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. 2021 Feb 20;12(1):199–228. doi: 10.1016/j.jcmgh.2021.02.004

Figure 2.

Figure 2

Distribution and metabolic consequences of L30P/R34P expression in HBs. (A) Expression of NFE2L2, KEAP1, GLUT1, GLUT2, GLUT4, PKM-1, PKM-2, and Cpt1a in 2 representative sets of total lysates from the indicated tissues. (B) Nuclear (N)/cytoplasmic (C) fractionation of the indicated tissues, n = 3–5 samples/group. GAPDH and histone H3 (H3) immunoblots were performed as controls for protein loading and the purity of each fraction. Numbers above the NFE2L2 and KEAP1 panels indicate the fraction of protein associated with each compartment as determined by densitometric scanning of bands. (C) Total OCRs of mitochondria from the indicated tissues in the presence of malate, ADP, pyruvate, glutamate, and succinate. (D) Complex I responses, calculated after addition of rotenone to the reactions in (C) without succinate. (E) Complex II responses as determined from residual activity after addition of rotenone. (F) Responses to pyruvate. (G) Responses to glutamate. (H) β-FAO responses after addition of malate, L-carnitine, and palmitoyl-CoA. (I) Quantification of mitochondrial DNA (mtDNA) in representative tissues. TaqMan reactions amplified a segment of the mt D-loop region.3,25Each point represents the mean of triplicate TaqMan reactions after normalizing to a control TaqMan reaction for the ApoE nuclear gene. (J) In vitro recovery from oxidative stress. Monolayer cultures of the indicated tumor cells expressing cyto-roGFP or mito-roGFP were exposed to 5 mmol/L hydrogen peroxide (bar) while being monitored by live cell confocal microscope.