Skip to main content
. Author manuscript; available in PMC: 2022 Nov 4.
Published in final edited form as: Mol Cell. 2021 Sep 30;81(21):4440–4456.e7. doi: 10.1016/j.molcel.2021.09.008

Figure 7. Protexin loss exposes a previously uncharacterized role for RNA polymerase activity and REV3 polymerase activity in fork protection.

Figure 7.

(A) WT U2OS were stably transfected with linearized plasmids expressing tagged vector, WT REV3 or REV3 D2781A/D2783A (lacking polymerase activity) and clones were selected. WT U2OS or the above clones were treated with control or an siRNA to endogenous REV3’s 3’ UTR as indicated for 48 h then treated with cisp for 18 h before immunoblotting. (B) WT and SCAI-null U2OS cells were treated with 2 μM cisp or vehicle for 10 h then with vehicle control or 10 μg/ml α-amanitin (POL2i) for 6 h before immunoblotting. (C). WT and SCAI-null U2OS were treated with EdU 15 minutes prior to treatment with vehicle or 5 μM cisp for 3 h. Cells were pre-extracted and stained for RPA2 and EdU (via click-it reaction). Nuclear intensity of EdU positive cells was measured by imageJ. Representative images shown. (D) WT U2OS were treated with EdU and either vehicle, 5 μM cisp for 3 h, α-Amanitin for 2 h or both (α-Amanitin added after 1 h cisp treatment) and processed as in (C). Representative images shown. (E) Quantification of the experiments in (C). T-tests, **** = p<0.0001. (F) Quantification of the experiments in (D). T-tests, **** = p<0.0001. (G) WT U2OS were treated with control or siRNA to FANCM for 48 h then with EdU and 5 μM cisp for 1 hr followed by vehicle or 100 μM DRB for 2 h before processing as in (C). Quantification shown. T-tests, **** = p<0.0001. (H) WT U2OS were treated with EdU and 5 μM cisp for 1 hr. Vehicle, α-Amanitin (POL2i) or ML-60218 (POL3i) were then added for 2 h before processing as in (C). Quantification shown. T-tests, **** = p<0.0001 (I) WT and SCAI-null U2OS treated with 1 μM cisp for 8 h were processed for PLAs using the indicated antibodies. Quantification shown in bottom panel. Mann Whitney test, ***=p<0.001. (J) SCAI-null U2OS transfected with the indicated siRNAs for 60 h, were treated with 1 μM cisp for 8 h and processed for PLAs as in (I). Quantification shown in bottom panel. Mann Whitney test, ***=p<0.001. (K) WT and SCAI-null U2OS transfected with the indicated siRNAs for 60 h, were treated with 1 μM cisp for 8 h, pre-extracted, fixed and processed for PLAs using the indicated antibodies. Quantification shown in bottom panel. Mann Whitney test, ***=p<0.001.