anti nucleolin  (Proteintech)

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–C HeLa I- Ppo I cells were pre-treated with ATM inhibitor (ATMi; KU-55933) or EHMT2 inhibitor (EHMT2i; UNC0638) for 1 h prior to I- Ppo I induction for 4 h. After fixation, cells were subjected to immunofluorescence with anti-C23 ( A ), anti-Ki-67 ( B ), anti-UBF ( C ) or anti-γH2AX antibodies, respectively. The dashed circles outline margins of the nuclei. Percentages of cells with the indicated protein-containing nucleolar caps were quantified from three experiments. D – F EHMT2-KO HeLa I- Ppo I cells were processed for immunofluorescence as described in ( A – C ). Cells were labeled with anti-C23 ( D ), anti-Ki-67 ( E ), anti-UBF ( F ) or anti-γH2AX antibodies, respectively. Nuclei were counterstained with DAPI. Percentages of cells with the nucleolar caps were quantified from three experiments. Bars represent mean ± SEM; ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

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: Immunofluorescence, Labeling

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: bioRxiv

Article Title: Nucleoli as drivers of nuclear remodelling in cardiomyocytes during Heart Failure

doi: 10.64898/2026.02.24.707499

Figure Lengend Snippet: Ai , Left, representative immunocytochemistry micrographs of detyrMT (green), actin (red) and nuclei (HOECHST, blue). White arrow indicates detyrMT inside the NI. Right, scatter plot of NI quantification in control, PTL and CytD-treated cardiomyocytes (N=3-5 rats, 44-106 nuclei). Aii , Line-scan schematic of cytoplasmic and nucleoplasmic CaTs in a cardiomyocyte. Aiii , Representative recordings of distinct subcellular regions, as indicated in (ii), baseline quantification in control, PTL and CytD at 1 and 2.5Hz (bottom) (N=3-4 rats, 32-36 cells). Bi , Left, representative immunocytochemistry micrographs of nucleolin (yellow) and nuclei (HOECHST, blue). White arrow indicates NI-nucleoli localization. Right, scatter plots of area, circularity (N=5 rats,162-209 nucleoli) and NI number (N=3 rats, 60-57 nuclei) in control and BMH21-treated cells. Ci , Left, representative immunohistochemistry micrographs α-actinin (red) and nuclei (HOECHST, blue). Right, scatter plot of NI quantification in donor and DCM tissue (N=8 patients, 99-120 cells). Di , Animal model schematic. Representative immunocytochemistry micrographs of nuclei (HOECHST, grey) in Dii and detyrMT (grey) in Diii . Scatter plots of NI quantification ( Dii ) in control, 8-weeks and 16-weeks MI cardiomyocytes (N=3-4 rats, 40-44 cells) and detyrMT area quantification in control and 8-weeks MI cardiomyocytes (N=3 rats, 35-42 cells). Div , Left, mechano-Scanning Ion Conductance Microscopy representative topography and Young’s modulus maps. Right, Young’s modulus quantification in control and 8-weeks MI (N=3 rats, 33-36 cells). Representative immunocytochemistry micrographs of nucleolin (magenta) and nuclei (HOECHST, blue) in Ei and nucleophosmin (magenta), γH2AX-DNA damage (green) and nuclei (HOECHST, blue) in Eii . Scatter plots of circularity (N=3-4 rats, 96-137 nucleoli) and chromocenter Manders Coefficient (M1,M2) (N=3 rats, 43-49 cells) in control and MI cardiomyocytes ( Ei ) and γH2AX foci area ( Eii ) (N=3 rats, 48-63 cells). Eiii , Left, representative heatmap. Right, Brillouin shift absolute difference in control and 8-weeks cardiomyocytes (N=3 rats, 36-37 cells). Statistics: nested t-test ( Bi , Diii-iv ), unpaired t-test Kolmogorov-Smirnov ( Bi , Ci , Ei-iii ), non-parametric Kruskal Wallis with Dunn’s multiple comparison ( Ai , Dii , Ei ).Normality tested with Shapiro-Wilk.Data represent mean±SEM. Scale bar 5µm.

Article Snippet: Primary antibodies used: anti-mouse α-actinin (Sigma, MA1-22863), anti-rabbit detyrosinated tubulin (Invitrogen, MA5-44591), anti-rabbit nucleolin (Cell Signaling, #14574), anti-mouse nucleophosmin (Abcam, ab10530), γH2AX (Cell Signaling, #5438).

Techniques: Immunocytochemistry, Control, Immunohistochemistry, Animal Model, Microscopy, Comparison