Dynamics of DUX4 and HSATII expression, and BMI1 aggregation in DUX4-expressing cells. (A) Representative immunofluorescence of DUX4 (gray) expression in −dox, briefly induced (4-h doxycycline treatment, “4-h fix”) and 24 h after brief doxycycline pulse (“24-h fix”) in iDUX4 cells (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (B) Fraction of nuclei DUX4+ for all conditions. Dots represent the average of fields taken. N ≥ 50 nuclei. (C) The ratio of DUX4 signal type (aggregates or pan-nuclear staining pattern) in +dox at either 4- or 24-h timepoint. N ≥ 50 nuclei. (D) HSATII RNA FISH signal (gray) in −dox, and pulse or continuous doxycycline induction and fixed 20 h after induction (scale bar = 20 μm). Images are representative of four independent experiments conducted on separate days. (E) Fraction nuclei that are HSATII RNA positive (HSATII+) in −dox, pulse, or continuous conditions. Dots represent the average of fields taken. N ≥ 50 nuclei. (F) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and BMI1 (magenta) signal in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Images are representative of one independent experiment. (G) BMI1 signal intensity measured within nuclei in −dox, +dox HSATII−, or HSATII+ nuclei, compared to BMI1 signal intensity measured within HSATII RNA foci or randomly drawn ROI within the nucleoplasm in +dox HSATII+ nuclei. Each dot represents either individual nuclei or individual foci, respectively. Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition or N ≥ 40 ROI. (H) Immunofluorescence of RNF2 (green) and BMI1 (magenta) signal in −dox or +dox (4-h pulse and fixed/analyzed 20-h post-induction) iDUX4 cells. Images are representative of three independent experiments conducted on separate days. (I) Fraction nuclei with BMI1 signal type (foci versus pan-nuclear staining pattern) within +dox cells. Dots represent each independent experiment. (J) Fraction nuclei with RNF2 foci co-occur with or contain only BMI1 foci in +dox iDUX4 cells. Fraction calculated represents fields taken from each independent experiment and N ≥ 50 nuclei. (K) Immunofluorescence of H2AUb (green) and BMI1 (magenta) signal in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Images are representative of two independent experiments conducted on separate days. (L) Nuclear H2AUb signal in −dox or +dox cells with BMI1 signal type. Dots represent individual nuclei for the representative experiment. N ≥ 25 nuclei per condition. (B, E, G, I, and L) Data represent means ± SD. Statistical differences between groups were analyzed employing either two-tailed paired t test or were assessed with one-way ANOVA Tukey’s multiple comparison test between each group and a control.
Figure S1.

Dynamics of DUX4 and HSATII expression, and BMI1 aggregation in DUX4-expressing cells. (A) Representative immunofluorescence of DUX4 (gray) expression in −dox, briefly induced (4-h doxycycline treatment, “4-h fix”) and 24 h after brief doxycycline pulse (“24-h fix”) in iDUX4 cells (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (B) Fraction of nuclei DUX4+ for all conditions. Dots represent the average of fields taken. N ≥ 50 nuclei. (C) The ratio of DUX4 signal type (aggregates or pan-nuclear staining pattern) in +dox at either 4- or 24-h timepoint. N ≥ 50 nuclei. (D) HSATII RNA FISH signal (gray) in −dox, and pulse or continuous doxycycline induction and fixed 20 h after induction (scale bar = 20 μm). Images are representative of four independent experiments conducted on separate days. (E) Fraction nuclei that are HSATII RNA positive (HSATII+) in −dox, pulse, or continuous conditions. Dots represent the average of fields taken. N ≥ 50 nuclei. (F) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and BMI1 (magenta) signal in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Images are representative of one independent experiment. (G) BMI1 signal intensity measured within nuclei in −dox, +dox HSATII−, or HSATII+ nuclei, compared to BMI1 signal intensity measured within HSATII RNA foci or randomly drawn ROI within the nucleoplasm in +dox HSATII+ nuclei. Each dot represents either individual nuclei or individual foci, respectively. Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition or N ≥ 40 ROI. (H) Immunofluorescence of RNF2 (green) and BMI1 (magenta) signal in −dox or +dox (4-h pulse and fixed/analyzed 20-h post-induction) iDUX4 cells. Images are representative of three independent experiments conducted on separate days. (I) Fraction nuclei with BMI1 signal type (foci versus pan-nuclear staining pattern) within +dox cells. Dots represent each independent experiment. (J) Fraction nuclei with RNF2 foci co-occur with or contain only BMI1 foci in +dox iDUX4 cells. Fraction calculated represents fields taken from each independent experiment and N ≥ 50 nuclei. (K) Immunofluorescence of H2AUb (green) and BMI1 (magenta) signal in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Images are representative of two independent experiments conducted on separate days. (L) Nuclear H2AUb signal in −dox or +dox cells with BMI1 signal type. Dots represent individual nuclei for the representative experiment. N ≥ 25 nuclei per condition. (B, E, G, I, and L) Data represent means ± SD. Statistical differences between groups were analyzed employing either two-tailed paired t test or were assessed with one-way ANOVA Tukey’s multiple comparison test between each group and a control.

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