Fig 2: Penfluridol decreases mitochondrial activity and mass in human non-small cell lung cancer (NSCLC) cells via targeting peroxisome-proliferator-activated receptor co-activator-1a (PGC-1a). (A) Fluorescent JC-1 images of the mitochondrial membrane potential in A549 cells treated with vehicle or penfluridol (5 µM) for 24 h. The red-to-green fluorescence ratio indicates a decrease in functional mitochondria with the membrane potential. (B) For the flow cytometric analysis, cells were stained with MitoTracker Red H2XRos (which fluoresces upon oxidation in respiring mitochondria). Representative histograms (left panel) and bar graph (n = 3) (right panel) of the flow cytometric analysis between vehicle- and penfluridol-treated A549 and HCC827 cells are depicted. (C,E) A549 cells were treated with vehicle or penfluridol (5 µM) for 24 h to detect mRNA levels of MTND6, MTCO1, MTCO2 (C), and PPARGC1A (E) using an RT-qPCR. Quantitative results of indicated mRNA levels were adjusted to GAPDH mRNA levels. * p < 0.05, *** p < 0.001, compared to the vehicle group. (D,F) A549 cells were treated with vehicle or penfluridol at indicated concentrations for 24 h. Cells were harvested and then subjected to a Western blot analysis to detect PGC-1a (D), phosphorylated (p)-AMPK, sirtuin 1 (SIRT1), and p53 levels (F). GAPDH, ß-actin, or AMP-activated kinase (AMPK) was used as an equal loading control. Western blots shown are representative results from 3 independent experiments. (G) Correlation analysis of the TCGA lung cancer database (TCGA, PanCancer Atlas) using the cBioPortal revealing a positive correlation between PPARGC1A and SIRT1.

Fig 3: Does-dependent p53 activation determines transition from mitochondrial compensation to mitochondrial dysfunction. A, Contractile speed of NMVMs determined by live cell imaging are shown as mean±SEM (n=20 cells per group). B, Representative micrographs of p53, PGC1-a, and PGC1-ß using immunoblotting are shown. Histone H3 used as loading control. (C through E) Densitometric quantifications are shown as mean±SEM (n=3 per group). F, Mitochondrial amount assayed by Mitotracker staining is shown as mean±SD (triplicates, n=30 NMVMs). G, Real-time mitochondrial respiration of NMVMs (n=3 per group). H and I, Basal and maximal OCR of NMVMs were quantified and are shown as mean±SEM (n=3 per group; 3 measurements per group). J, Representative micrographs of NMVMs examined by transmission electron micrographs. Scale bars are 2 µm and 0.4 µm at 5000 and 25000× magnification, respectively. Kruskal-Wallis test followed by Dunn’s multiple-comparisons test was used for panel F; 1-way ANOVA followed by Tukey’s multiple-comparisons test was used for remaining analyses. *P<0.05, **P<0.01, ***P<0.001 compared with vehicle group. HFpEF, heart failure with preserved ejection fraction; NMVM, neonatal mouse ventricular cardiomyocyte; OCR, oxygen consumptio rate; p53CKO, cardiomyocyte-specific p53 knockout; PGC1-a, proliferator-activated receptor gamma, coactivator 1 alpha; and PGC1-ß, proliferator-activated receptor gamma, coactivator 1 beta.

Fig 4: p53CKO animals fail to develop HEpEF upon HFD+L-NAME challenge. A and B, Representative immunoblots of p53, Pgc1-a, and Pgc1-ß expression in isolated AMVMs are shown. Histone H3 used as loading control. C and D, Densitometric quantifications of Pgc1-a and Pgc1-ß expression. Results are shown as mean±SEM (n=6 per male p53CKO group; n=5 per female p53CKO group). E, mtDNA copy number in AMVMs was determined by real-time quantitative polymerase chain reaction and is shown as mean±SEM (n=6 per male p53CKO group; n=5 per female p53CKO group). F, Mitochondrial amount was assayed using Mitotracker staining and is shown as mean±SD (n=6 per male p53CKO group; n=5 per female p53CKO group; 15 AMVMs per mouse). G, Real-time mitochondrial respiration of AMVMs (n=3 per group). H and I, Basal and maximal OCR of AMVMs were quantified and are shown as mean±SEM (n=3 per group; 3 measurements per mouse). Mann-Whitney test was used for panel F; 2-tailed unpaired Student's t test was used for remaining analyses. *P<0.05, **P<0.01, ***P<0.001 compared with chow group. AMVM indicates adult mouse ventricular cardiomyocyte; HFD, high-fat diet; HFpEF, heart failure with preserved ejection fraction; L-NAME, N?-Nitro-L-arginine methyl ester hydrochloride; OCR, oxygen consumption rate; p53CKO, cardiomyocyte-specific p53 knockout; PGC1-a, proliferator-activated receptor gamma, coactivator 1 alpha; and PGC1-ß, proliferator-activated receptor gamma, coactivator 1 beta.

Fig 5: p53-activation in HFpEF cardiomyocytes result in blocked mitochondrial biogenesis. A, Representative images of p53, Pgc1-a, and Pgc1-ß expression in AMVMs assayed by immunoblotting. Histone H3 used as loading control. (B through D) Densitometric quantifications of p53, PGC1-a, and PGC1-ß protein expression. Results are shown as mean±SEM (n=6 per group). One-way ANOVA followed by Tukey's multiple-comparisons test was used. *P<0.05, **P<0.01, ***P<0.001 compared with chow group in the same genotype are shown. AMVM indicates adult mouse ventricular cardiomyocyte; HFpEF, heart failure with preserved ejection fraction; mTRG2, second -generation telomerase RNA component knockout; PGC1-a, proliferator-activated receptor gamma, coactivator 1 alpha; PGC1-ß, proliferator-activated receptor gamma, coactivator 1 beta; and WT, wild type.