Figure S4.
Silencing of SLX4, XPF, MUS81, and FANCD2, and additional data for SLX4- and FANCD2-mediated accumulation of the DDR factors at the LacI-bound lacO.(A and B) U2OS 40–2-6 ER-LacI cells were transfected with control (siGL2) or SLX4 (siSLX4-3 or siSLX4-4)–targeting siRNAs for 48 h. (A) Confirmation of SLX4 knockdown. Cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Coomassie Brilliant Blue staining served as a loading control. (B) After siRNA treatment, cells were further treated with 1 µM 4-OHT for 2 h and then subjected to colocalization analysis as described in Fig. 1, C and D. Values were calculated from the sum scores of two independent experiments. **, P < 0.01; ***, P < 0.001 (χ2 test). Individual data points from the two independent experiments are also shown. (C–F) Cells were transfected with control siRNAs (a mixture of siGFP and siLuci) or siRNAs targeting XPF (siXPF-1 and siXPF-2) or MUS81 (siMUS81-1 or siMUS81-2) for 48 h and analyzed as described in A and B. ***, P < 0.001; n.s., not significant (χ2 test). (G and H) Cells were transfected with control (mixture of siGFP and siLuci) or FANCD2 (siFANCD2-1 or siFANCD2-2)-targeting siRNAs for 48 h. (G) Cells were then subjected to immunoblotting with anti-FANCD2 antibody. (H) At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for 6 h and then subjected to colocalization analysis as described in B. **, P < 0.01; ***, P < 0.001 (χ2 test). (I) U2OS 40–2-6 ER-LacI cells were treated with 10 µM ATR inhibitor (VE-821) or a vehicle (DMSO) for 6 h and 1 µM 4-OHT for the last 2 h (as in Fig. 3 E). Cells were then subjected to SDS-PAGE and immunoblotting to confirm that ATR-mediated Chk1 phosphorylation is inhibited by VE-821 treatment. (J) U2OS 40–2-6 ER-LacI cells were incubated with 1 µM 4-OHT and either DMSO as a vehicle or 50 µM mirin for 2 h. Colocalization frequencies of RPA foci with LacI foci are analyzed as described in B. n.s., not significant (χ2 test). (K) U2OS 40–2-6 ER-LacI cells were transfected with the indicated siRNAs. At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for a further 6 h and then analyzed as described in B. ***, P < 0.001 (χ2 test).

Silencing of SLX4, XPF, MUS81, and FANCD2, and additional data for SLX4- and FANCD2-mediated accumulation of the DDR factors at the LacI-bound lacO. (A and B) U2OS 40–2-6 ER-LacI cells were transfected with control (siGL2) or SLX4 (siSLX4-3 or siSLX4-4)–targeting siRNAs for 48 h. (A) Confirmation of SLX4 knockdown. Cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Coomassie Brilliant Blue staining served as a loading control. (B) After siRNA treatment, cells were further treated with 1 µM 4-OHT for 2 h and then subjected to colocalization analysis as described in Fig. 1, C and D. Values were calculated from the sum scores of two independent experiments. **, P < 0.01; ***, P < 0.001 (χ2 test). Individual data points from the two independent experiments are also shown. (C–F) Cells were transfected with control siRNAs (a mixture of siGFP and siLuci) or siRNAs targeting XPF (siXPF-1 and siXPF-2) or MUS81 (siMUS81-1 or siMUS81-2) for 48 h and analyzed as described in A and B. ***, P < 0.001; n.s., not significant (χ2 test). (G and H) Cells were transfected with control (mixture of siGFP and siLuci) or FANCD2 (siFANCD2-1 or siFANCD2-2)-targeting siRNAs for 48 h. (G) Cells were then subjected to immunoblotting with anti-FANCD2 antibody. (H) At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for 6 h and then subjected to colocalization analysis as described in B. **, P < 0.01; ***, P < 0.001 (χ2 test). (I) U2OS 40–2-6 ER-LacI cells were treated with 10 µM ATR inhibitor (VE-821) or a vehicle (DMSO) for 6 h and 1 µM 4-OHT for the last 2 h (as in Fig. 3 E). Cells were then subjected to SDS-PAGE and immunoblotting to confirm that ATR-mediated Chk1 phosphorylation is inhibited by VE-821 treatment. (J) U2OS 40–2-6 ER-LacI cells were incubated with 1 µM 4-OHT and either DMSO as a vehicle or 50 µM mirin for 2 h. Colocalization frequencies of RPA foci with LacI foci are analyzed as described in B. n.s., not significant (χ2 test). (K) U2OS 40–2-6 ER-LacI cells were transfected with the indicated siRNAs. At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for a further 6 h and then analyzed as described in B. ***, P < 0.001 (χ2 test).

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