Fixed cells were stained with the indicated antibodies. GUID:?80BE154B-1391-4B91-861F-483D6BD7A23C Physique S2: CSN-CRL4CDT2 depletion induces cell cycle block in G2 phase and re-replication. (A) HeLa cells were depleted of the indicated proteins and cell cycle distribution was MNS analyzed by MNS DNA content flow cytometry detection following propidium iodide staining. A representative FACS profile of many independent experiments with similar results is shown. (B) HeLa cells transfected with the indicate siRNAs were fixed and stained with antiCSer10-phospho-H3 main antibody, Alexa 488-conjugated secondary antibody, and PI. Cells were analyzed by FACS. The mitotic cells in square are positive for phospho-H3. (C) HeLa cells synchronized by DTB in mid S-phase and analyzed for IF in Physique 1C, were checked for cell cycle phase by FACS analysis and for protein depletion by immunoblotting.(TIF) pone.0060000.s002.tif (884K) GUID:?2DD9BD35-0D84-459A-9239-9E51CD7F3981 Physique S3: Either DDB1- or CDT2-depleted U2OS cells show DDR activation and cell cycle delay. U2OS cells depleted of the indicated proteins were harvested for further analysis. (A) Cells were fixed and stained with antibodies to H2AX phospho-S139 (H2AX) and 53BP1; the nucleus was counterstained with DAPI. A fluorescent image of a representative nucleus is usually shown. A Cell sample was employed to check protein depletion by immunoblotting. (B) The cell cycle distribution was analyzed by DNA content flow cytometry detection following propidium iodide staining. A representative FACS profile with percentage of cells in each cell cycle phase of three impartial experiments with comparable results is shown.(TIF) pone.0060000.s003.tif (1.1M) Rabbit Polyclonal to Actin-pan GUID:?F1BBCA1C-A6E9-40D7-927E-D9F24A854F49 Figure S4: CSN-CRL4CDT2 has CDT1-dependent and CDT1-independent functions. (A) HeLa cells were transfected with the indicated siRNAs ( MNS caffeine). Cell cycle distribution was analyzed by BrdU incorporation and DNA content circulation cytometry detection. (B) 48 hrs following the last transfection cycle with control (siLUC), CUL4A (siCUL4A), CUL4B (siCUL4B) or both CUL4A and CUL4B (siCUL4) siRNAs, HeLa cells were harvested and processed for SDS-PAGE. Immunoblotting was performed with the indicated antibodies. (C) 48 hrs following the last transfection cycle with control (siLUC) and CSN5 (siCSN5) siRNAs, HeLa cells were harvested and processed for SDS-PAGE. Immunoblotting was performed with the indicated antibodies.(TIF) pone.0060000.s004.tif (1.0M) GUID:?108ABBD6-AA16-4950-A958-A60ECCFC0E1D Physique S5: DDB1-depleted cells show DSBs. Alkaline comet assay on control and DDB1-depleted HeLa cells. A graphical representation of the imply percentage of cells with tail instant >3 as DNA damage parameter is shown. Mean value and error were calculated on three impartial experiments.(TIF) pone.0060000.s005.tif (551K) GUID:?1B847EE5-5CF7-475E-9F7C-34A25C5147F2 Physique S6: CSN depletion activates checkpoints. HeLa cells were harvested 48 hrs after the last transfection cycle with control (siLUC) or both CSN2 and CSN5 siRNA (siCSN). Total protein lysates were fractionated by SDS-PAGE and immunoblotted with the indicated antibody.(TIF) pone.0060000.s006.tif (882K) GUID:?9E33A6B7-89BE-4A16-8A69-48B593DAAE19 Figure S7: DDB1-depleted U2OS cells show a CDT1-dependent and a CDT1-impartial delay in G2. U2OS cells were harvested 48 hrs after the last transfection cycle with control (siLUC), DDB1 (siDDB1) or both DDB1 and CDT1 (siDDB1+siCDT1) siRNAs and subjected to further analysis (A) Cell cycle distribution was analyzed by BrdU incorporation and DNA content flow cytometry detection. In the upper panel is shown a dual parameter dot plot of PI versus BrdU-Alexa 488. In the lower panel is shown a histogram display of DNA content versus counts. (B) Total protein extracts were fractionated by SDS-PAGE and immunoblotted with the indicated antibody.(TIF) pone.0060000.s007.tif (1.0M) GUID:?429645F5-6EC2-45F4-96E4-157C6C2E30FB Physique S8: DDB1-depletd cells are a mix population of both ATM and RPA colocalizing foci cells and RPA only foci cells. U2OS cells were transfected with control or siDDB1. Fixed cells were stained with the indicated antibodies. Nuclei were stained by DAPI. (A) A fluorescent picture of a representative microscopic field is usually shown. The white arrow indicates cells with both pATM and RPA signal. The yellow arrow indicates MNS cells with RPA signal. (B) Cells with RPA only foci and RPA +pATM foci were counted and represented as bar graph. Mean value and error were calculated on three impartial experiments. At least 50 cells per impartial experiment were scored.(TIF) pone.0060000.s008.tif (1012K) GUID:?02E6876C-52DE-4425-B21A-F7F0D61A65E7 Figure S9: Inactivation of either HLTF/RAD18 or CRL4CDT2 induces cell mortality. (A) and (B) U2OS cells were transfected with the indicated siRNAs and both detached and adherent cells were harvested. Total protein lysates were analyzed by immunoblotting with the indicated antibodies. indicates caspase3-cleaved PARP1 fragment. * indicates a background band. (C) HeLa cells were transfected with the indicated siRNAs..