Fig 1: DCAF14 is a replication stress response protein(A) Immunoblots of lysates harvested from U2OS cells transfected with either non-targeting siRNA (siNT)or siRNA targeting DCAF14 (siDCAF14). CHK1 serves as loading control. Arrow denotes DCAF14 protein.(B–D) siNT- and siDCAF14-transfected U2OS cells were left either untreated or treated with 4 mM HU or 100 nM CPT for 4 h. For γH2AX and RPA staining, cells were pulsed with 5-ethynyl-2’-deoxyuridine (EdU) for 10 min prior to treatment. For native BrdU staining, cells were pulsed with 10 μM BrdU. The intensities of γH2AX and RPA were measured by quantitative imaging after preselecting EdU-positive nuclei. Mean ± SEM values are representative of three biological repeats. a.u., arbitrary units.(E) siNT-and siDCAF14-transfected U2OS cells were plated for colony-forming assays as indicated. Inset: panel depicts representative dishes for untreated and treated conditions. All percent viability calculations were derived relative to untreated cells (mean ± SD, n = 3).(F) siRNA-transfected U2OS cells were left untreated or treated with 0.2 μM aphidicolin (APH) or 100 nM CPT for 24 h. 53BP1 foci were measured in nuclei without detectable proliferating cell nuclear antigen (PCNA) staining. Mean ± SEM values are representative of three biological repeats.(G) DR-GFP U2OS reporter cells were transfected with indicated siRNAs and analyzed by flow cytometry following transfection with I-Scel expression vector. Percent GFP values were normalized to control (siNT), and values were plotted using three replicates (mean ± SD). For (B)–(D) and (F), p values were derived using Mann-Whitney tests. For(E) and (G), p values were derived using multiple t test and unpaired t test, respectively. At least 150 nuclei were analyzed for(B)–(D), and 100 nuclei were analyzed for (F).See also Figure S1.
Fig 2: DCAF14 is recruited to stalled replication forks(A) Schematic for iPOND-SILAC-mass spectrometry (MS) analyses to identify replication fork proteins in HU-treated cells.(B) The average abundance values for selected proteins and protein complexes are shown. Complete datasets with statistics are presented elsewhere (Dungrawala et al., 2015). n = 14.(C) In situ proximity ligation assay (PLA) was performed using antibodies targeting RPA32 and DCAF14. Cells were left either untreated or treated with 4 mM HU and 100 nM CPT for 4 h. Nuclei with ≥5 PLA foci were scored using 500–1,000 nuclei per sample. Mean ± SD values are representative of two biological repeats. p values were derived using one-way ANOVA. Inset: representative nuclei for HU-treated cells. Scale bar, 5 μm.
Fig 3: Loss of DCAF14 impairs fork stability and fork recovery(A) Schematic for DNA fiber assays. Representative fiber images for indicated samples are shown.(B and C) siNT and siDCAF14-transfected U2OS cells were pulsed using labeling scheme in presence of CPT. CldU and IdU lengths were measured, and lengths were plotted as histograms in (B) and as ratios in (C).(D) Parental U2OS cells or DCAF14 KO clones transfected with entry vector or DCAF14 cDNA were pulsed using labeling scheme in presence of CPT, and IdU/CldU ratios were plotted. Immunoblot depicts DCAF14 expression levels and KU70 represents loading control.(E) Whole-cell lysates for the indicated samples were immunoprecipitated using FLAG resin, and immunoprecipitates were probed with the indicated antibodies. Representative blot is shown from three biological repeats.(F) U2OS cells transfected with the indicated siRNAs were pulsed with CldU for 30 min followed by IdU with 100 nM CPT for 30 min, and IdU/CldU ratios were plotted.(G) Representative immunoblot depicting knockdown efficiencies for the indicated siRNAs is shown from two biological repeats. KU70 serves as loading control. Arrow represents neddylated species.(H) siNT- and siDCAF14-transfected U2OS cells were treated as shown. Fibers with both CldU and IdU labels were scored as a percentage of all fibers imaged.(I) Comet tails were measured for indicated cells with or without HU using neutral comet assay. Box-and-whiskers plots depicting 10–90 percentile are plotted using at least 100 nuclei. At least 120 fibers were analyzed, and representative mean ± SEM values from two biological replicates are plotted in (C), (D), (F), and (H). p values were derived using Mann-Whitney tests in (C), (D), (F), and (I). Unpaired t test was used in (H).See also Figure S2.
Fig 4: DCAF14 mediates replication fork protection(A) Schematic for fork degradation assays is depicted. Representative fiber images for indicated samples are shown.(B–E and G–J) Fork degradation assays were performed in U2OS cells using the indicated siRNAs. In (C), MRE11 inhibitor Mirin was used as indicated. In (I), cells were transfected with constructs expressing either wild-type RAD51 or K133R mutant RAD51. IdU/CldU ratios for individual experiments are plotted. Mean ± SEM values are representative of at least two biological repeats. At least 120 fibers were analyzed. p values were derived using Mann-Whitney tests.(F) Parental U2OS and DCAF14 KO cells (top) and siNT- and siDCAF14-transfected U2OS cells (bottom) were either left untreated or treated with 4 mM HU for 4 h. Chromatin extracts and whole-cell lysates were isolated as shown, separated using SDS-PAGE, and probed for the indicated antibodies by immunoblotting.See also Figures S3 and S4.
Fig 5: Selective release of BET proteins from chromatin in Huh7 cells by RVX-208.Salt extraction of chromatin releases BET proteins into the soluble (S) fraction in RVX-208 stimulated cells, whereas another dual bromodomain protein PHIP stays in the insoluble (pellet, P) fraction. The solubility of other transcription factors (HNF4a, NR2F2) is unaltered.
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