Fig 2: Didymin enhances mitochondrial biogenesis and function, promotes lipophagy, and inhibits apoptosis by activating Sirt1 in mice hepatocytes. A Western blot analysis of Sirt1 expression in hepatocytes (n = 3). B Western blot analysis of NRF1, TFAM, NDUFB8, SDHB, and MTCO2 in hepatocytes. C Transmission electron microscopy figures of hepatocytes, arrows show mitochondria (Scale bar = 0.6 μm). D Transmission electron microscopy figures of hepatocytes, arrows show autolysosomes (Scale bar = 0.6 μm). E Western blot analysis of LC3, Beclin1, P62, PLIN2, LAMP1, and ATG5 in hepatocytes (n = 3). F Western blot analysis of Cleaved PARP, Bax, Bcl2, Cleaved Caspase 3, and Caspase 3 proteins in hepatocytes (n = 3). G Immunofluorescence staining of γ-H2AX in hepatocytes (Scale bar = 50 μm). NRF1 nuclear respiratory factor 1, TFAM mitochondrial transcription factor A, PLIN2 perilipin 2, LAMP1 lysosomal associated membrane protein 1, Bax Bcl-2-associated X protein, Bcl2 B-cell lymphoma 2

Fig 3: Altered mitochondrial oxidative phosphorylation enzymes transcripts and estimation of mitochondrial vs nuclear DNA in liver of 12-week-old Ghr-mutant mice fed ad libitum. (A) Hepatic transcripts for enzymes involved in mitochondrial oxidative phosphorylation complex mt-CO1, Ndufa5 were elevated in Ghr-mutant males while ATP5a was elevated in all the mutant mice across both sexes; Uqcrc2 and Sdhb were elevated only in Ghr−/− male and female mice. Other mitochondrial function regulator genes mfn2 and nqo1 were elevated only in Ghr-mutant males as compared to wild-type (WT) mice. (B) Male Ghr−/−- and GhrBox1−/−-mutant mice livers have elevated levels of cytochrome C oxidase IV (COX IV), mitochondria transcription factor A (mTFAM) as well as OxPhos complexes I, III, and V. Additionally complex II was elevated in GhrBox1−/− mice and no change was evident in SDHA levels. (C) No change in mitochondrial numbers was evident in Ghr-mutant females as evident from lack of differences in mitochondrial vs nuclear genome. Data represented as mean ± SEM (n = 4 mice/genotype). *P less than .05; **P less than .01; ***P less than .001; ****P less than .0001 compared to wild-type (WT) of the same sex by analysis of variance.

Fig 4: Effects of caspase-2 on liver homogenate oxygen consumption and respiratory chain protein levels in mice. A Representative trace of oxygen consumption in mouse liver homogenate. B Maximal respiration levels in WT and Casp2−/− mice. C Representative immunoblots and relative protein levels of SDHA, SDHB, TIMM23, TOMM20, and CASP2 in liver samples from WT and Casp2.−/− female mice. Data are presented as mean ± SD, *P < 0.05, **P < 0.001

Fig 5: Identification of caspase-2-downstream proteins regulating mitochondria-related gene transcription. A Protein–protein interaction network of the predicted transcription factors and caspase-2, based on the STRING database. B Representative immunoblots and relative protein levels of CASP2 and p53 in SW620 WT and SW620 Casp2−/− cells. C Relative gene expression of Tp53, Sdhb, Timm21, Timm23, Tomm20, and Tomm40 in SW620 Casp2.−/− cells transfected with siRNA targeting Tp53 or negative control (NC). D Representative immunoblots and (E) relative protein levels of CASP2, p53, SDHA, SDHB, TIMM23, TOMM20, and TOMM40 in SW620 Casp2-KO and SW620 Casp2-KO + shp53 cells. Results are shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001