Fig. S7
Depletion of CEP164 or ZNF423(Zpf423) Results in Sensitivity to DNA Damaging Agents (A) Multicolor competition assay (MCA) (Smogorzewska et al., 2007). GFP-U2OS cells were transfected with the indicated siRNAs against CEP164 and ZNF423. Seventy-two hours after after transfection they were mixed with RFP-U2OS cells transfected with Luciferase (Luc) and DNA damage was induced using the indicated agents. After 7 days, percent of gfp-positive cells was measured using fluorescence-activated cell sorting and the resistance to DNA damage was calculated in comparison to untreated cells. DNA damage resistance was set at 100% for GFP-U2OS cells transfected with an siRNA against Luciferase (Luc). Cells transfected with siRNAs against ATM were used as a positive control. Experiments were done in triplicate. Standard deviations are indicated. Depletion of CEP164 (B) led to sensitivity to camptothecin (CPT) and gamma irradiation (IR). In addition, depletion with siRNA #3 led to sensitivity to mitomycin C (MMC). Furthermore, transfection of two separate ZNF423 siRNAs (#2 and #3), that resulted in ZNF423 mRNA depletion as assessed by RT-qPCR (C), caused sensitivity to MMC, CPT, and IR (L). An siRNA that did not result in ZNF423 mRNA depletion (C), siRNA #1 did not result in increased sensitivity to DNA damage. (B) Western blot analysis of CEP164 expression after siRNA transfection in cells used in the MCA assay shown in (A) (C) RT-qPCR in U2OS cells transfected with the three siRNAs against ZNF423 used in the MCA assay shown in (A). Reactions were performed in triplicate. Standard deviations are indicated. See also Figure 6. |
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