Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Schematic representation of the yeast two-hybrid screen performed using Gal4-DBD-Sam68 as bait and a Gal4-AD fusion cDNA library from LNCaP cells, b, Table reporting the Sam68-interacting factors identified by the screen, c, Five clones of the AH109 yeast strain transformed with the plasmid expressing Gal4-AD-XRN2 (1,929–2,842 nt) (clone 177) and Gal4-DBD-Sam68 fusion proteins, or both plasmids co-transformed with empty vectors as controls. Clones were plated in non-stringency (SD without Leu and Trp) and high-stringency (SD without Leu, Trp, His and Ade) medium and grown at 28 °C for four days, d, Scheme of the XRN2 structure with the position of the Sam68-interacting region (red box), e. Representative western-blot analysis of the reciprocal co-immunoprecipitation (co-IP) between endogenous Sam68 and XRN2 from LNCaP nuclear extracts using Sam68 (α-Sam68) or XRN2 (α-XRN2) antibodies (n = 3). Input = 0.25%. f, Representative western-blot analysis of the co-IP of endogenous Sam68 with XRN2, performed using LNCaP nuclear extracts (NE) in the presence (+) or absence (−) of RNaseA (n = 3). A representative agarose gel of RNA degradation is also shown (RNA). In e and f, non-immune rabbit immunoglobulins G (α-IgG) were used as a negative control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Two Hybrid Screening, cDNA Library Assay, Clone Assay, Transformation Assay, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Agarose Gel Electrophoresis, Negative Control

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polvadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Pearson’s correlation analyses of XRN2 and MYC expression in the PC Jenkins dataset (GSE46691). Pearson’s correlation coefficient (r; two-sided) and P value are reported (95% confidence interval), b, Dot plot showing the distribution of XRN2 expression in patients with PC (Jenkins dataset, GSE46691), classified into Sam68low (blue circles) and Sam68high (red squares) expression groups according to Z-score normalization. The median is shown as a solid horizontal line, c, Representative images of immunohistochemistry analyses of patients with PC (n = 20) with low and high expression of XRN2 and Sam68. Spearman’s correlation is reported (ρ = 0.653; P = 0.002). d, Violin plot showing the correlation between Sam68 and XRN2 expression with Gleason score, in patients with PC (Jenkins dataset, GSE46691). In b and d, statistical significance was calculated by the Mann-Whitney test (two-sided), and P values are reported (95% confidence interval).

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Bar graph showing the percentage of 3’UTR- and CDS-APA events annotated in the genes expressed in LNCaP cells (white columns) and the percentage of those that are differentially regulated in Sam68- and XRN2-depleted cells (gray columns). Statistical significance wascalculated by modified Fisher’s exact test (two-sided, 95% confidence interval), and the exact P values are reported. b,c, Representative western-blot (b) and densitometric analyses (c) of subcellular fractionation experiments (n = 3) performed in control (sh-scr), Sam68 (sh-Sam68) and XRN2 (sh-XRN2) stably depleted LNCaP cells. CE, total cell extract; Cyt, cytoplasmic fraction; Nuc, nucleoplasmic fraction; Chr, chromatin fraction. d,e, Western blot (d) and bar graphs showing qPCR analysis (e) of pA usage of the SCARB2 gene evaluated in cells knocked down for XRN2 targeting 3’UTR (sh-XRN2-3’UIR) and transfected with empty vector (EV), wild-type (WT) and catalytically inactive (D235A) XRN2 (n = 3). LNCaP cells stably depleted with a shRNA targeting CDS (sh-XRN2) were used as control. Fold change of distal (d-pA) relative to the proximal pA (p-pA) in the 3’UTR was calculated by the ACq method. The representative western blot (d) shows the expression of endogenous (XRN2) and recombinant (FLAG) proteins; β-actin was used as loading control. f,g, CLIP assays performed in LNCaP cells stably depleted for XRN2 (sh-XRN2) (n = 3) (f) or transfected as in d (n = 3) (g) using the Sam68 antibody or control IgCs. The RNA associated with Sam68 was quantified by qPCR using primers located upstream of regulated and non-regulated pAs and is represented as percentage (%) of input. Inc and e-g, statistical significance was calculated by unpaired Student’s t-test (two-sided). In c, sh-XRN2/Cyt P = 0.324, sh-XRN2/Nuc P = 0.058, sh-XRN2/Chr P = 0.035, sh-Sam68/Cyt P = 0.8119, sh-Sam68/Nuc P = 0.7612, sh-Sam68/Chr p = 0.6481. In e, sh-XRN2/EV p = 3.4 ×10−3, sh-XRN2-UTR/EVP = 2.1 × 10−3, sh-XRN2-UTR/XRN2WT P = 0.4198, sh-XRN2-UTR/XRN2D235A P = 0.2456. In f, Sam68(sh-scr-downreg/sh-scr-upreg) p = 4.34 ×10−5, Sam68downreg(sh-scr/sh-XRN2) P = 1.7 × 10−3, Sam68upreg(sh-scr/sh-XRN2) P = 3 × 10−4. In g, downregulated: Sam68(sh-scr+EV/sh-XRN2-3’UTR + EV) P = 2 × 10−3, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2WT) P = 0.0215, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2D235A) P = 0.1502, Sam68(sh-XRN2-3’UTR + XRN2WT/sh-XRN2-3’UTR + EV) P = 0.0252, Sam68(sh-XRN2-3’UTR + XRN2D235A/sh-XRN2-3’UTR + EV) P = 0.0157; upregulated: Sam68(sh-scr + EV/sh-XRN2-3’UTR + EV) P = 7.3 × 10−5, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2WT) P = 0.036, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2D235A) P = 0.031, Sam68(sh-XRN2-3’UTR + XRN2WT/sh-XRN2-3’UTR + E V) p = 3.3 × 10−3, Sam68(sh-XRN2-3’UTR + XRN2D235A/sh-XRN2-3’UTR + EV) P = 0.0141. In c and e-g, the bars represent mean + s.d. of three biological replicates; statistical value is reported as *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Modification, Western Blot, Fractionation, Control, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Expressing, Recombinant

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Pearson’s correlation analysis of XRN2 and MYC expression in the jenkins dataset (GSE46691). Pearson’s correlation coefficient (r; two-sided) and P values are reported (95% confidence interval), b, Distribution of XRN2 expression in patients with PC classified as MYCflow (blue circles) and MYChigh (red squares) groups according to Z-score normalization of expression data retrieved from the jenkins dataset (GSE46691). Statistical significance was calculated by Mann-Whitney test (two-sided), and the P value is reported, c, Representative semiquantitative (sq) PCR analysis of ChIP experiments (n = 3) performed in LNCaP cells using MYC antibody and IgG, or no antibody (−), as negative controls. MYC binding was evaluated on the XRN2 promoter. Binding to the sam68 promoter and 16q22 intergenic region were used as positive and negative control, respectively. A schematic representation of the indicated promoters and 16q22 intergenic region is also shown. MYC binding sites (boxes), and positions of primers used for PCR analyses (arrows) are reported. d,e, qPCR (d) and western-blot (e) analyses of MYC, XRN2 and Sam68 expression in LNCaP and 22Rv1 cells lines transfected with control (si-scr#l) and MYC (si-MYC#1) siRNAs (n = 3). Expression was reported as fold change (ΔΔCq) with respect to control. Data represent mean + s.d. of three biological replicates, and statistical significance was calculated by unpaired Student’s t-test (two-sided) (MYC/LNCaP P = 3.8 × 10−5, MYC/22Rv1 P = 5.1 × 10−6; XRN2/LNCaP P = 3.7 × 10−3, XRN2/22Rv1 P = 1.4 × 10−3; Sam68/LNCaP P = 8.4 × 10−5, Sam68/22Rv1P = 7.7 × 10−5). In d, statistical value is reported as **P < 0.01, ***P < 0.001. In e, β-actin was used as loading control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, MANN-WHITNEY, Binding Assay, Negative Control, Western Blot, Transfection, Control

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Meta-transcriptome profiles of Sam68 binding across mRNA transcripts retrieved from two replicates of CLIP-seq experiments (GSE85164). TSS, transcription start site; TES, transcription end site; RPM, reads per million, b, Representative western-blot analysesofthe co-IP ofSam68 and XRN2 with componentsoftheC/P complex from LNCaP nuclear extracts using Sam68 (α-Sam68) and XRN2 (α-XRN2) antibodies, or rabbit immunoglobulins G (α-IgG) as negative control (n = 2). c, Bar graphs representing the percentage of genes (left) and polyadenylation sites (pAs; right graph) undergoing APA regulation in Sam68 (si-Sam68)- and XRN2 (si-XRN2)-depleted LNCaP cells, d, Venn diagram showing the overlap between regulated APA events identified in Sam68- or XRN2-depleted cells. Statistical significance was calculated by hypergeometric test and the P value is shown. e, Venn diagram showing the number of unique and common up- (purple) and downregulated (orange) APA events identified in Sam68- and XRN2-depleted cells. f,g, Bar graphs showing qPCR analysis of pA usage evaluated in two representative genes undergoing 3’UTR-APA (f) and CDS-APA (g) regulation in cells knocked down for Sam68 (si-Sam68), XRN2 (si-XRN2) or both proteins. Fold change of distal (d-pA) (f) or intronic (g) pA relative to the proximal pA (p-pA) in the 3’UTR was calculated by the ΔCq method. Data represent mean + s.d. of three biological replicates. Statistical significance was calculated by unpaired Student’s t-test (two-sided). In f, SCARB2: si-Sam68/si-scr P = 1.5 × 10−3, si-XRN2/si-scr P = 2.0 × 10−3, si-Sam68si-XRN2/si-scr P = 0.017; FLNB: si-Sam68/si-scr P = 0.015, si-XRN2/si-scr P = 2.1 × 10−3, si-Sam68si-XRN2/si-scr P = 3 × 10−4. In g, RNF130: si-Sam68/si-scr P = 0.013, si-XRN2/si-scr P = 5.5 × 10−3, si-Sam68si-XRN2/si-scr P = 5.4 × 10−3; CEP70: si-Sam68/si-scr P = 4.3 × 10−3, si-XRN2/si-scr P = 0.0112, si-Sam68si-XRN2/si-scr P = 0.0147. In f and g, statistical values are reported as *P < 0.05; **P < 0.01; ***P < 0.001. UCSC genome browser tracks showing APA regulation of the events analyzed are also shown on the left side of each graph. Purple and orange boxes in the schemes indicate up- and downregulated events, respectively. Schematic representations of these CDS- and 3’UTR-APA events are shown in the upper panels.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Negative Control

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: Genome-wide regulation of APA by XRN2 and Sam68 in PC cells (Related to Fig. 4).

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Genome Wide

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Percentage and number of up- (purple) and downregulated (orange) 3’UTR-APA events regulated by Sam68 and XRN2 (pA position is shown as F, proximal-most; M, intermediate; L, distal-most), b, Changes of 3’UTR pA isoform abundance (ΔAbn) at both p-pA and d-pA sites in si-Sam68 and si-XRN2 cells. Mean values and number of pA events (n) are reported, c, Percentage of up- and downregulated canonical and non-canonical PAS sequences in 3’UTR-APA events regulated by Sam68 and XRN2. d, AAUAAA frequency profile in up- (purple), down- (orange) and unregulated (black) 3’UTR pAs evaluated between −100 and +100 nt from the CS (shading represents 95% confidence interval). Statistical significance (unpaired Student’s t-test, two-sided) was calculated between −15 and −25 nt (boxplot). e, A- and G-base frequency distribution in up- (purple), down-grange) and unregulated (black) pAs between −100 and +100 nt from the CS (0). f, Scheme of cis-elements and CS position. Hexamers enriched between −100 and +100 nt from the CS in up- and downregulated pAs with respect to unregulated pAs. Motif (H), number (N) and significance score (P) of hexamers are indicated. Significance score was calculated by –log10(P)xS, where P is based on the Fisher’s exact test and the S value was 1 or −1 for enrichment and depletion, respectively, g, APA isoform abundance (Abn) of si-Sam68/si-XRN2 up- (mean = 28.6) and downregulated (mean = 47.2) isoforms. Values refer to expression in control cells, h, Scheme of the FLNB minigene comprising the genomic region from the second-last exon to 200 nt downstream of the d-pA (source data). i,j, Semiquantitative (micrographs) and quantitative (bar graphs) analyses of pA usage in LNCaP transfected with the FLNB minigene and indicated plasmids (n = 3). Protein expression was evaluated by western blot, k, CLIP assays performed in sh-Sam68 and sh-XRN2 cells using CPSF30 antibody or IgGs (n = 3). Statistical significance was calculated by unpaired Student’s t-test, two-sided (b, g, i-k) and with Fisher’s exact test, two-sided (a, c). (l-k) Bar graphs represent mean + s.d. When not indicated, P values are reported as *P < 0.05, ***P < 0.001, ****P < 0.0001 (exact P values are reported in the source data). In the boxplots (b, d, g), the center line and box indicate the median and the 25th and 75th percentiles, respectively. Whiskers indicate ±1.5x interquartile range.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, Control, Transfection, Western Blot

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Enrichment of Gene Ontology (GO) terms (dot plot) in genes regulated by 3’UTR-APA upon depletion of Sam68 or XRN2. Dot size and color indicate the number of genes and statistical significance (Fisher’s exact test, two-sided), respectively, b, Cytometric analyses showing DNA content versus BrdU incorporation upon stable depletion of Sam68 (sh-Sam68) and XRN2 (sh-XRN2) in LNCaP cells. The bar graph shows the percentage of BrdU-positive (S phase) cells, c. Percentage (mean + s.d.) of BrdU-positive LNCaP cells described in b at the indicated time points after release from G1/S synchronization. d,e, Western blot (d) and qPCR (e) analyses of MCM10 and ORC2 expression level in sh-Sam68 and sh-XRN2 LNCaP cells (n = 3). f, PCR strategy used to evaluate 3’UTR-APA isoforms distribution on a 15–50% sucrose gradient, g, sqPCR analysis of the indicated p-pA and d-pA isoform abundance within the polysomal and non-polysomal fractions obtained from sucrose gradient. The graphs show the densitometric analysis of the band signal in each fraction, expressed as a percentage of that detected in all fractions, h, Relative luciferase activity (Renilla/Firefly ratio) of long and short MCM10 3’UTR in LNCaP cells. i, Representative western-blot analysis (n = 3) of the indicated proteins performed in LNCaP cells depleted for the indicated genes, j, Cytometric analyses showing DNA content versus BrdU incorporation in control (si-scr), si-MCMlO and si-ORC2 LNCaP cells. The bar graph shows the percentage of S-phase BrdU-positive cells, k, Kaplan-Meier curves comparing progression-free survival of494 patients with PC (Prostate Adenocarcinoma, TCGA, PanCancer Atlas; https://www.cbioportal.org) stratified according to MCM10 (right), ORC2 (middle) and MCM10/ORC2 (left) expression level. I, Schematic model showing the impact of the functional interaction between Sam68 and XRN2 on cell cycle regulation. The Sam68/XRN2 complex promotes 3’UTR shortening of cell cycle-related genes, increasing their mRNA translation efficiency and cell proliferation. Conversely, Sam68/XRN2 knockdown induces 3’UTR lengthening, reduces translation efficiency of transcripts and causes cell cycle arrest. In b, e, h and j, the bar graphs represent the mean + s.d. In b, c, e, g, h and j, statistical significance was calculated by unpaired Student’sf-test, two-sided (n = 3; *P < 0.05, **P < 0.01,***P < 0.001; NS, not significant; exactPvalues are reported in the source data). In d and I, β-actin was used as loading control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: BrdU Incorporation Assay, Western Blot, Expressing, Luciferase, Activity Assay, Control, Functional Assay, Knockdown

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polvadenvlation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP28 stabilizes the expression of RecQ family helicases and maintains the viability of triple negative breast cancer cells

doi: 10.1016/j.jbc.2021.101443

Figure Lengend Snippet: USP28 regulates RecQ helicases. A , immunoblot analysis of RECQL5 in MDA-MB-231, T47D, and 293T cells depleted of USP28. B , immunoblot analysis of RecQ family helicases in MDA-MB-231 cells depleted of USP28. C , analysis of RECQL5 stability in a cycloheximide chase experiment. 293T cells were transfected with Flag-RECQL5 first and then depleted of USP28 expression, treated with 100 μg/ml cycloheximide (CHX), and harvested at different time points for analysis. D , quantification of Flag-RECQL5 levels normalized to ACTIN in ( C ). E , overexpression of USP28 stabilizes RECQL5. 293T cells with USP28 or USP28 C171A overexpression were subjected to the same CHX chase experiment as in ( C ) except the endogenous RECQL5 was examined. F , quantification of RECQL5 levels normalized to Tubulin in ( E ). BLM, bloom syndrome gene product; USP, Ubiquitin-specific cysteine protease; WRN, werner syndrome gene product.

Article Snippet: The antibodies used in this study were as follows: USP28 (17707-1-AP, 1:1000 WB, Proteintech); RECQL1 (A300-450A, 1:1000 WB, Bethyl Lab, Montgomery); WRN (4666S,1:1000 WB,Cell Signaling); BLM (A300-110A,1:1000 WB, Bethyl Lab); RECQL4 (17008-1-AP,1:1000 WB, Proteintech); RECQL5 (A302-520A, 1:2000 WB, Bethyl Lab); Flag (F3165, 1:2000, Sigma); Ub (SC-8017, 1:200 WB, Santa Cruz Biotechnology); γH2AX (05-636, 1:500 IF, Millipore); Claspin (2800S, 1:1000 WB, Cell Signaling); c-MYC (SC-40, 1:200 WB, Santa Cruz); Phospho-Chk1-Ser345 (2348, 1:1000 WB, Cell Signaling); CHK1 (ab32531, 1:1000 WB, Abcam); GAPDH (60004-1-1g, 1:5000 WB, Proteintech); Tubulin (66240-1-1g, 1:5000 WB, Proteintech); ACTIN (66009-1-1g, 1:3000 WB, Proteintech).

Techniques: Western Blot, Transfection, Expressing, Over Expression, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP28 stabilizes the expression of RecQ family helicases and maintains the viability of triple negative breast cancer cells

doi: 10.1016/j.jbc.2021.101443

Figure Lengend Snippet: USP28 inhibition induces accumulation of DNA damage and activation of DNA damage checkpoint in TNBC cells. A , immunofluorescence staining and quantification of DNA damage marker γH2AX foci in USP28 -depleted MDA-MB-231 cells. Student’s t test: ∗∗∗, p < 0.001. The scale bar represents 10 μm. B , immunoblot analysis of CHK1 in MDA-MB-231 cells depleted of USP2 8. C , immunoblot analysis of CLASPIN in MDA-MB-231 cells depleted of USP28 . D , immunoblot analysis of relative proteins in HCC1806 cells treated with USP28 inhibitor AZ1 for 48 h. BLM, bloom syndrome gene product; TNBC, triple-negative breast cancer; USP, Ubiquitin-specific cysteine protease; WRN, werner syndrome gene product

Article Snippet: The antibodies used in this study were as follows: USP28 (17707-1-AP, 1:1000 WB, Proteintech); RECQL1 (A300-450A, 1:1000 WB, Bethyl Lab, Montgomery); WRN (4666S,1:1000 WB,Cell Signaling); BLM (A300-110A,1:1000 WB, Bethyl Lab); RECQL4 (17008-1-AP,1:1000 WB, Proteintech); RECQL5 (A302-520A, 1:2000 WB, Bethyl Lab); Flag (F3165, 1:2000, Sigma); Ub (SC-8017, 1:200 WB, Santa Cruz Biotechnology); γH2AX (05-636, 1:500 IF, Millipore); Claspin (2800S, 1:1000 WB, Cell Signaling); c-MYC (SC-40, 1:200 WB, Santa Cruz); Phospho-Chk1-Ser345 (2348, 1:1000 WB, Cell Signaling); CHK1 (ab32531, 1:1000 WB, Abcam); GAPDH (60004-1-1g, 1:5000 WB, Proteintech); Tubulin (66240-1-1g, 1:5000 WB, Proteintech); ACTIN (66009-1-1g, 1:3000 WB, Proteintech).

Techniques: Inhibition, Activation Assay, Immunofluorescence, Staining, Marker, Western Blot, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The deubiquitinase USP28 stabilizes the expression of RecQ family helicases and maintains the viability of triple negative breast cancer cells

doi: 10.1016/j.jbc.2021.101443

Figure Lengend Snippet: RECQ family helicases are required for the proliferation of TNBC cells. A , the growth curve analysis of MDA-MB-231 cells depleted of RECQ helicases. Each helicase was knocked down via two independent shRNAs. B , a schematic model illustrating the function of USP28 in helping replication stress relief in TNBC cells through RecQ family helicases, thereby maintaining the viability of TNBC cells. BLM, bloom syndrome gene product; TNBC, triple-negative breast cancer; USP, Ubiquitin-specific cysteine protease; WRN, werner syndrome gene product.

Article Snippet: The antibodies used in this study were as follows: USP28 (17707-1-AP, 1:1000 WB, Proteintech); RECQL1 (A300-450A, 1:1000 WB, Bethyl Lab, Montgomery); WRN (4666S,1:1000 WB,Cell Signaling); BLM (A300-110A,1:1000 WB, Bethyl Lab); RECQL4 (17008-1-AP,1:1000 WB, Proteintech); RECQL5 (A302-520A, 1:2000 WB, Bethyl Lab); Flag (F3165, 1:2000, Sigma); Ub (SC-8017, 1:200 WB, Santa Cruz Biotechnology); γH2AX (05-636, 1:500 IF, Millipore); Claspin (2800S, 1:1000 WB, Cell Signaling); c-MYC (SC-40, 1:200 WB, Santa Cruz); Phospho-Chk1-Ser345 (2348, 1:1000 WB, Cell Signaling); CHK1 (ab32531, 1:1000 WB, Abcam); GAPDH (60004-1-1g, 1:5000 WB, Proteintech); Tubulin (66240-1-1g, 1:5000 WB, Proteintech); ACTIN (66009-1-1g, 1:3000 WB, Proteintech).

Techniques: Ubiquitin Proteomics

Journal: Nucleic Acids Research

Article Title: Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1

doi: 10.1093/nar/gkac1240

Figure Lengend Snippet: MLH1, MSH2 and MSH6 function in the same pathway, but function independently of EXO1, for suppression of RMDs. ( A ) Shown is the effect of siRNAs targeting MLH1 (siMlh1) on three RMD events (9.1 kb DSB/repeat distance, RMD-GFP, 1% GFP-GFP, 3% RMD-GFP) in WT, Mlh1 − / − , Msh2 − / − and Exo1 − / − mESCs. Frequencies are normalized to transfection efficiency and parallel siCTRL (= 1). n = 6. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005, **** P < 0.0001, unpaired t -test for siCTRL versus siMlh1, and unpaired t -test using Holm−Sidak correction for effect of siMlh1 in WT versus the other genetic backgrounds. ( B ) Shown is the analysis as in (A), but using the 16 bp DSB/repeat distance. n = 6. Statistics as in (A). ( C ) Shown is the analysis as in (A), but for effects of siRNAs targeting MSH6 (siMsh6). n = 6. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005, **** P < 0.0001, unpaired t -test for siCTRL versus siMsh6, and unpaired t -test using Holm−Sidak correction for effect of siMsh6 in WT versus the other genetic backgrounds. ( D ) Shown is the analysis in (C), but using the 16 bp DSB/repeat distance n = 6. Statistics as in (C). Data are represented as mean values ± SD.

Article Snippet: Blots were probed with antibodies for CtIP (Active Motif 61141), BLM (Bethyl Laboratories A300-110A), MLH1 (Abcam, ab92312), MSH6 (Proteintech, 18120–1AP), MSH2 (Bethyl Laboratories, A300-452), EXO1 (Bethyl Laboratories, A302-640A), TOP3α (Proteintech, 14525I-AP), FLAG (Sigma, A8592) and ACTIN (Sigma, A2066).

Techniques: Transfection

Journal: PLoS ONE

Article Title: The Discovery of a Reciprocal Relationship between Tyrosine-Kinase Signaling and Cullin Neddylation

doi: 10.1371/journal.pone.0075200

Figure Lengend Snippet: (A) MA5.8ζ IL-2 secretion at 12 h normalized to the number of live cells measured by annexin V and propidium iodide staining and (B) transcription at 12 h with 4 µg/mL α-CD3 and the indicated concentrations of OPT (n = 3). (C–G) IL-2 secretion by MA5.8ζ cells, cells expressing control shRNA, and cells expressing an shRNA for (C) CUL1, (D) CUL2, (E) CUL3, (F) CUL4a/b and (G) CUL5 after 48 h of stimulation with indicated concentrations of α-CD3. Data are averaged from two independent experiments, where each experiment examined three independently created lines for each shRNA. (H) IL-2 secretion at 48 h by MA5.8ζ cells and cells expressing either control shRNA, CUL2 shRNA or CUL3 shRNA with 1 µg/mL α-CD3 and 200 nM MLN4924. Data are averaged as in (C–G). Values represent mean ± s.e.m.; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Rabbit polyclonal antibodies against CUL3 (A301-109A) and CUL5 (A302-173A) were purchased from Bethyl Laboratories, Inc.

Techniques: Staining, Expressing, Control, shRNA

Journal: Nucleic Acids Research

Article Title: Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1

doi: 10.1093/nar/gkac1240

Figure Lengend Snippet: Components of mismatch repair and the BLM-TOP3α-RMI1/2 (BTR) complex suppress RMDs, whereas several other factors promote these events. ( A ) Shown are effects of siRNAs targeting 55 factors on frequency of RMDs at both 16 bp and 9.1 kb DSB/repeat distances for 1%RMD-GFP. Frequencies are normalized to transfection efficiency and parallel siCTRL (= 1). Genes are ranked by the fold-effect relative to siCTRL at 9.1 kb. All siRNAs tested n = 2, and those with ≥1.5-fold effect from these trials were tested a total of n = 4. Grey: n = 2, black: n = 4, red: n = 4 and also ≥1.5-fold effect relative to siCTRL. ( B ) Shown are the effects of the siRNAs targeting 55 factors on frequency of RMDs at both 16 bp and 9.1 kb DSB/repeat distances for RMD-GFP. Colors and analysis as in (A). ( C ) Ratio of divergent versus identical RMD frequencies. For the data shown in (A) and (B), shown is the ratio of RMD frequencies from 1% RMD-GFP divided by RMD-GFP. Grey: n = 2, black: n = 4. As above, genes are ranked by the fold-effect relative to siCTRL at 9.1 kb. Data are represented as mean values ± SD.

Article Snippet: Blots were probed with antibodies for CtIP (Active Motif 61141), BLM (Bethyl Laboratories A300-110A), MLH1 (Abcam, ab92312), MSH6 (Proteintech, 18120–1AP), MSH2 (Bethyl Laboratories, A300-452), EXO1 (Bethyl Laboratories, A302-640A), TOP3α (Proteintech, 14525I-AP), FLAG (Sigma, A8592) and ACTIN (Sigma, A2066).

Techniques: Transfection

Journal: Nucleic Acids Research

Article Title: Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1

doi: 10.1093/nar/gkac1240

Figure Lengend Snippet: TOP3α suppresses RMDs in a manner that is distinct from MLH1. ( A ) Shown are the frequencies of the RMD events depicted in the diagram in Figure (i.e. six different DSB/repeat distances with RMD-GFP, 1% RMD-GFP and 3% RMD-GFP) for WT mESCs transfected with siCTRL and EV, siTop3a and EV, and siTop3α and TOP3α expression vector. n = 6. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005, **** P < 0.0001, unpaired t -test with Holm−Sidak correction. ( B ) Immunoblotting analysis of TOP3α and ACTIN in WT mESCs transfected with either siCTRL EV, siTop3α EV or siTop3a with TOP3α expression vector. Endogenous mouse TOP3α was not detected, likely due to the immunogen being human TOP3α. Also shown is immunoblotting analysis of BLM and ACTIN in WT mESCs transfected with either siCTRL, siBlm or siTop3α. ( C ) Shown is the frequency of top strand base retention performed as in Figure , at the 16 bp DSB/repeat distance for WT (siCTRL) and WT siTop3α. WT (siCTRL) values are the same as in Figure . n = 3. * P ≤ 0.05, unpaired t -test. ( D ) Shown is the effect of siRNAs targeting TOP3α (siTop3α) on three RMD events (9.1 kb DSB/repeat distance, RMD-GFP, 1% GFP-GFP, 3% RMD-GFP) in WT, Mlh1 − / − and Msh2 − / − , mESCs. Frequencies are normalized to transfection efficiency and parallel siCTRL (= 1). n = 6. ** P ≤ 0.005, *** P ≤ 0.0005, **** P < 0.0001, unpaired t -test for siCTRL versus siTop3α, and unpaired t -test using Holm−Sidak correction for effect of siTop3α in WT versus the other genetic backgrounds. ( E ) Shown is the analysis as in (D), but using the 16 bp DSB/repeat distance. n = 6. Statistics as in (D), except with ** P ≤ 0.005. Data are represented as mean values ± SD. The † symbol notes that endogenous mouse Top3α is not readily detected by this antibody raised against the human protein.

Article Snippet: Blots were probed with antibodies for CtIP (Active Motif 61141), BLM (Bethyl Laboratories A300-110A), MLH1 (Abcam, ab92312), MSH6 (Proteintech, 18120–1AP), MSH2 (Bethyl Laboratories, A300-452), EXO1 (Bethyl Laboratories, A302-640A), TOP3α (Proteintech, 14525I-AP), FLAG (Sigma, A8592) and ACTIN (Sigma, A2066).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Nucleic Acids Research

Article Title: Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1

doi: 10.1093/nar/gkac1240

Figure Lengend Snippet: Assay system for examining RMDs that are affected by CtIP and BLM. ( A ) Shown is the RMD-GFP reporter, which is integrated into the Pim1 locus in chromosome 17 of mESCs, such that repair of two DSBs by an RMD leads to GFP + cells. The two repeats shown as ‘R’, the 5’ repeat being endogenous sequence and the 3’ repeat is fused to GFP. 1%RMD-GFP has 1% sequence divergence between the repeats. RMDs are induced by creating two DSBs: one 268 bp downstream of the 5’ repeat, and the other either 16 bp or 9.1 kb upstream of the 3’ repeat, which we refer to as the DSB / repeat distance. ( B ) Shown are the effects of siRNAs targeting BLM (siBlm) and CtIP (siCtIP) for 4 RMD reporter assays, and an NHEJ assay (EJ7-GFP, No Indel EJ). Repair frequencies are normalized to transfection efficiency, and parallel non-targeting siRNA (siCTRL = 1). n = 4. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005, **** P < 0.0001, Statistics are one-way ANOVA using Tukey's multiple comparisons test.

Article Snippet: Blots were probed with antibodies for CtIP (Active Motif 61141), BLM (Bethyl Laboratories A300-110A), MLH1 (Abcam, ab92312), MSH6 (Proteintech, 18120–1AP), MSH2 (Bethyl Laboratories, A300-452), EXO1 (Bethyl Laboratories, A302-640A), TOP3α (Proteintech, 14525I-AP), FLAG (Sigma, A8592) and ACTIN (Sigma, A2066).

Techniques: Sequencing, NHEJ Assay, Transfection