tp53bp1  (Novus Biologicals)

Journal: Cell Death & Disease

Article Title: The EHMT2-MBLAC2 axis suppresses ribosomal DNA transcription in response to nucleolar DNA damage

doi: 10.1038/s41419-026-08616-1

Figure Lengend Snippet: A The cellular localization of MBLAC2 in HeLa I- Ppo I cells following rDNA DSBs induction. HeLa I- Ppo I cells were transfected with EGFP-MBLAC2 and were treated with Shield-1 and 4-OHT for 4 h. Fixed cells were labeled with anti-53BP1, anti-γH2AX or anti-C23 antibodies. Nuclei were counterstained with DAPI. Enlarged images show the details of the indicated proteins. Quantification of relative signal intensities of EGFP-EHMT2, 53BP1, γH2AX and C23 was performed by ImageJ. The white lines in the enlargements of the representative images indicate the lines for analysis. The edge of the indicated nucleolar caps was labeled with dotted circle. B Analysis of nucleolar transcription activity by EU incorporation assay in MBLAC2-inactivated HeLa I- Ppo I cells following rDNA DSBs induction. Cells transduced with control (shCTR) or two independent MBLAC2-targeted shRNAs were induced for rDNA DSBs for 4 h. EU nucleolar intensity was subsequently determined by EU incorporation assay. At least 200 cells exhibiting well-circumscribed nucleoli were quantitatively assessed across two independent experiments. Quantification of relative EU nucleolar intensity is shown in Tukey boxplots. C Nucleolar EU intensities were analyzed in HeLa I- Ppo I cells treated with the EHMT2 gRNA or MBLAC2 siRNA after I- Ppo I induction. Relative nucleolar EU intensity was quantified from at least two independent experiments. Immunoblot of MBLAC2 and EHMT2 in the HeLa I- Ppo I cells induced with the indicated gRNA or siRNA. D Colony survival of HeLa I- Ppo I cells transfected with CTR siRNA and siRNA targeting MBLAC2 or EHMT2 following I- Ppo I induction, respectively. Cells were induced for rDNA DSBs for 5 min. After washing with PBS twice, cells were allowed to grow for two weeks before harvest and Coomassie blue staining. The relative outgrowth of the colonies between groups were quantified and plotted. The protein expression of MBLAC2 and EHMT2 in HeLa I- Ppo I cells transfected with the indicated siRNAs were examined by immunoblot. E Immunoblot of MBLAC2 and EHMT2 in the HeLa I- Ppo I cells transduced with control gRNA (CTR gRNA) and two EHMT2 gRNAs (EHMT2 KO1 and EHMT2 KO2). F shRNA-mediated MBLAC2 knockdown efficiency was measured by Western blotting. G The EHMT2-MBLAC2 interaction was confirmed by Co-immunoprecipitation (Co-IP). Flag-DYRK1B was used as the positive control. H HeLa I- Ppo I cells transduced with control gRNA (CTR gRNA) and two EHMT2 gRNAs (EHMT2 KO1 and EHMT2 KO2) were treated with cycloheximide for 0, 5, and 10 h. The protein expression of MBLAC2 and EHMT2 in HeLa I- Ppo I cells was examined by immunoblot. I The relative MBLAC2 protein level in HeLa I-PpoI cells treated with cycloheximide as depicted in ( A ) was measured. Data were derived from three independent experiments. J HeLa I-PpoI cells were transduced with control gRNA (CTR gRNA) and two EHMT2 gRNAs (EHMT2 KO1 and EHMT2 KO2). Fold change of MBLAC2 mRNA in I- Ppo I cells treated with cycloheximide was determined by RT-qPCR. Quantification of MBLAC2 mRNA fold change was from three independent experiments. K Proposed working model of EHMT2-MBLAC2 axis in promoting rDNA DSB-induced transcriptional suppression. Bars represent mean ± SEM; ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Antibodies used for immunofluorescence (IF) staining: anti-γH2AX (EMD Millipore, #JBW301); Anti-53BP1 (Novusbio, #NB100-304); Fibrillarin(CST, 2639T); anti-Ki-67 (Abcam, #ab15580); anti-C23 (Nucleolin; CST, #14574); anti-UBF (F-9) (Santa Cruz, #sc-13125); anti-BRCA1 (Santa Cruz, #sc-6954); Anti-Rad51 (Santa Cruz, # sc-398587); anti-Myc (Abclonal, #AE070); anti-GFP (Proteintech, #66002-1); Alexa Fluor 594 AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch, #115-585-166); Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch, #111-545-003); Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch, #115-545-003); Alexa FluorTM Plus 405 Anti-Rabbit (Invitrogen, # A48254 ); Alexa FluorTM Plus 405 Anti-Mouse (Invitrogen, #A48255).

Techniques: Transfection, Labeling, Activity Assay, Transduction, Control, Western Blot, Staining, Expressing, shRNA, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Positive Control, Derivative Assay, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Ultra-low-input rG4-seq reveals the RNA G-quadruplex regulome in gene expression and genome integrity

doi: 10.1093/nar/gkag040

Figure Lengend Snippet: Loss of DHX36 leads to R-loop accumulation and DNA damage. ( A and B ) Representative images (A) and quantification (B) of R-loops detected by S9.6 antibody in control and Dhx36 fl/fl ;SKO oocytes. DNA was stained with DAPI (blue). Data are presented as mean ± SEM; P < 0.0001 (Student’s t -test); scale bars: 10 μm. ( C ) Western blot analysis showing levels of DHX36, phosphorylated CHK1 (p-CHK1), and γH2AX in control and Dhx36 fl/fl ;SKO oocytes. β-Actin serves as loading control. ( D ) Representative images showing co-localization of R-loops (S9.6, green) with DNA damage markers RAD51 and 53BP1 (red) in control and Dhx36 fl/fl ;SKO oocytes; scale bars: 10 μm. ( E ) Representative images showing R-loops (S9.6) and DNA double-strand breaks (γ-H2AX) in control and Dhx36 fl/fl ;SKO oocytes with or without RNaseH1 mRNA or α-amanitin treatment; scale bars: 10 μm.

Article Snippet: Primary antibodies used were: SRSF1/ASF/SF2 (12929-2-AP, Proteintech, rabbit, IF), RNASEH1 (15606-1-AP, Proteintech, rabbit, IF/ICC), Phospho-Histone H2A.X (Ser139) (20E3, 9718S, CST, rabbit, IF), 53BP1 (ET1704-05, HUABIO, rabbit, IF), YBX2/MSY2 (R1510-36, HUABIO, rabbit, IF), DHX36 (ab70269_50 μl, Abcam, WB), Anti-DNA G-quadruplex (G4) (clone 1H6, MABE1126, Merck Millipore, mouse, IF), Anti-GFP (ab290, Abcam, rabbit, LACE-seq), Anti-FLAG M2 (F1804-50UG, Sigma, LACE-seq), HA-tag (MF5) (H1003-100 μl, Lablead, mouse, LACE-seq), RanBP16/exportin 7 (GTX638871, Genetex, rabbit, IF), RNA pol II (39 097, Active Motif, mouse, IF/Cut&Tag), RNA pol II CTD phospho Ser2 (Clone 3E10, 61 984, Active Motif, rat, IF), Anti-DNA-RNA Hybrid (clone S9.6, MABE1095, Merck Millipore, mouse, IF), SFRS9 (A12538, Abclonal, rabbit, IF), HEXIM1 (ab240647, Abcam, rabbit, Cut&Tag), Cdk9 (ab239364, Abcam, rabbit, IF/Cut&Tag), p300 (F-4, sc-48343, Santa Cruz Biotechnology, mouse, IF), Cyclin T1 ( R24032 , Zen-bio, rabbit, IF), TCEA1 ( A20972 , Abclonal, rabbit, IF), and DDX4/MVH antibodies (ab13840 and ab27591, Abcam, rabbit and mouse respectively, IF).

Techniques: Control, Staining, Western Blot

Journal: Journal of medicinal chemistry

Article Title: Discovery of Potent Isoquinolinequinone N -Oxides to Overcome Cancer Multidrug Resistance.

doi: 10.1021/acs.jmedchem.4c00705

Figure Lengend Snippet: Figure 10. Effect of compound 25 on the NCI-H460 cellular expression of proteins involved in proliferation, cell death and DNA damage, analyzed by Western Blotting. (A) Representative blots of TP53BP1, PARP-1, cyclin D1, p21, cyclin E, p-histone H2A.X, p53, and procaspase-3, after cells treatment for 48 h with medium, DMSO (vehicle), doxorubicin (positive control), GI50 concentration (0.35 μM), or 2 × GI50 concentration (0.7 μM) of compound 25. V1, % of vehicle used at the GI50 concentration of compound; V2, % of vehicle used at 2 × GI50 concentration of compound. Doxo: doxorubicin at 50 nM. (B) Graphical representation of the expression of proteins analyzed. β-actin was used as loading control. Results are the mean ± SEM from 3 independent experiments. * p ≤0.05, ** p ≤0.01 when comparing DMSO vs compound treatment.

Article Snippet: The membranes were incubated for 90 min at RT with the following primary antibodies: caspase-3 (1:200; sc-56053), cyclin D1 (1:200; sc-8396), cyclin E (1:100; sc-377100), p21 (1:200; sc-6246), p53 (1:200; sc-126), PARP-1 (1:200; sc-8007), p-Histone H2A.X (1:200; sc-517348), TP53BP1 (1:100; sc-515841), and β-actin (1:200; sc13118) from Santa Cruz Biotechnology, Dallas, Texas, USA.

Techniques: Expressing, Western Blot, Positive Control, Concentration Assay, Control