Figure 5.
Knockdown of FAM43A activates CHK2 and RNR-dependent mtDNA synthesis. (A) Representative immunoblots of pCHK2 (Thr68), CHK2, and ACTIN (loading control) in cells transfected with the indicated siRNA and collected 96 h after transfection. (B) Quantification of A. The optical density of pCHK2 Thr68 and CHK2 were normalized to loading, and the ratio of pCHK2 Thr68 to total CHK2 was quantified as a measure of CHK2 activation (n = 3 biological replicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05. (C) Representative immunoblots of CHK2, p53R2, and ACTIN (loading control) in cells transfected with the indicated siRNA and collected 96 h after transfection. (D) Representative immunoblots of pCHK2 Thr68, CHK2, TFAM, and ACTIN (loading control) in cells transfected with the indicated siRNA. 48 h after transfection, cells were treated with ddC and collected 96 h after transfection for analysis. (E) Quantification of (D). The optical density of pCHK2 Thr68 and CHK2 were normalized to loading, and the ratio of pCHK2 Thr68 to total CHK2 was quantified as a measure of CHK2 activation (n = 2 biological replicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05; **, P < 0.01. (F) Representative immunoblots of RRM1, p53R2, TFAM, and ACTIN (loading control) in cells transfected with the indicated siRNA. After 48 h transfection, cells were treated with ddC and collected 96 h after transfection for analysis. (G–J) Relative mtDNA copy number of IMR90 cells transfected with the indicated siRNA and collected for analysis 96 h after transfection as measured by qPCR using ND1 primers, normalized to nuclear B2M. Data points represent the average of technical triplicates from three biological replicates. Data from G–I were normalized to the same control (siCtrl) sample. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ns, P > 0.05; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F5.

Knockdown of FAM43A activates CHK2 and RNR-dependent mtDNA synthesis. (A) Representative immunoblots of pCHK2 (Thr68), CHK2, and ACTIN (loading control) in cells transfected with the indicated siRNA and collected 96 h after transfection. (B) Quantification of A. The optical density of pCHK2 Thr68 and CHK2 were normalized to loading, and the ratio of pCHK2 Thr68 to total CHK2 was quantified as a measure of CHK2 activation (n = 3 biological replicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05. (C) Representative immunoblots of CHK2, p53R2, and ACTIN (loading control) in cells transfected with the indicated siRNA and collected 96 h after transfection. (D) Representative immunoblots of pCHK2 Thr68, CHK2, TFAM, and ACTIN (loading control) in cells transfected with the indicated siRNA. 48 h after transfection, cells were treated with ddC and collected 96 h after transfection for analysis. (E) Quantification of (D). The optical density of pCHK2 Thr68 and CHK2 were normalized to loading, and the ratio of pCHK2 Thr68 to total CHK2 was quantified as a measure of CHK2 activation (n = 2 biological replicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05; **, P < 0.01. (F) Representative immunoblots of RRM1, p53R2, TFAM, and ACTIN (loading control) in cells transfected with the indicated siRNA. After 48 h transfection, cells were treated with ddC and collected 96 h after transfection for analysis. (G–J) Relative mtDNA copy number of IMR90 cells transfected with the indicated siRNA and collected for analysis 96 h after transfection as measured by qPCR using ND1 primers, normalized to nuclear B2M. Data points represent the average of technical triplicates from three biological replicates. Data from G–I were normalized to the same control (siCtrl) sample. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ns, P > 0.05; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F5.

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