KDM2 proteins and PRC1 components are required for 53BP1 foci formation and/or H2AUb signaling following DNA damage. (A) Quantification of total RNF2 protein levels in control knockdown (siCTRL) or in RNF2-depleted cells (siRNF2). The blot shows three biological replicates. Protein levels were normalized to loading control (GAPDH). (B) Representative immunofluorescence images of H2AUb signal (gray) within siCTRL cells or siRNF2 cells either left untreated or treated briefly with 10 μM etoposide. Scale bar = 20 μm. Images are representative of three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (C) Quantification of nuclear H2AUb signal intensity in siCTRL or siRNF2 cells with or without etoposide treatment. Dots represent the number of nuclei measured. Nuclei are indicated for representative experiment and N ≥ 25 nuclei per condition. (D) Representative immunofluorescence images of γH2A.X (green) and 53BP1 (magenta) within siCTRL cells or siRNF2 cells either left untreated or treated briefly with 10 μM etoposide. Scale bar = 20 μm. Images are representative from three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (E) Quantification of the number of 53BP1 foci per nucleus in siCTRL or siRNF2 cells with or without etoposide treatment. Nuclei are indicated for representative experiment and N ≥ 25 nuclei per condition. (F) Western blot of whole cell lysate probing for total KDM2A and KDM2B protein levels in control knockdown (siCTRL), KDM2A-depleted cells (siKDM2A), or KDM2B-depleted (siKDM2B) cells. Blot shows two biological replicates. Protein levels were normalized to loading control (GAPDH). (G) Representative immunofluorescence images of γH2A.X (green) and 53BP1 (magenta) within siCTRL, siKDM2A, or siKDM2B cells either left untreated or treated briefly with 10 μM etoposide. Scale bar = 20 μm. Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (H) Quantification of the number of 53BP1 foci per nucleus in siCTRL, siKDM2A, or siKDM2B cells with or without etoposide treatment. Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition. (A, C, E, and H) Data represent means ± SD. Statistical differences between groups were analyzed with one-way ANOVA Tukey’s multiple comparison or Dunnett’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData FS5.
Figure S5.

KDM2 proteins and PRC1 components are required for 53BP1 foci formation and/or H2AUb signaling following DNA damage. (A) Quantification of total RNF2 protein levels in control knockdown (siCTRL) or in RNF2-depleted cells (siRNF2). The blot shows three biological replicates. Protein levels were normalized to loading control (GAPDH). (B) Representative immunofluorescence images of H2AUb signal (gray) within siCTRL cells or siRNF2 cells either left untreated or treated briefly with 10 μM etoposide. Scale bar = 20 μm. Images are representative of three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (C) Quantification of nuclear H2AUb signal intensity in siCTRL or siRNF2 cells with or without etoposide treatment. Dots represent the number of nuclei measured. Nuclei are indicated for representative experiment and N ≥ 25 nuclei per condition. (D) Representative immunofluorescence images of γH2A.X (green) and 53BP1 (magenta) within siCTRL cells or siRNF2 cells either left untreated or treated briefly with 10 μM etoposide. Scale bar = 20 μm. Images are representative from three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (E) Quantification of the number of 53BP1 foci per nucleus in siCTRL or siRNF2 cells with or without etoposide treatment. Nuclei are indicated for representative experiment and N ≥ 25 nuclei per condition. (F) Western blot of whole cell lysate probing for total KDM2A and KDM2B protein levels in control knockdown (siCTRL), KDM2A-depleted cells (siKDM2A), or KDM2B-depleted (siKDM2B) cells. Blot shows two biological replicates. Protein levels were normalized to loading control (GAPDH). (G) Representative immunofluorescence images of γH2A.X (green) and 53BP1 (magenta) within siCTRL, siKDM2A, or siKDM2B cells either left untreated or treated briefly with 10 μM etoposide. Scale bar = 20 μm. Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (H) Quantification of the number of 53BP1 foci per nucleus in siCTRL, siKDM2A, or siKDM2B cells with or without etoposide treatment. Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition. (A, C, E, and H) Data represent means ± SD. Statistical differences between groups were analyzed with one-way ANOVA Tukey’s multiple comparison or Dunnett’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData FS5.

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