Fig 2: Transcriptional regulation of NRF1 on NF-κB and TFAM is up-regulated under hypoxia. Primary microglia were infected with lentivirus followed by hypoxia treatment for 24 h. (A) The protein level of NRF1 was measured by western blotting. (B–D) mRNA levels of Rela, Il1b, and Tnfa were determined by real-time PCR (*P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA). (E) TNF-α secretion by microglia to the supernatant of culture medium was measured by ELISA (**P < 0.01 and ***P < 0.001 by two-way ANOVA). (F) Enrichments of the Rela promoter by NRF1 were measured by ChIP–qPCR (*P < 0.05 and **P < 0.01 by Student's t-test). (G and H) The mRNA level of Tfam (G) and mtDNA copies (H) were determined by real-time PCR (*P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA). (I) Enrichments of the Tfam promoter by NRF1 were measured by ChIP–qPCR (*P < 0.05 by Student's t-test). n.s. indicates no significance.

Fig 3: TFAM Knockdown and Overexpression in CD4 T cells from PLHIV and HS. (A) Western blot analysis of mtTFA and β-actin expression in HS CD4 T cells with or without TFAM KD. (B) Representative OCR summary data for non-mitochondrial, basal respiration, maximal respiration, spare respiration capacity, proton leak, and ATP production in HS CD4 T cells with or without TFAM KD. (C) Genomic DNA was isolated to determine levels of mtDNA relative to nuDNA by qPCR. DNA content from mtTFA KD was normalized to the control group. KD = knockdown. (D) Western blot analysis of mtTFA and β-actin expression in PLHIV CD4 T cells with or without TFAM OE. (E) Representative OCR summary data in CD4 T cells from PLHIV following TFAM OE. OE = overexpression. (F) A model depicting the mechanisms and outcomes of CD4 T cell inflammaging in HIV latency.

Fig 4: Rg3 suppresses the production of mitochondrial ROS induced by DEX. C2C12 myotubes were treated with DEX with vehicle or Rg3 for additional one day (D4). (A) Mitochondrial ROS production analysis. Fluorescence intensity is given as arbitrary units and values presented as means ± SD (n=5). Rg3/DEX-treated group was compared with DEX-treated group, **P<0.002; one-way ANOVA with post Bonferroni's multiple comparison test. (B) Mitochondrial membrane potential analysis. Fluorescence ratio (590 to 530 nm) was used for quantitative analysis. Values presented are as means ± SD (n=4). Rg3/DEX-treated group was compared with DEX-treated group, **P<0.002; one-way ANOVA with post Bonferroni's multiple comparison test. (C) The determination of ATP content. The total ATP content was measured using ATP determination kit. Values presented are as means ± SD (n=4). Rg3/DEX-treated group was compared with DEX-treated group, *P<0.033; one-way ANOVA with post Bonferroni's multiple comparison test. (D) Model of the effect and molecular mechanisms of ginsenoside Rg3 on DEX-induced myotube atrophy. Rg3 protects DEX-induced myotube atrophy through the activation of Akt/mTOR signaling and suppresses muscle-specific ubiquitin ligases. In addition, Rg3 efficiently prevents DEX-triggered mitochondrial dysfunction of myotubes through PGC1-α activities and its mitochondrial biogenetic transcription factors, NRF1 and Tfam. Rg3, ginsenoside Rg3; ROS, reactive oxygen species; DEX, dexamethasone; PGC1-α, peroxisome proliferator-activated receptor γ coactivator 1-α; NRF1, nuclear respiratory factor 1; Tfam, mitochondrial transcription factor A.

Fig 5: Regulation of senescence-associated secretory phenotype and mitochondrial homeostasis by miR-184. A, B, The changes in mRNA expression levels of CD44, interleukin 6 (IL-6), VEGF, and monocyte chemotactic protein-1 (MCP-1) in mature CD44−/dull cultured human corneal endothelial cell (hCEC) subpopulations (SPs) (A) and immature CD44+++ hCEC SPs (B) by transfection of an miRNA (miR)-184 mimic. The relative expression of mRNA was analyzed using 18S mRNA for normalization. Transfection of miR mimics at 30 nM was conducted using a DharmaFECT Duo reagent (GE Healthcare Dharmacon) in accordance with the manufacturer’s protocol. C, Changes in mitochondrial membrane potentials of mature CD44−/dull hCECs following transfection with either an miR-184 or miR-24-3p inhibitor. Seventy-two hours after transfection, the cells were incubated with JC-1 dye, and relative mitochondrial membrane potentials were observed using fluorescence microscopy. D, Qualitative analyses of JC-1 and Western blotting (WB). The changes in expression levels of PGC1α, VDAC1, TFAM, TOMM20, COXIV, and β-actin in mature CD44−/dull hCECs due to transfection using the miR-184 or miR-24-3p inhibitor. Forty-eight hours after transfection, expression levels were assessed using WB. Proteins were loaded at 25 μg protein/lane.