Figure 4.
FAM43A responds to mtDNA depletion in a CHK2-dependent manner. (A) Representative immunoblots of phosphorylated CHK2 (pCHK2 Thr68), CHK2, and ACTIN (loading control) in untreated (UNT), ddC-treated (96 h), and 4- and 8-days following removal of ddC (Recovery). (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; ns, P > 0.05; **, P < 0.01. (C) Representative spinning disk images of Hoechst (blue), γH2AX (green), and 53BP1 (magenta) in UNT, Doxorubicin-treated (1uM for 4 h), and ddC-treated cells. The merge of these signals is shown in the right column. Scale bar: 10 µm. (D and E) Quantification of γH2AX and (E) 53BP1 foci/cell in UNT (n = 59 nuclei from two biological replicates), Doxorubicin-treated (1 µM for 4 h) (n = 10 nuclei from two biological replicates), and ddC-treated cells (n = 49 nuclei from two biological replicates) from (C). Doxorubicin was used as a positive control for nuclear DNA damage. Error bars represent mean ± SEM, multiple Unpaired Student’s t test; ns, P > 0.05; ****, P > 0.0001. (F) Representative immunoblots of CHK2, p53R2, TFAM, and ACTIN (loading control) of IMR90 cells transfected with the indicated siRNA. 48 h after transfection, cells were treated with ddC for 48 h and collected for analysis. (G) IMR90 cells were transfected with the indicated siRNA. 96 h after transfection, cells were treated with ddC for 48 h. The relative mRNA expression of FAM43A was measured by RT-qPCR normalized to ACTIN (n = 3 technical triplicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; ***, P < 0.001; **, P < 0.01; ns, P > 0.05. (H) Representative immunoblots of phosphorylated CHK2 (pCHK2 Thr68), CHK2, and ACTIN (loading control) in UNT and cells treated with ddC only or ddC and CHK2 inhibitor (10 µM) for 48 h. (I) Relative FAM43A mRNA expression in UNT and cells treated with ddC only or ddC and CHK2 inhibitor for 48 h (n = 3 technical triplicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; **, P < 0.01: ***, P < 0.001. (J) Representative Airyscan images of endogenous FAM43A (green) and Nuclei (Hoechst; blue) in UNT and ddC-treated (96 h) IMR90 cells ± CHK2 Inhibitor (48 h). Insets show the morphological change of FAM43A. Scale bar: 10 µm. (K and L) Quantification of the fluorescence intensity of FAM43A signal and (L) number of FAM43A aggregates per cell in UNT and cells treated with ddC for 96 h ± CHK2 Inhibitor (n = 15 from two biological replicates per condition). Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ns, P > 0.05; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F4.

FAM43A responds to mtDNA depletion in a CHK2-dependent manner. (A) Representative immunoblots of phosphorylated CHK2 (pCHK2 Thr68), CHK2, and ACTIN (loading control) in untreated (UNT), ddC-treated (96 h), and 4- and 8-days following removal of ddC (Recovery). (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; ns, P > 0.05; **, P < 0.01. (C) Representative spinning disk images of Hoechst (blue), γH2AX (green), and 53BP1 (magenta) in UNT, Doxorubicin-treated (1uM for 4 h), and ddC-treated cells. The merge of these signals is shown in the right column. Scale bar: 10 µm. (D and E) Quantification of γH2AX and (E) 53BP1 foci/cell in UNT (n = 59 nuclei from two biological replicates), Doxorubicin-treated (1 µM for 4 h) (n = 10 nuclei from two biological replicates), and ddC-treated cells (n = 49 nuclei from two biological replicates) from (C). Doxorubicin was used as a positive control for nuclear DNA damage. Error bars represent mean ± SEM, multiple Unpaired Student’s t test; ns, P > 0.05; ****, P > 0.0001. (F) Representative immunoblots of CHK2, p53R2, TFAM, and ACTIN (loading control) of IMR90 cells transfected with the indicated siRNA. 48 h after transfection, cells were treated with ddC for 48 h and collected for analysis. (G) IMR90 cells were transfected with the indicated siRNA. 96 h after transfection, cells were treated with ddC for 48 h. The relative mRNA expression of FAM43A was measured by RT-qPCR normalized to ACTIN (n = 3 technical triplicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; ***, P < 0.001; **, P < 0.01; ns, P > 0.05. (H) Representative immunoblots of phosphorylated CHK2 (pCHK2 Thr68), CHK2, and ACTIN (loading control) in UNT and cells treated with ddC only or ddC and CHK2 inhibitor (10 µM) for 48 h. (I) Relative FAM43A mRNA expression in UNT and cells treated with ddC only or ddC and CHK2 inhibitor for 48 h (n = 3 technical triplicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; **, P < 0.01: ***, P < 0.001. (J) Representative Airyscan images of endogenous FAM43A (green) and Nuclei (Hoechst; blue) in UNT and ddC-treated (96 h) IMR90 cells ± CHK2 Inhibitor (48 h). Insets show the morphological change of FAM43A. Scale bar: 10 µm. (K and L) Quantification of the fluorescence intensity of FAM43A signal and (L) number of FAM43A aggregates per cell in UNT and cells treated with ddC for 96 h ± CHK2 Inhibitor (n = 15 from two biological replicates per condition). Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ns, P > 0.05; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F4.

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