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: Immunity, Inflammation and Disease

Article Title: WTAP Contributes to Periodontitis Pathogenesis by Promoting PDLSC Senescence and Impairing Osteogenic Differentiation via m6A‐Dependent Regulation of TP53BP1

doi: 10.1002/iid3.70335

Figure Lengend Snippet: WTAP modifies TP53BP1 via m6A. (a) The m6A sites of TP53BP1 were predicted by the SRAMP website. (b, c) TP53BP1 expression in P‐PDLSCs transfected with shNC and shWTAP was measured by RT‐qPCR and western blot, respectively (Unpaired t ‐test). (d) MeRIP was used to determine the m6A levels on TP53BP1 mRNA in P‐PDLSCs transfected with shNC and shWTAP (Unpaired t ‐test). (e) TP53BP1 mRNA enrichment in P‐PDLSCs treated with anti‐IgG + shNC, anti‐IgG + shWTAP, anti‐WTAP+shNC, and anti‐WTAP + shWTAP was examined by RIP (one‐way ANOVA). (f) TP53BP1 mRNA expression in P‐PDLSCs co‐treated with shNC or shWTAP and Act D for 0, 2, 4, and 6 h was detected by RT‐qPCR (one‐way ANOVA). (g) Luciferase activities in the WT‐TP53BP1 + shNC, WT‐TP53BP1 + shWTAP, MUT‐TP53BP1 + shNC, and MUT‐TP53BP1+shWTAP groups were measured by dual‐luciferase reporter assay (one‐way ANOVA). All experiments were conducted at least three independent times. * p < 0.05.

Article Snippet: Wild‐type (WT) and mutant (MUT) TP53BP1 3′UTR luciferase reporter plasmids (WT‐TP53BP1 and MUT‐TP53BP1) were constructed by Sangon Biotech (Shanghai) Co. Ltd. (Shanghai, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay

Journal: Immunity, Inflammation and Disease

Article Title: WTAP Contributes to Periodontitis Pathogenesis by Promoting PDLSC Senescence and Impairing Osteogenic Differentiation via m6A‐Dependent Regulation of TP53BP1

doi: 10.1002/iid3.70335

Figure Lengend Snippet: TP53BP1 downregulation mitigates WTAP‐caused P‐PDLSC senescence and oxidative stress. (a) RT‐qPCR was used to measure the mRNA levels of TP53BP1 in P‐PDLSCs transfected with shNC and shTP53BP1 (Unpaired t ‐test). (b) Western blot was applied to determine the protein levels of TP53BP1 in P‐PDLSCs transfected with shNC and shTP53BP1 (Unpaired t ‐test). (c–h) The P‐PDLSCs were treated with oeNC + shNC, oeWTAP + shNC, and oeWTAP + shTP53BP1. (c–e) The MDA, SOD, and ROS levels were examined using the corresponding kits (one‐way ANOVA). (f) The γ‐H2AX positive cells were detected using IF (Scale bar = 100 μm; one‐way ANOVA). (g) SA‐β‐gal positive cells were measured using the corresponding kit (Scale bar = 100 μM; one‐way ANOVA). (h) Western blot was applied to determine the protein levels of p53 and p16 (one‐way ANOVA). All experiments were conducted at least three independent times. * p < 0.05.

Article Snippet: Wild‐type (WT) and mutant (MUT) TP53BP1 3′UTR luciferase reporter plasmids (WT‐TP53BP1 and MUT‐TP53BP1) were constructed by Sangon Biotech (Shanghai) Co. Ltd. (Shanghai, China).

Techniques: Quantitative RT-PCR, Transfection, Western Blot

Journal: Immunity, Inflammation and Disease

Article Title: WTAP Contributes to Periodontitis Pathogenesis by Promoting PDLSC Senescence and Impairing Osteogenic Differentiation via m6A‐Dependent Regulation of TP53BP1

doi: 10.1002/iid3.70335

Figure Lengend Snippet: WTAP represses the osteogenic differentiation of P‐PDLSCs via TP53BP1. The P‐PDLSCs were treated with oeNC+shNC, oeWTAP+shNC, and oeWTAP + shTP53BP1. (a) ALP staining of P‐PDLSCs induced for 7 days (Scale bar = 100 μM; one‐way ANOVA). (b) ARS staining of P‐PDLSCs induced for 28 days (Scale bar = 100 μM; one‐way ANOVA). (c) Western blot was used to determine the protein levels of OPN, OCN, and RUNX2 (one‐way ANOVA). All experiments were conducted at least three independent times. * p < 0.05.

Article Snippet: Wild‐type (WT) and mutant (MUT) TP53BP1 3′UTR luciferase reporter plasmids (WT‐TP53BP1 and MUT‐TP53BP1) were constructed by Sangon Biotech (Shanghai) Co. Ltd. (Shanghai, China).

Techniques: Staining, Western Blot

Journal: Cell Reports Methods

Article Title: Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

doi: 10.1016/j.crmeth.2025.101270

Figure Lengend Snippet: Induction of resistance in vitro induces a reduction in PARP trapping as assessed by the EGFP-PARP1-mCherryFP FRET biosensor (A–C) (A) Schematic of the treatment protocol to induce in vitro resistance to olaparib or rucaparib. OVCAR4 EGFP-PARP1-mCherryFP cells were treated daily with 20 μM olaparib or rucaparib for 8 weeks. Resistance was assessed by SRB assay to (B) rucaparib or (C) olaparib and compared to vehicle-treated parental OVCAR4 EGFP-PARP1-mCherryFP cells. t tests were performed to confirm IC 50 values, n = 3 biological repeats. (D and E) (D) Live-cell FLIM-FRET HCA assays were performed to assess changes in donor lifetime of the EGFP-PARP1-mCherryFP FRET biosensor upon 1 h treatment with rucaparib or (E) olaparib. Single-cell FLIM data show population-level distributions of mean-weighted fluorescence lifetime. Data are shown as mean ± SEM, n = 3 biological repeats, and statistical significance was assessed using one-way ANOVA. (F) Change in FRET biosensor donor fluorescence lifetime of vehicle-exposed (left) or olaparib-exposed (right) cells upon treatment with rucaparib at 0, 10, 30, or 50 μM for 1 h. One-way ANOVA (Kruskal-Wallis) with Dunn’s multiple comparison test was used to assess statistical significance between treatment conditions. (G and H) (G) RT-qPCR analysis of PARG and (H) TP53BP1 relative mRNA expression normalized to GAPDH expression. n = 3 biological repeats. (I) Representative images of rucaparib and EGFP fluorescence in OVCAR4 EGFP-PARP1-mCherryFP cells following vehicle, rucaparib, or olaparib exposure in vitro for 9 weeks. (J) Quantification of average intracellular rucaparib intensity of images in (I), each point is a single cell average and error bars represent population median. n = 3 biological repeats.

Article Snippet: TaqMan primer probes ( GAPDH (Hs02786624_g1), PARG ( Hs00608254_m1 ), TP53BP1 (Hs00996827_m1), ABCB1 (Hs00184500_m1), Thermo Fisher Scientific).

Techniques: In Vitro, Sulforhodamine B Assay, Fluorescence, Comparison, Quantitative RT-PCR, Expressing

Journal: Cell Reports Methods

Article Title: Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

doi: 10.1016/j.crmeth.2025.101270

Figure Lengend Snippet: In vivo resistance generation to olaparib leads to a reduction in PARP trapping (A) OVCAR4 EGFP-PARP1-mCherryFP cells were injected intraperitoneally (I.P.) into female CD1 nude mice and allowed to grow for 21 days before mice were treated with vehicle or olaparib (5 mg/kg) daily for 14 days. Mice were culled upon reaching humane endpoint, and omental tumors and ascites were harvested. (B) Representative images showing OVCAR4 EGFP-PARP1-mCherryFP cells grown in 2D monolayers before injecting into CD1 nude mice (left) and the ascites harvested from the peritoneal cavity of these mice after 90 or 92 days for vehicle- and olaparib-treated groups, respectively. Images were acquired using the Incucyte S3 with 10× objective. (C) Quantification of average spheroid area per condition. (D) Viability assays were performed on the ascites cells grown in 2D, treated with olaparib for 72 h. Survival scores are normalized to vehicle-treated controls. Error bars represent SEM, n = 3 biological replicates. (E) Representative IHC images of MDR1 (brown stain) from OVCAR4 EGFP-PARP1-mCherryFP tumors, scale bars: 1 mm. (F) OVCAR4 EGFP-PARP1-mCherryFP omental tumors harvested at day 90 (vehicle) or 92 (olaparib) were stained for MDR1 by IHC. The number of MDR1-positive tumor cells was quantified using QuPath. Statistical significance was tested using an unpaired t test. (G and H) (G) RT-qPCR analysis of PARG and (H) TP53BP1 relative mRNA expression normalized to GAPDH expression. n = 3 biological repeats. (I) Representative images of rucaparib intrinsic fluorescence (left), EGFP-PARP1 (middle), and merged image (right) in the vehicle- or olaparib-exposed populations, scale bars: 25 μm. (J) Quantification of median rucaparib signal per cell. n = 3 biological repeats. (K) Fluorescence lifetimes of EGFP measured in FLIM assays of OVCAR4 EGFP-PARP1-mCherryFP cells that had been harvested from mice ascites of each group. Cells retrieved from vehicle- and olaparib-treated mice were incubated with olaparib (0–50 μM) for 1 hour prior to fluorescence microscopy. Error bars show mean and SEM value, n = 3 biological replicates. One-way ANOVA (Kruskal-Wallis) with Dunn’s multiple comparison test was used to assess statistical significance between treatment conditions.

Article Snippet: TaqMan primer probes ( GAPDH (Hs02786624_g1), PARG ( Hs00608254_m1 ), TP53BP1 (Hs00996827_m1), ABCB1 (Hs00184500_m1), Thermo Fisher Scientific).

Techniques: In Vivo, Injection, Staining, Quantitative RT-PCR, Expressing, Fluorescence, Incubation, Microscopy, Comparison