Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase 10 Regulates DNA Mismatch Repair and May Involve the Deacetylation of MutS Homolog 2 *

doi: 10.1074/jbc.M114.612945

Figure Lengend Snippet: Multiple HDACs interact with MSH2. A, several Class II HDACs interact with MSH2 in 293T cells. 293T cells were transiently transfected with plasmids expressing HA-MSH2 and FLAG (F)-tagged Class II HDACs as indicated. HA-MSH2 immunoprecipitation (IP) with anti-HA-agarose beads was performed followed by immunoblotting (IB) with an anti-FLAG antibody (upper panel). Total cell lysates were subjected to immunoblot analyses with anti-FLAG and anti-HA antibodies (middle and lower panels). B, all Class I HDACs interact with MSH2 in 293T cells. 293T cells were transiently transfected with plasmids expressing HA-MSH2 and FLAG-tagged Class I HDACs as indicated. HA-MSH2 immunoprecipitation with anti-HA-agarose beads was performed followed by immunoblotting with an anti-FLAG antibody (upper panel). Total cell lysates were subjected to immunoblot analyses with anti-FLAG and anti-HA antibodies (middle and lower panels). C, multiple HDACs interact with endogenous MSH2 in 293T cells. 293T cells were transiently transfected with plasmids expressing the indicated FLAG-tagged Class I and II HDACs. Immunoprecipitation of HDACs with anti-FLAG M2-agarose beads was performed followed by immunoblot analyses with anti-MSH2 antibody to detect endogenous MSH2 (upper panel). The blot was then stripped and reprobed with anti-FLAG antibody (middle panel). Levels of MSH2 were confirmed by Western blot analysis of the cell lysates using an anti-MSH2 antibody (lower panel). D, interaction between endogenous HDAC10 and MSH2 in HeLa cells. HeLa cell lysates were first immunoprecipitated with anti-IgG or anti-MSH2 antibody and then subjected to immunoblot analysis using an anti-HDAC10 antibody. The blot was then stripped and reprobed with an anti-MSH2 antibody.

Article Snippet: 293T cells were transiently transfected with plasmids expressing FLAG-MSH2 and treated with TSA for 12 h. Highly acetylated MSH2 was immunoprecipitated by anti-FLAG-agarose beads and followed by incubating with 3 μg of BSA (as a control) or recombinant HDAC10 (BML-SE559-0050, Enzo Life Sciences) at 37 °C for 2 h. After washing the anti-FLAG-agarose beads with LS buffer (0.1% NP-40, 10% glycerol in PBS), the FLAG-MSH2 immunoprecipitates were subjected to the immunoblotting analysis with the anti-MSH2Ac-K73 antibody ( upper panel ).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase 10 Regulates DNA Mismatch Repair and May Involve the Deacetylation of MutS Homolog 2 *

doi: 10.1074/jbc.M114.612945

Figure Lengend Snippet: HDAC10 deacetylates MSH2 at Lys-73. A, the level of Lys-73 acetylation (Ac) was dramatically increased by treatment with TSA but not NIC or NaB. HeLa cells were treated with HDAC inhibitors, including TSA, NaB, and NIC, for 12 h as indicated. Total lysates were subjected to immunoblot (IB) analysis using the anti-MSH2Ac-K73 antibody (upper panel). The blot was stripped and reprobed with anti-MSH2 antibody (lower panel). B, FLAG-HDAC10 deacetylates Lys-73 in MSH2. 293T cells were transiently transfected with plasmids expressing HA-MSH2 and FLAG (F)-tagged HDACs as indicated. Total cell lysates were immunoprecipitated (IP) with anti-HA-agarose beads followed by immunoblotting with anti-MSH2Ac-K73 antibody (first panel). The blot was then stripped and reprobed with anti-HA antibody (second panel). Total cell lysates were subjected to immunoblotting with anti-MSH2 (third panel) or anti-FLAG (fourth panel) antibody. C, HDAC10 deacetylates MSH2 at Lys-73 in vitro. 293T cells were transiently transfected with plasmids expressing FLAG-MSH2 and treated with TSA for 12 h. Highly acetylated MSH2 was immunoprecipitated by anti-FLAG-agarose beads and followed by incubating with 3 μg of BSA (as a control) or recombinant HDAC10 (BML-SE559-0050, Enzo Life Sciences) at 37 °C for 2 h. After washing the anti-FLAG-agarose beads with LS buffer (0.1% NP-40, 10% glycerol in PBS), the FLAG-MSH2 immunoprecipitates were subjected to the immunoblotting analysis with the anti-MSH2Ac-K73 antibody (upper panel). The blot was then stripped and reprobed with the anti-FLAG antibody (lower panel). D, HDAC10 overexpression reduces Lys-73 acetylation in HeLa cells. FLAG-HDAC10 was stably overexpressed in HeLa cells. Total lysates prepared from control and FLAG-HDAC10-overexpressing cells were subjected to immunoblot analysis with anti-MSH2Ac-K73, anti-MSH2, and anti-FLAG antibodies (upper, middle, and lower panels, respectively). E, HDAC10 knockdown increases Lys-73 acetylation in MSH2. The lentiviral system was used to transduce HDAC10-specific shRNA (TRCN0000004861, Thermo Fisher Scientific) or scramble shRNA into HeLa cells. Lentivirus-transduced cells were selected for puromycin resistance, and the shRNA stably expressing cells were lysed in NETN buffer (100 mm NaCl, 20 mm Tris-Cl (pH 8.0), 0.5 mm EDTA, 0.5% NP-40). The total cell lysates were immunoprecipitated with the anti-MSH2 antibody followed by immunoblotting analysis with the anti-MSH2Ac-K73 antibody (first panel), anti-HDAC10 antibody (second panel), anti-MSH2 antibody (third panel), or anti-β-actin antibody (fourth panel).

Article Snippet: 293T cells were transiently transfected with plasmids expressing FLAG-MSH2 and treated with TSA for 12 h. Highly acetylated MSH2 was immunoprecipitated by anti-FLAG-agarose beads and followed by incubating with 3 μg of BSA (as a control) or recombinant HDAC10 (BML-SE559-0050, Enzo Life Sciences) at 37 °C for 2 h. After washing the anti-FLAG-agarose beads with LS buffer (0.1% NP-40, 10% glycerol in PBS), the FLAG-MSH2 immunoprecipitates were subjected to the immunoblotting analysis with the anti-MSH2Ac-K73 antibody ( upper panel ).

Techniques: Western Blot, Transfection, Expressing, Immunoprecipitation, In Vitro, Recombinant, Over Expression, Stable Transfection, Transduction, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase 10 Regulates DNA Mismatch Repair and May Involve the Deacetylation of MutS Homolog 2 *

doi: 10.1074/jbc.M114.612945

Figure Lengend Snippet: HDAC10 promotes DNA MMR activity. A, TSA, but not NaB or NIC, reduces DNA MMR activity. HeLaS3 cells were treated with 1.3 μm TSA, 10 mm NaB, or 10 mm NIC for 8 h. Nuclear extracts prepared from these cells (50, 100, or 200 μg) were used in the DNA MMR assay. B, HDAC10 promotes DNA MMR activity. FLAG-HDAC10 was stably overexpressed in HeLaS3 cells. Nuclear extracts of control and FLAG (F)-HDAC10-expressing stable cells were used in the DNA MMR assay. Data represent three independent experiments. C, repaired bands in panel B were quantified by densitometry and graphed. Values represent mean ± S.D. D, HDAC10 knockdown reduces DNA MMR activity. HDAC10 was stably knocked down in HeLa cells as in Fig. 3E. The substrate only (as a negative control) and the substrate with nuclear extracts of HeLa cells (as a positive control), sh Scramble-, or shHDAC10-transduced HeLa cells were used in the DNA MMR assay (upper panel). Data represent three independent experiments. The lower panel is the quantitative result of repair percentage in each group (lower panel). Values represent mean ± S.D. *, p < 0.05. E, MSH2K73R does not promote DNA MMR activity. MSH2WT and MSH2K73R were stably overexpressed in HeLaS3 cells. Nuclear extracts of control, MSH2WT-, and MSH2K73R-expressing stable cells were used in the DNA MMR assay (lower panel). For standardization of MMR assay, HelaS3 nuclear lysates were used for MMR assay at the indicated concentration. As a negative control for the assay, HEC59 cells that lack MSH2 were used (upper panel). The MMR assay was performed as outlined under “Experimental Procedures.”

Article Snippet: 293T cells were transiently transfected with plasmids expressing FLAG-MSH2 and treated with TSA for 12 h. Highly acetylated MSH2 was immunoprecipitated by anti-FLAG-agarose beads and followed by incubating with 3 μg of BSA (as a control) or recombinant HDAC10 (BML-SE559-0050, Enzo Life Sciences) at 37 °C for 2 h. After washing the anti-FLAG-agarose beads with LS buffer (0.1% NP-40, 10% glycerol in PBS), the FLAG-MSH2 immunoprecipitates were subjected to the immunoblotting analysis with the anti-MSH2Ac-K73 antibody ( upper panel ).

Techniques: Activity Assay, Stable Transfection, Expressing, Negative Control, Positive Control, Concentration Assay