for human mtDNA-encoded Cox II gene and for human nuclear DNA-encoded GAPDH were used for the analysis. The results are presented as the ratio of mtDNA relative to nuclear DNA. Results are presented as fold changes relative to parental HCT116 cells. n = 3. (C) RT-qPCR analysis of the expression of the mtDNA-encoded genes, the mitochondrial biogenesis markers COX I (left) and COX II (right) in HCT116 cells with OE or KO SMYD5. Results are presented as fold changes relative to parental HCT116 cells. n = 3. (D) Representative TEM images (left) of IECs in sections of colon tissues from Smyd5fl/fl and Smyd5ΔIEC mice and quantitative analyses of the number of mitochondria per cell (right). The “n” refers to the number of mice per genotype. n = 3 mice per group. Yellow asterisks indicate mitochondria. Scale bars: 500 nm. (E) Representative TEM images (left) of IECs from Smyd5fl/fl and Smyd5ΔIEC mice (n = 3 mice for each group). Right: Quantitative analyses of mitochondrial perimeter, circularity, and the number of mitochondria per unit area (μm2). For mitochondrial perimeter and circularity, n = 30 mitochondria per genotype group. For mitochondria counts per μm2, n = 30 random visual fields per genotype group. (F) OCR measured by a Seahorse XF96 analyzer (left) in HCT116 cells with SMYD5 OE, KO, or unchanged (parental). Glucose (25 mmol/L) and pyruvate (1 mmol/L) were supplied as substrates. Oxygen consumption was measured under basal conditions, after the sequential addition of oligomycin, the pharmacologic uncoupler carbonyl cyanide phenylhydrazone (FCCP), the Complex III and I inhibitors antimycin A, and rotenone. Right: Quantitation of basal and maximal respiration capacity. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001; n = 3.