Fig 2: The contents of lactic acid and TCA related metabolites in mitochondria after acute lactate injection and the colocalization of mitochondria and LDHA. (A) The relative abundance of Lactic acid, Succinic acid, Malic acid, and Oxalacetic acid in mitochondria. P: after acute PBS injection (n=3); L: after acute lactate injection (n=3). The data are presented as the mean±SD, and significant differences between the two groups were analyzed with the independent-samples t-test. * p<0.05. (B) Confocal laser scanning microscopic imaging of immunolabeled LDH (green), and HSP60 (red). Antibodies against HSP60 were used as a label of the mitochondria.

Fig 3: Whole-cell DNA- and Exchange-PAINT enabled by SDC microscopy. a Schematic drawing of the cell and the detection volume for z positions 0 µm (left), 1 µm (middle), and 4 µm (right). b 500-nm slice of the microtubule network in a HeLa cell at the coverslip surface. Left: super-resolved DNA-PAINT image. Right: super-resolved (SR) and diffraction-limited (DL) zoomed-in image of the highlighted area. c 500-nm slice of the same cell ~1 µm away from the coverslip surface. d 500 nm slice of the same cell ~4 µm away from the coverslip surface. e–g Three-target Exchange-PAINT of Alpha-Tubulin (green), TOM20 (red), and HSP60 (blue). e Left: DNA-PAINT super-resolution image of a 500-nm slice at the coverslip surface. Right: super-resolved (SR) and diffraction-limited (DL) zoomed-in image of the highlighted area. f 500 nm slice of the same cell ~1 µm away from the coverslip surface. g 500-nm slice of the same cell ~4 µm away from the coverslip surface. Scale bars, 5 µm (b–g), 500 nm (insets in b and e)

Fig 4: Minocycline attenuates translation and reduces ribosomal load of highly translated mRNAs.(A) Autoradiograph (top) monitoring 35S incorporation over 1 hr of HeLa cells treated for 6 hr with cycloheximide or increasing concentrations of minocycline. Coomassie gel (bottom) is shown as a loading control. Total of four independent experiments. (B) Polysome profile analysis of 12 hr water- (black) or minocycline-treated (blue) HeLa cells. High molecular weight polysomes (P) and 80S monosomes (M) regions are shaded in gray. An increase in monosomes and a decrease in high-molecular-weight polysomes are indicative of translation attenuation. Total of three independent experiments. (C) Immunoblots probing for HSP60 and HSP70 expression of water- or minocycline-treated (100 µM) HeLa cells after a 1 hr, 43°C heat shock. Total of three independent experiments. (D) qRT-PCR analysis of HSP60 and HSP70 mRNA expression in HeLa cells treated with or without minocycline (100 µM) before and after a 1 hr, 43°C heat shock. Minocycline treatment was initiated 12 hr prior to the heat shock. Data are represented as mean ± S.E.M. Significance determined by the Mann-Whitney t-test. Total of three independent experiments. (E) Immunoblots probing for BiP expression of water- or minocycline-treated (100 µM) HEK293DAX cells engineered to allow UPRER activation by trimethoprim treatment (TMP). Water or 100 µM minocycline was added alone or in combination with 20 uM trimethoprim for 12 hr prior to cell lysis. Samples were normalized by RNA concentration. Total of three independent experiments. (F) Bar graph shows change in protein aggregate formation, as measured by the ProteoStat assay, before and after a 1 hr, 43°C heat shock in control (black) and minocycline-treated (blue) HeLa cells. Data are represented as mean ± S.E.M. Significance determined by the Mann-Whitney t-test. Total of four independent experiments. (G) Autoradiograph (top) monitoring 35S incorporation of HeLa cells treated with the ISR inhibitor ISRIB (200 nM), minocycline (100 µM), thapsigargin (thaps, 200 nM) and a combination of the three. Coomassie gel (bottom) is shown as a loading control. Total of two independent experiments. Asterisks indicate significance *<0.05, **<0.01, ***<0.001, n.s. not significant. Figure 5. Lifespan extension by minocycline depends on cytoplasmic translation.

Fig 5: The genetic ablation of PA200 results in deficient mitochondrial function. (A) Oxygen consumption rate (OCR) was measured using the Seahorse XF 96 to analyze mitochondrial function. Control and shPA200 cells were seeded at a density of 35,000 cell/well in Seahorse XF 96 plates. The following day, the basal OCR was determined for 30 min after sequentially injecting mitochondrial drugs, including 1.5 µM oligomycin (Olig), 1 µM FCCP, and 1 µM of rotenone/antimycin-A cocktail (Anti/Rot). Data were analyzed using Wave Desktop software and presented as mean values ± SD (n = 3). Statistical analysis was performed using unpaired t-tests. (* indicates p < 0.05) (B) Calculated basal respiration, proton leak-linked respiration, maximal mitochondrial respiratory capacity, spare respiratory capacity, and ATP-coupled respiration. Data were normalized to total protein (pmol/min/µg protein). Data were analyzed using Wave Desktop software and presented as mean values ± SD (n = 3). Statistical analysis was performed using unpaired t-tests. (** indicates p < 0.01 and ns indicates not significant). (C) Total cell lysates from control and shPA200 cells were analyzed by western blot. OXPHOS antibody cocktail was used to detect four subunits of the electron transport chain (ETC) complexes, CI-NDUFB8, CII-SDHB, CIII-UQCRC2, and CIV-MTCOI, and one subunit for the ATP synthase, CV-ATP5A. Images were taken using a ChemiDoc Imager, and the density of the protein was normalized to the mitochondrial Hsp 60. (D) Statistical histogram of CI-NDUFB8 subunit from total lysates of PA200-deficient and control cells. Data were analyzed using Image Lab V 6.1 software and presented as mean values ± SD (n = 4). Groups were compared using unpaired t-tests. (*** indicates p < 0.001). (E) Mitochondria fractionation from control and shPA200 cells were performed and analyzed by western blot. The OXPHOS antibody cocktail was used to detect four subunits of the electron transport chain (ETC) complexes, CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MTCOI, and one subunit for the ATP synthase, CV-ATP5A. Images were taken using a ChemiDoc Imager and the density of the protein was normalized to the mitochondrial Hsp 60. (F) Statistical analysis of the CI-NDUFB8 subunit from mitochondrial sufractions of shPA200 and control cells. Data were analyzed using Image Lab V 6.1 software and presented as mean values ± SD (n = 4). Groups were compared using unpaired t-tests. (** indicates p < 0.01).