figure s1. in absence of rvb1, treatment with various dna damaging agents results in increased...
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Figure S1. In absence of Rvb1, treatment with various DNA damaging agents results in increased phosphoH2AX. HeLa cells were transfected with indicated siRNA for 72 hrs and either left untreated or treated with indicated DNA damaging agents. Mitomycin C (MMC), cisplatin (CIS), camptothecin (CPT) or etoposide (ETP) were added for 12 hrs after 60 hrs of siRNA transfection. Cell lysates were either immnublotted with indicated antibody (A) or Immunofluorescence of the fixed cells with anti-phosphoH2AX antibody was performed (B).
- MMC CIS CPT ETP
H2AXpS139
H2AXpS139
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DAPI
siRVB1-A
siControl
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Jha S et al Fig S1
Figure S2. Increased phosphoH2AX foci formation after Rvb1 depletion can be rescued by expression of siRNA resistant Rvb1. HeLa cells (either stably expressing Flag-Rvb1 without 3’UTR or not) were transfected with indicated siRNA for 72 hrs and either left untreated or UV-irradiated (20 J/m2) before fixation. Mitomycin C (MMC) or etoposide (ETP) were added for 12 hrs after 60 hrs of siRNA transfection. Immunofluorescence of the fixed cells with anti-phosphoH2AX antibody.
- UV MMC ETP
H2AXpS139
H2AXpS139
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DAPI
siRVB1-B
siRVB1-B
siControl
H2AXpS139
DAPI
- Flag-Rvb1
+ Flag-Rvb1
Jha S et al Fig S2
Figure S3. Increase of DNA damage cannot account for the increase of phosphoH2AX accumulation at 60 min.HeLa cells were either UV-irradiated (20 J/m2) or left un-irradiated after 72 hrs of indicated siRNAs transfection. Cells were harvested at 60 min after radiation and single cell gel electrophoresis was performed in alkaline or neutral gels.The tail moment is directly proportional to the length and intensity of the comet tails. (A) Quantitation of single strand and double strand DNA breaks on alkaline agarose gels. Graphs represents the percentage of cells categorized by their tail moments at indicated time points. (B) Quantitation of double strand DNA breaks on neutral gels.
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Jha S et al Fig S3
Figure S4. Loss of Rvb1 does not affect repair of cyclobutane pyrimidine dimers. HeLa cells were transfected with indicated siRNA for 72 hrs and either left untreated or UV-irradiated (20 J/m2). DNA was extracted from the cells harvested at indicated time post UV-irradiation and ELISA with anti-CPD antibody was performed. Percentage recovery from cyclobutane pyrimidine dimer is plotted.
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Jha S et al Fig S4
Figure S5. Both wild type and mutant Tip60 interact with Rvb1. 293T cells were transfected with either vector or plasmids expressing FLAG-TIP60 wild type (Tip60wt) or FLAG-TIP60 mutant (Tip60mut). Immunoprecipitates of Flag-tagged proteins were immunoblotted with anti-Rvb1 or anti-Flag antibodies.
Jha S et al Fig S5
Figure S6. Tip60 acetylates histone H4. 293T cells transfected with vector or plasmid expressing FLAG-TIP60. Tip60 complex was immunopecipitated using anti-Flag antibody. Equal amounts of immune-complexes were incubated for 0 or 10 min with core histones in the HAT assay. Acetylated histone H4 detected by immunoblotting with anti-acetyl-lysine and anti-histone H4 antibodies. Coomassie stained (CB) gel and immunoblot with anti-Flag antibody shows equal amount of histones and Tip60 complex used in the indicated reactions respectively.
Jha S et al Fig S6
Figure S7. (A) Tip60 complex mediated histone acetyl transferase (HAT) activity. Flag-Tip60 immunoprecipitates prepared as in Fig. S6 after transfection of indicated plasmids. Equal amounts of immune-complexes were incubated for 0 or 10 min with core histones in the HAT assay. Acetylated histone H4 detected by immunoblotting with anti-acetyl-lysine and anti-histone H4 antibodies. (B) Quantitation of HAT activity of Tip60 complex in (A). Anti-acetyl-lysine signal was quantitated using Scion image software. Mean S.D. of 3 experiments.
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Jha S et al Fig S7
Figure S8. Purification of recombinant Rvb1. Rvb1 was expressed as a His-tagged protein in bacteria and purified using TALON metal affinity resin under native conditions (Clonetech). CB and IB represents Coomassiae blue stained gel and immunoblot with anti-His antibody respectively.
Jha S et al Fig S8
Figure S9. rTip60 mediated histone acetyltransferase (HAT) activity. His-Flag tagged Tip60 was expressed and purified using TALON metal affinity resin under native conditions (Clonetech). HAT assay was performed using increasing amount of rTip60 rRvb1 as indicated. Transfer of acetyl groups to the histones was detected using anti-acetyl antibody. Immunoblots with anti-Flag and anti-Rvb1 are shown for amount of rTip60 and rRvb1 proteins respectively.
Jha S et al Fig S9