Figure 3.
FAM43A inhibits mitochondrial biogenesis and mtDNA replication. (A) Relative FAM43A mRNA level in IMR90 cells transfected with the indicated siRNA and collected 96 h after transfection. Expression was measured by RT-qPCR and normalized to ACTIN. Data points represent the average of three biological replicates. Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05: ***, P < 0.001. (B–D) (B) Mitochondrial mass (Mitotracker Green staining, MTG), (C) Mitochondrial membrane potential (TMRE staining) and (D) MTG/TRME ratio of IMR90 cells measured 96 h after transfection with the indicated siRNA. Data points represent the mean of three technical replicates from three biological replicates and normalized to the value of the control siRNA (siCtrl), which was given a value of 1.0. Error bars represent mean ± SD, multiple unpaired Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (E) Relative mtDNA copy number of IMR90 cells transfected with the indicated siRNA and collected 96 h after transfection measured by qPCR with ND1 primers, normalized to nuclear B2M. Data points represent the mean of three technical replicates from three biological replicates. Error bars represent mean ± SD, multiple Unpaired Student’s t test; **, P < 0.01. (F) Relative Fam43a mRNA level of mouse embryonic fibroblasts (MEFs) transfected with the indicated siRNA and collected 96 h after transfection. Expression was measured by RT-qPCR and normalized to ACTIN (n = 3 technical replicates). Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ****, P < 0.0001. (G) mtDNA copy number of MEFs transfected with the indicated siRNA. Expression was measured by qPCR (n = 3 technical replicates) using ND1 primers normalized to nuclear Tert. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; **, P < 0.01. (H) Representative immunoblots of PGC-1α, TFAM, and ACTIN (loading control) of IMR90 cells transfected with the indicated siRNA for 96 h. (I) Quantification of the relative optical density of PGC-1α from three independent experiments treated as in H. Error bars represent mean ± SD, multiple Unpaired Student’s t test; **, P < 0.01. ***, P < 0.001. (J) IMR90 cells were treated with ddC for 96 h and then transfected with the indicated siRNA (siFAM43A #1). Samples were collected at the end of ddC treatment (first two points) and 10- and 14-days after transfection. Data points represent the mean of three technical replicates from three biological replicates. Error bars represent mean and error ± SD, multiple unpaired Student’s t test; **, P < 0.01; ***. P < 0.001. (K) Quantification of Pulse EdU labeling (4 h) in IMR90 cells at 96 h after transfection with control (n = 18 from two biological replicates) or FAM43A siRNAs (n = 18–19 from two biological replicates). Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; **, P < 0.01; ****. P < 0.0001. (L) Representative spinning disk images of Alexa488-EdU (green), DNA (blue), HSP60 (red), and Hoechst (gray). Insets show the colocalization between EdU and DNA (bottom left) and the merge of all signals (bottom right). Scale bar: 10 µm. Source data are available for this figure: SourceData F3.

FAM43A inhibits mitochondrial biogenesis and mtDNA replication. (A) Relative FAM43A mRNA level in IMR90 cells transfected with the indicated siRNA and collected 96 h after transfection. Expression was measured by RT-qPCR and normalized to ACTIN. Data points represent the average of three biological replicates. Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05: ***, P < 0.001. (B–D) (B) Mitochondrial mass (Mitotracker Green staining, MTG), (C) Mitochondrial membrane potential (TMRE staining) and (D) MTG/TRME ratio of IMR90 cells measured 96 h after transfection with the indicated siRNA. Data points represent the mean of three technical replicates from three biological replicates and normalized to the value of the control siRNA (siCtrl), which was given a value of 1.0. Error bars represent mean ± SD, multiple unpaired Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (E) Relative mtDNA copy number of IMR90 cells transfected with the indicated siRNA and collected 96 h after transfection measured by qPCR with ND1 primers, normalized to nuclear B2M. Data points represent the mean of three technical replicates from three biological replicates. Error bars represent mean ± SD, multiple Unpaired Student’s t test; **, P < 0.01. (F) Relative Fam43a mRNA level of mouse embryonic fibroblasts (MEFs) transfected with the indicated siRNA and collected 96 h after transfection. Expression was measured by RT-qPCR and normalized to ACTIN (n = 3 technical replicates). Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ****, P < 0.0001. (G) mtDNA copy number of MEFs transfected with the indicated siRNA. Expression was measured by qPCR (n = 3 technical replicates) using ND1 primers normalized to nuclear Tert. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; **, P < 0.01. (H) Representative immunoblots of PGC-1α, TFAM, and ACTIN (loading control) of IMR90 cells transfected with the indicated siRNA for 96 h. (I) Quantification of the relative optical density of PGC-1α from three independent experiments treated as in H. Error bars represent mean ± SD, multiple Unpaired Student’s t test; **, P < 0.01. ***, P < 0.001. (J) IMR90 cells were treated with ddC for 96 h and then transfected with the indicated siRNA (siFAM43A #1). Samples were collected at the end of ddC treatment (first two points) and 10- and 14-days after transfection. Data points represent the mean of three technical replicates from three biological replicates. Error bars represent mean and error ± SD, multiple unpaired Student’s t test; **, P < 0.01; ***. P < 0.001. (K) Quantification of Pulse EdU labeling (4 h) in IMR90 cells at 96 h after transfection with control (n = 18 from two biological replicates) or FAM43A siRNAs (n = 18–19 from two biological replicates). Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; **, P < 0.01; ****. P < 0.0001. (L) Representative spinning disk images of Alexa488-EdU (green), DNA (blue), HSP60 (red), and Hoechst (gray). Insets show the colocalization between EdU and DNA (bottom left) and the merge of all signals (bottom right). Scale bar: 10 µm. Source data are available for this figure: SourceData F3.

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