DUX4 expression impacts DNA damage response factor recruitment to sites of damage. (A) Combined RNA-FISH and immunofluorescence of HSATII RNA (green), γH2A.X (red), and BRCA1 (cyan) signal in −dox, treated with 10 μM etoposide for 30 min and immediately fixed (+eto), briefly induced with 2 μg/ml doxycycline for 4 h and fixed 20 h after induction (+dox) iDUX4 cells (scale bar = 20 μm). Arrow indicates nuclei with γH2A.X foci and HSATII RNA but lacks BRCA1 foci formation at sites of damage. Asterisk indicates nuclei with γH2A.X foci and BRCA1 foci formation at sites of damage in HSATII− nuclei. Images are representative of two independent experiments conducted on separate days. (B) Fraction nuclei with BRCA1 foci formation at DNA damage sites (γH2A.X foci) in −dox, +eto and +dox either in HSATII− or HSATII+ nuclei. Dots represent fields taken from representative experiments. N ≥ 50 nuclei per condition. (C) Western blot of whole cell lysate probing BRCA1 total protein in +eto, −dox, pulsed, or continuous dox-treated cells. The blot shows two biological replicates. Note that I uses the same GAPDH control image because the same western membrane was used to probe for BRCA1 and RAP80. (D) Immunofluorescence of γH2A.X (red) and RNF168 (cyan) signal in −dox, treated with 10 μM etoposide for 30 min and immediately fixed (+eto), briefly induced with 2 μg/ml doxycycline for 4 h and fixed 20 h after induction (+dox), or briefly induced with 2 μg/ml doxycycline for 4 h and at 20 h after induction treated with 10 μM etoposide for 30 min and then immediately fixed (+dox +eto) iDUX4 cells (scale bar = 20 μm). Images are representative from two independent experiments conducted on separate days. (E) Fraction nuclei with RNF168 foci formation at DNA damage sites (γH2A.X foci) in +eto, +dox pulse (+dp), and +dp +eto cells. Dots represent the average of fields taken from a representative experiment. N ≥ 50 nuclei per condition. (F) Western blot of whole cell lysate probing RNF168 total protein in +eto, −dox, pulsed, or continuous dox treated cells. The blot shows two biological replicates. Note that Fig. 6 F uses the same GAPDH control image because the same western membrane was used to probe for RAD51 and RNF168. (G) Immunofluorescence of γH2A.X (green) and RAP80 (magenta) signal in −dox, +eto, +dox, and +dox +eto iDUX4 cells, (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (H) Fraction nuclei with RAP80 foci formation at DNA damage sites (γH2A.X foci) in +eto, +dox pulse (+dp), and +dp +eto cells. Dots represent the average of fields taken from a representative experiment. N ≥ 50 nuclei per condition. (I) Western blot of whole cell lysate probing RAP80 total protein in +eto, −dox, pulsed, or continuous dox treated cells. The blot shows two biological replicates. Note that C uses the same GAPDH control image because the same western membrane was used to probe for BRCA1 and RAP80. (B, E, and H) Data represent means ± SD. Statistical differences between groups were analyzed with one-way ANOVA Tukey’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData FS3.
Figure S3.

DUX4 expression impacts DNA damage response factor recruitment to sites of damage. (A) Combined RNA-FISH and immunofluorescence of HSATII RNA (green), γH2A.X (red), and BRCA1 (cyan) signal in −dox, treated with 10 μM etoposide for 30 min and immediately fixed (+eto), briefly induced with 2 μg/ml doxycycline for 4 h and fixed 20 h after induction (+dox) iDUX4 cells (scale bar = 20 μm). Arrow indicates nuclei with γH2A.X foci and HSATII RNA but lacks BRCA1 foci formation at sites of damage. Asterisk indicates nuclei with γH2A.X foci and BRCA1 foci formation at sites of damage in HSATII− nuclei. Images are representative of two independent experiments conducted on separate days. (B) Fraction nuclei with BRCA1 foci formation at DNA damage sites (γH2A.X foci) in −dox, +eto and +dox either in HSATII− or HSATII+ nuclei. Dots represent fields taken from representative experiments. N ≥ 50 nuclei per condition. (C) Western blot of whole cell lysate probing BRCA1 total protein in +eto, −dox, pulsed, or continuous dox-treated cells. The blot shows two biological replicates. Note that I uses the same GAPDH control image because the same western membrane was used to probe for BRCA1 and RAP80. (D) Immunofluorescence of γH2A.X (red) and RNF168 (cyan) signal in −dox, treated with 10 μM etoposide for 30 min and immediately fixed (+eto), briefly induced with 2 μg/ml doxycycline for 4 h and fixed 20 h after induction (+dox), or briefly induced with 2 μg/ml doxycycline for 4 h and at 20 h after induction treated with 10 μM etoposide for 30 min and then immediately fixed (+dox +eto) iDUX4 cells (scale bar = 20 μm). Images are representative from two independent experiments conducted on separate days. (E) Fraction nuclei with RNF168 foci formation at DNA damage sites (γH2A.X foci) in +eto, +dox pulse (+dp), and +dp +eto cells. Dots represent the average of fields taken from a representative experiment. N ≥ 50 nuclei per condition. (F) Western blot of whole cell lysate probing RNF168 total protein in +eto, −dox, pulsed, or continuous dox treated cells. The blot shows two biological replicates. Note that Fig. 6 F uses the same GAPDH control image because the same western membrane was used to probe for RAD51 and RNF168. (G) Immunofluorescence of γH2A.X (green) and RAP80 (magenta) signal in −dox, +eto, +dox, and +dox +eto iDUX4 cells, (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (H) Fraction nuclei with RAP80 foci formation at DNA damage sites (γH2A.X foci) in +eto, +dox pulse (+dp), and +dp +eto cells. Dots represent the average of fields taken from a representative experiment. N ≥ 50 nuclei per condition. (I) Western blot of whole cell lysate probing RAP80 total protein in +eto, −dox, pulsed, or continuous dox treated cells. The blot shows two biological replicates. Note that C uses the same GAPDH control image because the same western membrane was used to probe for BRCA1 and RAP80. (B, E, and H) Data represent means ± SD. Statistical differences between groups were analyzed with one-way ANOVA Tukey’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData FS3.

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