Figure S4.
Non-canonical CHK2 signaling activates RNR-dependent mtDNA synthesis in FAM43A-knockdown cells. (A and B) Expression of RRM2B and (B) RRM1 measured by RNA sequencing of IMR90 cells treated with ddC for the indicated time relative to untreated (UNT) control cells (n = 3 biological replicates). (C) 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 CHK2 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. (D) Representative immunoblots of phosphorylated CHK2 (pCHK2 Thr68), CHK2, and ACTIN (loading control) in WT and FAM43A knockout (FKO) H292 cells. (E) Quantification of pCHK2 Thr68/CHK2 ratio from three independent experiments conducted as in 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. Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05. (F) Representative immunoblots of p53R2 (top band) and ACTIN (loading control) in WT and FKO H292 cells. (G) Representative Spinning disk imaging of Nuclei (Hoechst; blue), γH2AX (green), and 53BP1 (magenta) in cells transfected with the indicated siRNA for 96 h. Merge of the signals is shown in the right-hand column. Control (siCtrl) cells were treated with Doxorubicin (1 µM for 4 h) as a positive control for nuclear DNA damage. Scale bar: 10 µm. (H and I) Quantification of γH2AX and (I) 53BP1 foci/cell in G. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05; ****, P > 0.0001. n = 15–25 nuclei/condition. (J) Relative expression of FAM43A, RRM1, and RRM2B in cells transfected with the indicated siRNA (siCtrl and siFAM43A #1) and collected 96 h after transfection (n = 3 technical triplicates). Expression was measured by RT-qPCR and normalized to ACTIN. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05; ***, P < 0.001; ****, P < 0.0001. (K) Relative expression of FAM43A and RRM2B normalized to ACTIN as measured by RT-qPCR. Cells were transfected with control or siRNA against RRM2B. 48 h after transfection, cells were transfected with siRNA against FAM43A and collected 96 h later. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (L) Relative expression of FAM43A and RRM1 in cells transfected with the indicated siRNA as in K. Expression was measured by RT-qPCR and normalized to ACTIN. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ns, P > 0.05; ****, P < 0.0001. (M) Relative expression of FAM43A and RRM2 of cells transfected with the indicated siRNA as in K. Expression was measured by RT-qPCR and normalized to ACTIN. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ***, P < 0.001; ****, P < 0.0001. (N) Relative expression of FAM43A and CHK2 of cells transfected with the indicated siRNA as in K. Expression was measured by RT-qPCR and normalized to ACTIN. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ****, P < 0.0001. The expression of FAM43A was normalized to the same control (siCtrl) sample in panels L–N. Source data are available for this figure: SourceData FS4.

Non-canonical CHK2 signaling activates RNR-dependent mtDNA synthesis in FAM43A-knockdown cells. (A and B) Expression of RRM2B and (B) RRM1 measured by RNA sequencing of IMR90 cells treated with ddC for the indicated time relative to untreated (UNT) control cells (n = 3 biological replicates). (C) 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 CHK2 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. (D) Representative immunoblots of phosphorylated CHK2 (pCHK2 Thr68), CHK2, and ACTIN (loading control) in WT and FAM43A knockout (FKO) H292 cells. (E) Quantification of pCHK2 Thr68/CHK2 ratio from three independent experiments conducted as in 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. Error bars represent mean ± SD, multiple Unpaired Student’s t test; *, P < 0.05. (F) Representative immunoblots of p53R2 (top band) and ACTIN (loading control) in WT and FKO H292 cells. (G) Representative Spinning disk imaging of Nuclei (Hoechst; blue), γH2AX (green), and 53BP1 (magenta) in cells transfected with the indicated siRNA for 96 h. Merge of the signals is shown in the right-hand column. Control (siCtrl) cells were treated with Doxorubicin (1 µM for 4 h) as a positive control for nuclear DNA damage. Scale bar: 10 µm. (H and I) Quantification of γH2AX and (I) 53BP1 foci/cell in G. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05; ****, P > 0.0001. n = 15–25 nuclei/condition. (J) Relative expression of FAM43A, RRM1, and RRM2B in cells transfected with the indicated siRNA (siCtrl and siFAM43A #1) and collected 96 h after transfection (n = 3 technical triplicates). Expression was measured by RT-qPCR and normalized to ACTIN. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05; ***, P < 0.001; ****, P < 0.0001. (K) Relative expression of FAM43A and RRM2B normalized to ACTIN as measured by RT-qPCR. Cells were transfected with control or siRNA against RRM2B. 48 h after transfection, cells were transfected with siRNA against FAM43A and collected 96 h later. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (L) Relative expression of FAM43A and RRM1 in cells transfected with the indicated siRNA as in K. Expression was measured by RT-qPCR and normalized to ACTIN. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ns, P > 0.05; ****, P < 0.0001. (M) Relative expression of FAM43A and RRM2 of cells transfected with the indicated siRNA as in K. Expression was measured by RT-qPCR and normalized to ACTIN. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ***, P < 0.001; ****, P < 0.0001. (N) Relative expression of FAM43A and CHK2 of cells transfected with the indicated siRNA as in K. Expression was measured by RT-qPCR and normalized to ACTIN. Data points represent the average of technical triplicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ****, P < 0.0001. The expression of FAM43A was normalized to the same control (siCtrl) sample in panels L–N. Source data are available for this figure: SourceData FS4.

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