Journal: Nucleic Acids Research

Article Title: Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

doi: 10.1093/nar/gkab019

Figure Lengend Snippet: LMNB1 acetyl mimic preserves laminar integrity during HSV-1 infection. ( A ) HSV-1 replication cycle demonstrating the temporal progression of viral infection and highlighting nuclear events. ( B ) Lamin B1 (LMNB1) has five domains, and lysine 134 (K134) falls in the Coil 1B domain. ( C ) mCherry-LMNB1 morphology in uninfected cells expressing WT, K134Q or K134R LMNB1. Representative immunofluorescence (IF) images are shown, scale bar represents 5 μm. ( D ) Relative input vDNA (viral genomes) ± SD, determined using the ΔΔCT method with GAPDH as an internal control, present at 1 hpi in WT, K134Q and K134R expressing cells, n = 3 biological replicates. ( E ) Viral genomes/μl at time points throughout the HSV-1 replication cycle. Individual genome amplification plots for WT, K134Q and K134R expressing cells are shown on the right. n = at least two biological replicates. ( F ) Relative viral DNA (viral genomes) ± SD present in the nuclear fraction of the 18 hpi WT, K134Q and K134R expressing cells. Unpaired t -test, n = 3 biological replicates. ( G ) Effect of LMNB1 mutants on HSV-1 viral titers, average ± SEM, one-way ANOVA, Tukey post-hoc test, n = 3 biological replicates. ( H, J, L ) Representative IF images of MRC5 cells expressing WT, K134Q or K134R mCherry-LMNB1 and infected with HSV-1 at ( H ) 8 hpi, ( J ) 12 hpi and ( L ) 18 hpi. Scale bar represents 5 μm. Left: mCherry-LMNB1, white arrows indicate breaks. Right: merged image of mCherry-LMNB1 (magenta), ICP4 (marker of viral infection, green) and DAPI (blue). ( I, K, M ) Representative IF images of linearized and binarized mCherry-LMNB1 at ( I ) 8 hpi, ( K ) 12 hpi and ( M ) 18 hpi, breaks indicated by white arrows. Scale bar represents 5 μm. ( N, O, P ) Quantification of number of breaks in the nuclear lamina in MRC5 cells expressing WT, K134Q or K134R mCherry-LMNB1 at ( N ) 8 hpi, ( O ) 12 hpi and ( P ) 18 hpi. Average ± SEM, one-way ANOVA, Bonferroni post-hoc test, n (number of cells) is indicated in the figure. ( Q ) Proposed model for the function of LMNB1 K134 acetylation during HSV-1 infection. K134 acetylation may stabilize LMNB1, causing it to incur fewer breaks and thereby inhibit viral capsid nuclear egress.

Article Snippet: LMNB1 sequence specific TrueGuide sgRNA (Invitrogen #A35533) targeting the sequence 5′-GCACGAGACCCGTTTAGTAG-3′ (Invitrogen #A35526) was used.

Techniques: Infection, Expressing, Immunofluorescence, Amplification, Marker

Journal: Nucleic Acids Research

Article Title: Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

doi: 10.1093/nar/gkab019

Figure Lengend Snippet: LMNB1 K134 is highly conserved and K134 acetyl mimic impedes the G1 to S cell cycle transition. ( A ) LMNB1 K134 conservation among 18 species arranged by phylogenetic similarity. Lysines are highlighted in orange and arginines are highlighted in blue. ( B ) LMNB1 K134 conservation with the other human lamins (LMNA/C and LMNB2). Lysines are highlighted in orange and arginines are highlighted in blue. ( C ) Cell cycle phase was determined by flow cytometry using the combination of three distinct markers (DAPI, EdU incorporation and histone 3 serine 10 phosphorylation). ( D, F ) To determine which stage of the cell cycle LMNB1 K134 acetylation may affect, cell cycle analysis was performed by flow cytometry of MRC5 cells stably expressing WT, K134Q or K134R mCherry-LMNB1. Representative contour plots are shown with each cell cycle stage indicated. ( E, G ) Average percentage of cells ± SEM in each phase of the cell cycle. ( E ) left: G1 phase, middle: S phase, right: G2 phase ( G ) M phase. ( H ) Average percentage of cells ± SEM in (left) early S phase and (right) mid/late S phase, n = 3 biological replicates, one-way ANOVA, Bonferroni post-hoc test, average is indicated in each bar.

Article Snippet: LMNB1 sequence specific TrueGuide sgRNA (Invitrogen #A35533) targeting the sequence 5′-GCACGAGACCCGTTTAGTAG-3′ (Invitrogen #A35526) was used.

Techniques: Flow Cytometry, Cell Cycle Assay, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

doi: 10.1093/nar/gkab019

Figure Lengend Snippet: K134 acetyl mimic expression reduces G1/S checkpoint resolution. ( A, C ) To determine which stage of the cell cycle LMNB1 K134 acetylation may affect in the presence of DSBs, cell cycle analysis was performed by flow cytometry of MRC5 cells stably expressing WT, K134Q or K134R mCherry-LMNB1 that were treated with bleomycin to induce DSBs. Representative contour plots are shown with each cell cycle stage indicated. ( B, D ) Average percentage of cells ± SEM in each phase of the cell cycle. ( B ) Left: G1 phase, middle: S phase, right: G2 phase ( D ) M phase. ( E ) Average percentage of cells ± SEM in (left) early S phase and (right) mid/late S phase, n = 3 biological replicates, one-way ANOVA, Bonferroni post-hoc test, average is indicated in each bar. ( F ) Representative western blot showing pRb (S780) phosphorylation levels in cells stably expressing WT, K134Q or K134R mCherry-LMNB1 across a time course of recovery from bleomycin treatment. ( G ) Proposed hypothesis that LMNB1 acetylation may affect progression through the G1/S checkpoint. As the G1/S checkpoint is regulated by assessing DNA damage, we propose that LMNB1 K134 acetylation triggers a stall in this checkpoint.

Article Snippet: LMNB1 sequence specific TrueGuide sgRNA (Invitrogen #A35533) targeting the sequence 5′-GCACGAGACCCGTTTAGTAG-3′ (Invitrogen #A35526) was used.

Techniques: Expressing, Cell Cycle Assay, Flow Cytometry, Stable Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

doi: 10.1093/nar/gkab019

Figure Lengend Snippet: K134 acetyl mimic expression alters LMNB1 protein associations and decreases association with chromatin. ( A ) Workflow for miniTurbo followed by mass spectrometry identification of changes in protein-protein associations with WT, K134Q or K134R LMNB1 during DNA repair after DNA damage induced by bleomycin treatment. ( B ) Nuclear proteins that exhibited a two-fold change in at least one comparison (K134Q/WT, K134Q/K134R or K134R/WT). Increased abundance is shown in yellow, while decreased abundance is shown in blue. ( C, F ) Representative IF images of ( C ) LMNB1-H3K4me3 (euchromatin marker) and ( F ) LMNB1-H3K9me3 (heterochromatin marker) proximity ligation assays (PLA) before (upper) or after (lower) bleomycin treatment. In the merged image, mCherry-LMNB1 (yellow), PLA (grey), DAPI (blue), scale bar represents 5 μm. ( D, E, G, H ) Quantification of the number of PLA puncta present ( D, G ) before or ( E, H ) after bleomycin treatment. Violin plot showing median (solid line) and interquartile range (doted lines), one-way ANOVA, Tukey post-hoc test, n (number of cells) is indicated in the figure.

Article Snippet: LMNB1 sequence specific TrueGuide sgRNA (Invitrogen #A35533) targeting the sequence 5′-GCACGAGACCCGTTTAGTAG-3′ (Invitrogen #A35526) was used.

Techniques: Expressing, Mass Spectrometry, Marker, Ligation

Journal: Nucleic Acids Research

Article Title: Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

doi: 10.1093/nar/gkab019

Figure Lengend Snippet: LMNB1 acetyl mimic impedes DNA repair and dissipation of γH2AX DNA damage foci and impedes cNHEJ. ( A ) Assessment of recovery from DNA damage by alkaline comet assay. Average ±95% confidence interval. Total number of comets analyzed ( n , combined from two biological replicates) is indicated. Kruskal–Wallis test was used to determine significance. Representative IF images of different amounts of DNA damage (% tail DNA) are shown. Scale bar represents 10 μm. ( B ) Workflow for experimental setup. Untreated cells were collected prior to the start of the treatment. All other samples were treated with bleomycin for 1 h to induce DSBs. Afterward, the media was replaced and the 0 h post-treatment (hpt) sample was collected. Samples were collected every hour for 6 hpt. ( C ) Representative IF images of γH2AX foci (green) in MRC5 cells stably expressing WT, K134Q or K134R mCherry-LMNB1 (magenta) at the indicated time points post-bleomycin treatment. Scale bar represents 5 μm. ( D ) Average γH2AX fluorescence intensity ± SEM at each time point post-bleomycin treatment. The number of cells (n) is indicated, one-way ANOVA, Bonferroni post-hoc test. ( E ) Left: Average γH2AX fluorescence intensity ± SEM (shaded) over the course of recovery from bleomycin treatment. Right: Average number of γH2AX foci ± SEM (shaded) over the course of recovery from bleomycin treatment. ( F ) Left: Schematic of EJ7 reporter strategy for assessing cNHEJ activity. Cells are transfected with CRISPR gRNAs and Cas9 that induce blunt end double strand breaks on either side of a spacer inserted at the coding sequence for G67 in GFP. If repaired through cNHEJ, the coding sequence of GFP is repaired and the cells will fluoresce green. Right: Average percentage of mCherry-LMNB1 WT, K134Q or K134R expressing cells that have successfully repaired ± SEM, n = 3 biological replicates, two-sided Student's t -test. ( G ) Left: Schematic of EJ5 reporter strategy for assessing all end joining (EJ). Cells are transfected with a plasmid for the I-SceI rare-cutting endonuclease. If repaired through EJ, the promoter and the coding region of GFP will be in frame and the cells will fluoresce green. Right: Average percentage of mCherry-LMNB1 WT, K134Q or K134R expressing cells that have successfully repaired ± SEM, n = 3 biological replicates, two-sided Student's t -test. ( H ) Schematic depicting some examples of how the I-SceI DSBs in the EJ5 reporter cells may be repaired. These include full repair, trimming of the sticky ends (indicated by blue and magenta) or end resection further into the I-SceI region beyond the sticky ends. ( I ) Quantification of the percentage of DSBs that were repaired by various types of repair in the mCherry-LMNB1 WT, K134Q and K134R EJ5 stable cell lines. ( J ) Relative abundance of the endogenous LMNB1 K134 acetylation in cells synchronized in G2 phase (CDK1i) compared to unsynchronized (untreated) cells. Mean ± SD.

Article Snippet: LMNB1 sequence specific TrueGuide sgRNA (Invitrogen #A35533) targeting the sequence 5′-GCACGAGACCCGTTTAGTAG-3′ (Invitrogen #A35526) was used.

Techniques: Alkaline Single Cell Gel Electrophoresis, Stable Transfection, Expressing, Fluorescence, Activity Assay, Transfection, CRISPR, Sequencing, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

doi: 10.1093/nar/gkab019

Figure Lengend Snippet: LMNB1 acetyl mimic delays 53BP1 foci formation after DNA damage. ( A ) Representative IF images of 53BP1 foci (green) in MRC5 cells stably expressing WT, K134Q or K134R mCherry-LMNB1 (magenta) at the indicated time points post-bleomycin treatment. Scale bar represents 5 μm. ( B ) Average number of 53BP1 foci ± SEM at each time point post-bleomycin treatment. The number of cells (n) is indicated, one-way ANOVA, Bonferroni post-hoc test. Baseline number of foci is indicated by the horizontal dashed line. ( C ) Average percentage of 53BP1 foci at or below baseline numbers (number of foci present in untreated cells) over the course of recovery from bleomycin treatment. ( D ) Representative IF images of the three 53BP1 phenotypes (diffuse, mixed, foci). Scale bar represents 5 μm. ( E ) (left) Percentage of WT, K134Q or K134R cells with distinct 53BP1 foci or diffuse/mixed 53BP1 over the course of recovery from bleomycin treatment (shown as percentage out of total). (right) Percentage of cells with (top) distinct 53BP1 foci or (bottom) diffuse/mixed 53BP1 over the course of recovery from bleomycin treatment. ( F ) Representative IF images of LMNB1–53BP1 proximity ligation assay 1 hpt after bleomycin treatment. Nuclei are outlined. In the merged image, mCherry-LMNB1 (magenta), PLA (grey), DAPI (blue), scale bar represents 5 μm. ( G ) Quantification of the number of PLA puncta. Violin plot showing median (solid line) and interquartile range (doted lines), one-way ANOVA, Tukey post-hoc test, n (number of cells) is indicated in the figure. ( H ) Proposed model for the effect of LMNB1 acetylation on impeding the recruitment of the NHEJ repair factor 53BP1 to sites of DSBs.

Article Snippet: LMNB1 sequence specific TrueGuide sgRNA (Invitrogen #A35533) targeting the sequence 5′-GCACGAGACCCGTTTAGTAG-3′ (Invitrogen #A35526) was used.

Techniques: Stable Transfection, Expressing, Proximity Ligation Assay

Journal: Nucleic Acids Research

Article Title: Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

doi: 10.1093/nar/gkab019

Figure Lengend Snippet: Proposed model for how LMNB1 K134 acetylation can stabilize the nuclear lamina and act as a molecular toggle for DNA DSB repair at the nuclear periphery. ( A ) In the presence of WT LMNB1, the lamina is accessible and DNA damage repair factors can be recruited to the nuclear periphery. 53BP1 foci can activate the cNHEJ pathway for rapid DNA repair and subsequently, cell cycle progression. During herpesvirus infection, the nuclear lamina can be disrupted to facilitate viral capsid nuclear egress. ( B ) In cells expressing LMNB1 K134 acetyl mimic, intra-laminar associations are favored and the lamina is inaccessible to damaged chromatin. Because 53BP1 recruitment to damaged DNA is impaired, the G1/S checkpoint is persistently activated, and DNA repair may be slowly accomplished by compensatory pathways. During herpesvirus infection, the nuclear lamina is stabilized, impeding viral capsid nuclear egress.

Article Snippet: LMNB1 sequence specific TrueGuide sgRNA (Invitrogen #A35533) targeting the sequence 5′-GCACGAGACCCGTTTAGTAG-3′ (Invitrogen #A35526) was used.

Techniques: Infection, Expressing

Journal: iScience

Article Title: Leveraging Vγ9Vδ2 T cells against prostate cancer through a VHH-based PSMA-Vδ2 bispecific T cell engager

doi: 10.1016/j.isci.2024.111289

Figure Lengend Snippet:

Article Snippet: For the generation of a LNCaP-derived PSMA knock-out cell line, LNCaP cells were co-transfected with four predesigned synthetic CRISPR guide RNAs (CRISPR646020_SGM, CRISPR971863_SGM, CRISPR646032_SGM and CRISPR646028_SGM, Thermo-Fisher Scientific) and the recombinant Streptococcus pyogenes Cas9 (wt) protein (Thermo-Fisher Scientific) using Lipofectamine (Thermo-Fisher Scientific), according to manufacturer’s recommendations (TrueGuide Synthetic sgRNA).

Techniques: Blocking Assay, Control, Labeling, Recombinant, Software, FCAP Assay, Isolation, Cell Isolation, Modification

Journal: EMBO Molecular Medicine

Article Title: BMP-9 mediates fibroproliferation in fibrodysplasia ossificans progressiva through TGF-β signaling

doi: 10.1038/s44321-024-00174-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Customized RNA was synthesized by Invitrogen (TrueGuide sgRNA; A35534 ).

Techniques: Transgenic Assay, Recombinant, Cell Counting, Cell Cycle Assay, Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Western Blot, Reverse Transcription, Control, Software, Real-time Polymerase Chain Reaction

Journal: Communications Biology

Article Title: Deep genomic characterization highlights complexities and prognostic markers of pediatric acute myeloid leukemia

doi: 10.1038/s42003-023-04732-2

Figure Lengend Snippet: a Bubble plots showing significant biological processes positively (red) and negatively (blue) associated with TP53 alterations by GSEA. The color of the bubbles indicates the −log10 (FWER-adjusted P value). NES normalized enrichment score. b TP53 -altered AML cell lines are more BUB1B -dependent, aneuploid, and etoposide-resistant. Gene effect describes how vital a particular gene is when the gene is knocked down in a cell line. A more negative score implies that a cell line is more dependent on that gene. Boxes represent the interquartile ranges and the black line inside the boxes indicates the median. The whiskers show the maximum and minimum except for outliers (circles, more than 1.5 × interquartile range outside of the box) and extremes (asterisk, more than 3 × interquartile range distant). Data were obtained from the DepMap. P values were calculated by the Mann–Whitney U -test. c Confirmation of BUB1B and CIT knockdown by quantitative RT-PCR and immunoblotting after 72 h of siRNA (si) transfection. mRNA levels were normalized to GAPDH . Consistent immunoblotting results were obtained from two experiments. d Effects of BUB1B and CIT knockdown on THP-1 and MOLM-13 proliferation. After 72 h of siRNA transfection, cell proliferation was monitored by CellTiter-Glo assays (RLU) and trypan blue cell counting (Relative cell number). Proliferation was relative to the 72-h post-transfection time point. RLU relative luminescence. e BUB1B knockdown induced apoptosis. Representative flow cytometric analysis of propidium iodide-stained cells after 5 days of BUB1B knockdown. Consistent results were obtained from 4 independent experiments (sub-G1: 40.2, 33.4, 37.1, and 41.1% in THP-1; 4.2, 3.1, 7.0, and 8.6% in MOLM-13. t = 14.98, df = 6, P = 5.6 ×10 −6 by t -test). f BUB1B knockdown induced a pro-apoptotic gene expression signature. Quantitative RT-PCR analysis was performed after 5 days of siRNA transfection. Expression levels were relative to the negative siRNA group. g CRISPR/Cas9 disruption of TP53 in MOLM-13 cells. Two clones showing heterozygous disruptions of TP53 by fragment analysis and Sanger sequencing. h Quantitative RT-PCR indicates comparable BUB1B knockdown in the negative control and CRISPR clones. i Proliferation of the negative control and CRISPR clones after BUB1B knockdown was assessed by CellTiter-Glo assays. Proliferation was relative to the negative siRNA transfection. Data in charts ( c , d , f , h , i ) are expressed as mean ± SE from three independent experiments. The number of values used to calculate the statistics (one-way ANOVA followed by Dunnett’s test in d , f , h , i ) in each group is indicated. For the post hoc Dunnett’s test, the control category was the negative si group ( d , f ) and the negative control CRISPR clone ( i ).

Article Snippet: The TrueGuide Synthetic sgRNA targeting TP53 (CRISPR718498_SGM) (Thermo Fisher Scientific) and TrueCut Cas9 Protein v2 (Thermo Fisher Scientific) were used to generate a ribonucleoprotein complex according to the manufacturer’s instructions.

Techniques: MANN-WHITNEY, Quantitative RT-PCR, Western Blot, Transfection, Cell Counting, Staining, Expressing, CRISPR, Clone Assay, Sequencing, Negative Control