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. Author manuscript; available in PMC: 2024 Feb 5.
Published in final edited form as: Cell Rep. 2023 Dec 5;42(12):113523. doi: 10.1016/j.celrep.2023.113523

Figure 4. FANCD2-dependent MiDAS is impaired when PCNA K164 cannot be ubiquitinated.

Figure 4.

(A) Representative images of phospho-H3 S10-stained nuclei (blue), EdU foci (green), and FANCD2 (red) foci from APH-treated RPE-1 wild-type and PCNAK164R cell lines. EdU foci almost exclusively (>95%) colocalized with FANCD2 foci. Scale bar, 5 μm.

(B) FANCD2 foci quantification from two biological replicates in RPE-1 wild-type (blue), PCNAK164R (maroon), RAD18−/− (salmon), and RAD18−/−;PCNAK164R (pink) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ***p < 0.001. FANCD2 foci overlapped with EdU foci in 98% of nuclei.

(C) EdU foci quantification from two biological replicates in RPE-1 wild-type (blue), PCNAK164R (maroon), RAD18−/− (salmon), and RAD18−/−;PCNAK164R (pink) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ***p < 0.001.

(D) FANCD2 foci quantification from two biological replicates in 293T wild-type (blue) and PCNAK164R cells (maroon) treated with 450 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by one-way ANOVA with Tukey’s multiple comparison test with ***p < 0.001.

(E) EdU foci quantification from two biological replicates in 293T wild-type (blue) and PCNAK164R cells (maroon) treated with 450 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by one-way ANOVA with Tukey’s multiple comparison test with ***p < 0.001.

(F) Western blot analyses of chromatin associated PCNA, with or without 40 J/m2 UV treatment, with histone H2AX as the loading control from RPE-1 wildtype, PCNAK164R, and reverted PCNAK164 cells.

(G) FANCD2 foci quantification from two biological replicates in RPE-1 wild-type (blue), PCNAK164R (maroon), and reverted PCNAK164 (purple) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ****p < 0.001.

(H) EdU foci quantification from two biological replicates in RPE-1 wild-type (blue), PCNAK164R (maroon), and reverted PCNAK164 (purple) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ****p < 0.001.