Journal: Molecular Cancer

Article Title: MutS homologue hMSH5: role in cisplatin-induced DNA damage response

doi: 10.1186/1476-4598-11-10

Figure Lengend Snippet: Analysis of cisplatin-induced hMSH5 association with chromatin and nuclear foci formation . ( A ) 293T/f-hMSH5 and 293T/f-hMSH5 Y742F cells were treated with 20 μM cisplatin, and cross-linked bulk chromatin was immunoprecipitated by an α-acetyl-histone H3 antibody 5 hrs post-treatment. The levels of chromatin-associated hMSH5 and its levels of phosphorylation were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Equal levels of acetyl-histone H3 and histone H4 were present in the immunoprecipitates. ( B ) Analysis of hMSH4 chromatin association following cisplatin treatment. Chromatin was prepared from 293T/f45 cells treated with cisplatin. The levels of chromatin-associated hMSH5 and hMSH4 were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Mouse IgG was used as a negative control. kDa , molecular weight ( Mr ) in thousands. ( C ) Examination of γ-H2AX foci formation 24 hrs post cisplatin exposure. 293T, 293T/f-hMSH5, 293T/f-hMSH5 Y742F , and 293T cells subjected to hMSH5 RNAi were used for this analysis. Cells possessing greater than 15 foci/nucleus were graphically displayed. ( D ) Immunoblotting analysis of the effectiveness of hRad51 or hMSH5 knockdown in 293T cells transfected with pmH1P-Bsd/hRad51 sh-1 or pmH1P-Bsd/hMSH5 sh-2. α-Tubulin was used as a loading control. kDa , molecular weight ( Mr ) in thousands. ( E ) Analysis of hMSH5 and hRad51 knockdown on cisplatin-induced hRad51 and hMSH5 nuclear foci formation. Cells were subjected to 10 μM cisplatin for 2 hrs and were analyzed for hMSH5 foci formation 6 hrs post cisplatin removal. Cells that possessed five or more nuclear foci for hMSH5 or hRad51 were scored. Error bars represent standard deviations of the means of three independent measurements. Statistically significant differences between knockdown and control cells were indicated with asterisks (p < 0.05, Student t -test).

Article Snippet: Antibodies used in the experiments included α-acetyl histone H3 (Upstate), mouse IgG (Upstate), α-p-Tyr (Cell Signaling), α-hMSH5 [ ], α-hMSH4 [ ], and α-histone H4 (Cell Signaling).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Molecular Weight, Transfection

Journal: American Journal of Cancer Research

Article Title: Short chain fatty acids exhibit selective estrogen receptor downregulator (SERD) activity in breast cancer

doi:

Figure Lengend Snippet: Effects of SCFAs on HDAC1 and HDAC6 expression. MCF-7 cells expressing wild-type ERα (A), ERα-D538G (B) and ERα-Y537S (C) were treated with SCFAs for 24 hours and whole cell lysates were analyzed by western blots for changes in histone acetylation and effects on total H3 and H4 are also given. MCF-7 cells expressing wild-type ERα (D), ERα-D538G (E) and ERα-Y537S (F) were transfected with oligonucleotides targeted against HDAC6 or HDAC1 and after 72 hours whole cell lysates were analyzed by western blots. MCF-7 cells expressing wild-type ERα (G), ERα-D538G (H) and ERα-Y537S (I) were treated with SCFAs by western blots as outlined above and in the Methods.

Article Snippet: Estrogen Receptor α (D6R2W), HDAC6 (D2E5), Acetyl-Histone H3 (Lys9/Lys14), Acetyl-Histone H3 (Lys27), Acetyl-Histone H4 (Lys8) antibodies were purchased from Cell Signaling (Boston, MA); HDAC1 (10E2) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Expressing, Western Blot, Transfection

Journal: bioRxiv

Article Title: A cobB like protein in Oryza sativa indica regulates the mitochondrial machinery under stress conditions

doi: 10.1101/2022.04.17.488584

Figure Lengend Snippet: (A) GST pull down assay was performed to detect the binding of HIS-GST-tagged OscobB with recombinant histones H3 and H4 (NEB). Here, 12% SDS-PAGE gel shows OscobB can physically bind to both the histones H3 and H4. GST protein alone was used as a negative control in lanes 8 and 9 suggesting that the histones are not non-specifically binding with GST. (B) Detection of deacetylase activity of OscobB: Deacetylation assay of histone H3 from rice leaf nuclear extract was performed at 28°C. The reaction mixture containing OscobB was collected at different time points (0-105 mins). Sample reaction was stopped by boiling them with sample dye and then loading on a 12% SDS-PAGE gel. Western blot analysis was performed using H3Ac primary antibody to detect the extent of deacetylation by OscobB. Here the loading control is histone H3. (C) Detection of the deacetylation of specific lysine residues in histone H3 by OscobB. Lane 1- Empty pET28a vector, lane 2- purified recombinant OscobB, lane 3- purified HsSIRT6. Anti-H3 antibody was used as loading control for all the western blots. Empty vector and HsSIRT6 were used as negative and positive control, respectively. Coomassie brilliant blue gel shows the reaction mix containing purified OscobB along with histone H3 in lane 2 and purified HsSIRT6 along with histone H3 in lane 3. (D) Saturation kinetics for the OscobB enzyme activity: MM plot showing the OscobB deacetylation of H3K9Ac and H3K18Ac from rice leaf nuclear extract using varied concentrations of NAD + (0-600 μM). The non-linear regression plots were calculated using Graphpad Prism 8.3.0. The error bar depicts the S.D.; n=3. Further kinetic parameters ( K m and k cat values) were calculated using these plots.

Article Snippet: The histone H3Ac antibody sampler kit (9927T) and acetylated Histone H4 antibody sampler kit (ab218056) used were purchased from Cell Signaling Technology (CST) and abcam (USA), respectively.

Techniques: Pull Down Assay, Binding Assay, Recombinant, SDS Page, Negative Control, Histone Deacetylase Assay, Activity Assay, Western Blot, Plasmid Preparation, Purification, Positive Control