human sam68  (Bethyl)

Journal: Communications Biology

Article Title: An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

doi: 10.1038/s42003-024-06129-1

Figure Lengend Snippet: a Immunoblot analysis of Sam68 and FADD after CRISPR-mediated knockout (KO) in the MCF-10A background. TWT = Targeted Wild-Type. b Quantification of DNA release from MCF-10A KO cell lines in culture. Individual cell lines shown, with data representing mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 biologically independent samples. Electropherograms were individually run at least n = 3 times and representative traces were selected. c Quantification of DNA release from Sam68 KO MCF-10A cell lines rescued by Sam68-GFP overexpression. Parental and TWT were grouped and labeled control, and two Sam68 KO3 and KO4 were grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 4 for all lines except n = 3 for Sam68 TWT before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. d Quantification of DNA release from FADD KO MCF-10A cell lines rescued by FADD-GFP overexpression. Parental and TWT were grouped and labeled control, and two FADD KO cell lines were grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 for all GFP-expressing lines and n = 4 for each FADD-GFP expressing line before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. e Cell growth assay of KO MCF-10A cell lines. Parental and TWT were grouped and labeled control. All four Sam68 KO cell lines and both FADD KO cell lines are respectively grouped. Data represent mean cell concentration ± SEM; n = 3 for each cell line before combining. f Annexin V and Propidium Iodide (PI) assay of KO MCF-10A cell lines. Parental and TWT were grouped and labeled control. All four Sam68 KO cell lines and both FADD KO cell were grouped, respectively. Data represent mean signal (RFU for PI; RLU for Annexin) ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 6 biologically independent samples for each cell line before combining. g Cytotoxic assay of MCF-10A KO panel treated with 1 ng/mL TRAIL ligand. Parental and TWT were grouped and labeled control. All four Sam68 mutant cell lines and both FADD mutant cell lines were grouped, respectively. Data represent mean percent survival ± SEM as normalized to vehicle of each cell line condition; n = 3 biologically independent samples for each cell line before combining. h Quantification of DNA release from MCF-10A KO panel treated with 1 ng/mL TRAIL ligand. Parental and TWT were grouped and labeled control. All four Sam68 mutant cell lines and both FADD mutant cell lines were respectively grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 biologically independent samples for each cell line before combining. i Quantification of DNA release from MCF-10A KO cell lines treated with 20 μg/mL ZVAD-FM-K. Parental MCF-10As, Sam68 KO3/4 mutant cell lines, and both FADD mutant cell lines were respectively grouped. Data represent mean fold change ± SEM in DNA release internally normalized to cell concentration for each cell line and then normalized to control vehicle; n = 3 biologically independent samples for all untreated lines and FADD KO1, n = 4 for treated lines for each cell line before combining. All statistics were ANOVA with Dunnett’s multiple comparison test at endpoint.

Article Snippet: Lentiviral expression vectors with CMV promoters driving GFP-tagged human Sam68 and FADD were purchased from Origene (PS100093, RC200263L4, RC201805L4).

Techniques: Western Blot, CRISPR, Knock-Out, Concentration Assay, Control, Over Expression, Labeling, Expressing, Growth Assay, Mutagenesis, Comparison

Journal: Communications Biology

Article Title: An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

doi: 10.1038/s42003-024-06129-1

Figure Lengend Snippet: a Quantification of DNA release from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 or FADD. Data represent mean fold change ± SEM in DNA release normalized to cell concentration for each cell line, then overall to GFP control; n = 4 biologically independent samples. b Fragmentation pattern of MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 and FADD. Electropherograms were individually run at least n = 3 times and representative traces were selected. c Quantification of Annexin V signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 and FADD. Data represent mean fold change ± SEM in RLU signal normalized to cell concentration at collection for each cell line, then overall to GFP control; n = 4 biologically independent samples for MDA-MB-231 and HCT116, n = 6 for MDA-MB-468. d Quantification of DNA release from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL ligand. Data represent mean fold change ± SEM in DNA release normalized to cell concentration for each treatment, then overall to vehicle control; n = 4 biologically independent samples. e Fragmentation pattern of MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL ligand. Electropherograms were individually run at least n = 3 times and representative traces were selected. f Quantification of Annexin V signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL Ligand. Data represent mean fold change ± SEM RLU signal normalized to cell concentration for each treatment, then overall to vehicle control; n = 6 biologically independent samples. g Quantification of Propidium Iodide signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL Ligand. Data represent mean fold change ± SEM RFU signal normalized to cell concentration for each treatment, then overall to vehicle control; n = 6 biologically independent samples. All statistics were ANOVA with Dunnett’s multiple comparison test at endpoint.

Article Snippet: Lentiviral expression vectors with CMV promoters driving GFP-tagged human Sam68 and FADD were purchased from Origene (PS100093, RC200263L4, RC201805L4).

Techniques: Over Expression, Concentration Assay, Control, Comparison

Journal: Communications Biology

Article Title: An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

doi: 10.1038/s42003-024-06129-1

Figure Lengend Snippet: a Immunoblot analysis of Sam68 and FADD after CRISPR-mediated knockout (KO) in the MCF-10A background. TWT = Targeted Wild-Type. b Quantification of DNA release from MCF-10A KO cell lines in culture. Individual cell lines shown, with data representing mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 biologically independent samples. Electropherograms were individually run at least n = 3 times and representative traces were selected. c Quantification of DNA release from Sam68 KO MCF-10A cell lines rescued by Sam68-GFP overexpression. Parental and TWT were grouped and labeled control, and two Sam68 KO3 and KO4 were grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 4 for all lines except n = 3 for Sam68 TWT before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. d Quantification of DNA release from FADD KO MCF-10A cell lines rescued by FADD-GFP overexpression. Parental and TWT were grouped and labeled control, and two FADD KO cell lines were grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 for all GFP-expressing lines and n = 4 for each FADD-GFP expressing line before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. e Cell growth assay of KO MCF-10A cell lines. Parental and TWT were grouped and labeled control. All four Sam68 KO cell lines and both FADD KO cell lines are respectively grouped. Data represent mean cell concentration ± SEM; n = 3 for each cell line before combining. f Annexin V and Propidium Iodide (PI) assay of KO MCF-10A cell lines. Parental and TWT were grouped and labeled control. All four Sam68 KO cell lines and both FADD KO cell were grouped, respectively. Data represent mean signal (RFU for PI; RLU for Annexin) ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 6 biologically independent samples for each cell line before combining. g Cytotoxic assay of MCF-10A KO panel treated with 1 ng/mL TRAIL ligand. Parental and TWT were grouped and labeled control. All four Sam68 mutant cell lines and both FADD mutant cell lines were grouped, respectively. Data represent mean percent survival ± SEM as normalized to vehicle of each cell line condition; n = 3 biologically independent samples for each cell line before combining. h Quantification of DNA release from MCF-10A KO panel treated with 1 ng/mL TRAIL ligand. Parental and TWT were grouped and labeled control. All four Sam68 mutant cell lines and both FADD mutant cell lines were respectively grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 biologically independent samples for each cell line before combining. i Quantification of DNA release from MCF-10A KO cell lines treated with 20 μg/mL ZVAD-FM-K. Parental MCF-10As, Sam68 KO3/4 mutant cell lines, and both FADD mutant cell lines were respectively grouped. Data represent mean fold change ± SEM in DNA release internally normalized to cell concentration for each cell line and then normalized to control vehicle; n = 3 biologically independent samples for all untreated lines and FADD KO1, n = 4 for treated lines for each cell line before combining. All statistics were ANOVA with Dunnett’s multiple comparison test at endpoint.

Article Snippet: Lentiviral expression vectors with CMV promoters driving GFP-tagged human Sam68 and FADD were purchased from Origene (PS100093, RC200263L4, RC201805L4).

Techniques: Western Blot, CRISPR, Knock-Out, Concentration Assay, Control, Over Expression, Labeling, Expressing, Growth Assay, Mutagenesis, Comparison

Journal: Communications Biology

Article Title: An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

doi: 10.1038/s42003-024-06129-1

Figure Lengend Snippet: a Quantification of DNA release from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 or FADD. Data represent mean fold change ± SEM in DNA release normalized to cell concentration for each cell line, then overall to GFP control; n = 4 biologically independent samples. b Fragmentation pattern of MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 and FADD. Electropherograms were individually run at least n = 3 times and representative traces were selected. c Quantification of Annexin V signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 and FADD. Data represent mean fold change ± SEM in RLU signal normalized to cell concentration at collection for each cell line, then overall to GFP control; n = 4 biologically independent samples for MDA-MB-231 and HCT116, n = 6 for MDA-MB-468. d Quantification of DNA release from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL ligand. Data represent mean fold change ± SEM in DNA release normalized to cell concentration for each treatment, then overall to vehicle control; n = 4 biologically independent samples. e Fragmentation pattern of MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL ligand. Electropherograms were individually run at least n = 3 times and representative traces were selected. f Quantification of Annexin V signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL Ligand. Data represent mean fold change ± SEM RLU signal normalized to cell concentration for each treatment, then overall to vehicle control; n = 6 biologically independent samples. g Quantification of Propidium Iodide signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL Ligand. Data represent mean fold change ± SEM RFU signal normalized to cell concentration for each treatment, then overall to vehicle control; n = 6 biologically independent samples. All statistics were ANOVA with Dunnett’s multiple comparison test at endpoint.

Article Snippet: Lentiviral expression vectors with CMV promoters driving GFP-tagged human Sam68 and FADD were purchased from Origene (PS100093, RC200263L4, RC201805L4).

Techniques: Over Expression, Concentration Assay, Control, Comparison

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: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: Reverse/β-turn peptidomimetic compounds are direct interactors of Sam68 (A) Chemical structure of HAT inhibitor C646 and bromodomain ligand I-CBP112, as well as β-turn peptidomimetics ICG-001 and CWP232228. (B) Western blot analysis of CBP-catalyzed H3K14ac and H3K18ac histone acetylation marks in C646 (0.25 μM), I-CBP112 (0.25 μM), and CWP232228 (0.1 μM) t-hESCs versus control DMSO. Total histone H3 and GAPDH were used as loading control. Relative OD signal quantification versus H3 intensity is presented (C646: n = 3, I-CBP112: n = 5, CWP232228: n ≥ 3, ∗: p = 0.0183, ∗∗: p = 0.0078, ∗∗∗: p ≤ 0.00033, two-tailed t test). Data are represented as mean ± SEM (error bars). (C) Dose-response experiment assessing the impact of bromodomain ligand-based (C646 and I-CBP112), and peptidomimetic (CWP232228) inhibition of CBP on t-hESC growth (C646, I-CPB112: n = 4; CWP232228: n = 3). (D) Early endoderm differentiation assay performed in t-hESCs in the presence of CWP232228 (0.1 μM, n = 6), I-CBP112 (0.25 μM, n = 3), or C646 (0.25 μM, n = 3) versus control DMSO (n = 6) and basal culture media (n = 3). Bar graph represents relative counts of FOXA2-positive (early endoderm marker)/OCT4-negative cells in DMSO, CWP232228, I-CBP112, and C646-treated t-hESCs versus basal culture media (one-way ANOVA, ∗∗∗: p < 0.0001). Data are represented as mean ± SEM (error bars). Scale bar: 100 μm. (E) Pro-drug CWP232228 is converted into its active form CWP231904 via hydrolysis of the phosphate group by serum/cellular alkaline phosphatase. (F) Affinity pull-down experiments using CWP231904-conjugated magnetic beads performed on whole hESC lysates. Physical interaction between CBP, Sam68, beta-Catenin, ETS, MYB, GATA2, and PTMA with immobilized CWP231904 was assessed by immunoblotting. Each protein was previously shown as a member of the CBP interactome showing selective enrichment in human primary AML versus healthy blood ( Benoit et al., 2017 ). Excess of soluble compounds (CWP and ICG001, 100 μM) were used to compete with immobilized CWP231904. Whole-cell lysate was used as input, and amine-functionalized beads were used as negative control (n = 2). The heatmap presents mean background-corrected OD signal for each putative interactor tested (gray: not tested).

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Western Blot, Two Tailed Test, Inhibition, Differentiation Assay, Marker, Magnetic Beads, Negative Control

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: In silico screening of peptidomimetics with enhanced binding affinity for Sam68 (A) Representation of Sam68 interacting with SH3 domain in Src kinase family proteins via proline-rich motifs located in N-terminal P1-P2 and between residues 275 and 374 (P3, P4, P5). Small molecule UCS15A is known to disrupt SH3-mediated interaction of Src with Sam68 P3-5 domains ( Sharma et al., 2001 ). (B) 2D representation of UCS15A and the peptidomimetic ICG-001 in silico predicted binding pocket in Sam68 275-374 peptide (red, oxygen; blue, nitrogen). Common residues involved in both small-molecule-binding pockets are highlighted in red. Predicted hydrogen bond length is represented by dashed lines (Å). (C) Schematic representation of the in silico structure-activity relationship analysis pipeline (PyRx) used to identify β-turn peptidomimetic molecules with enhanced binding affinity for Sam68 275-374 domain. “A” and “B” represent the positions of distinct substituents added to reverse-turn mimetic cores. (D) Dose-response curves assessing selective toxicity of peptidomimetics ICG-001, CWP232228, and PRI-724 in HT29 human colorectal cancer cell line versus normal intestinal progenitor cells HIEC (n ≥ 4, 48-h treatments). (E) Compound ranking based on predicted Keq for each β-turn analog (black dots). Only molecules presenting a standard deviation below 0.1 for a minimum of three analysis runs, with an exhaustiveness (“E”) level of “8” were plotted. Dots corresponding to ICG-001, CWP231904, PRI-724-OH, and YB-0159 were highlighted in red. Random structure ranking is represented by green dots. See also . (F) Structure of YB-0158, a phosphate-stabilized prodrug of YB-0159. (G) Docked poses of CWP231904 (left) and YB-0159 (right) in human Sam68 257-374 fragment (red, oxygen; blue, nitrogen). Glycine 305 is highlighted in red, where distinct hydrogen bond (gray dashed line) was predicted between YB-0159 and Sam68. The inset in the right pose represents a higher magnification view of the predicted hydrogen bond formation between YB-0158 and Gly305. See also .

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: In Silico, Binding Assay, Activity Assay, Standard Deviation

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: YB-0158 alters Sam68 biology in human cancer cells (A) Dose-response experiment assessing growth inhibition caused by peptidomimetics analogs CWP232228 and YB-0158 in t-hESCs (n = 3, 48-h treatments). Calculated EC 50 for each small molecule is presented in the inset table. See also . (B) Dose-response experiment assessing growth inhibition caused by peptidomimetic analogs CWP232228 and YB-0158 in HT29 colorectal cancer cells (n = 2, 48-h treatments). Calculated EC 50 for each small molecule is presented in the inset table. (C) Co-immunoprecipitation (IP) assessing changes in interaction levels between Src and Sam68 in response to CWP232228 (1.5 μM) and YB-0158 (0.3 μM) in HT29 cells (48 h) (n = 3, ∗: p = 0.021, ∗∗: p = 0.0088, two-tailed t test). Data are represented as mean ± SEM (error bars). Mouse IgGs were used as negative control for pull down. (D) Immunofluorescence staining of Sam68 in DMSO, CWP232228, and YB-0158-treated t-hESCs (48 h, n = 9). Quantification of nuclear Sam68 was performed by high-content imaging and presented as relative levels versus DMSO (∗∗: p < 0.01, ∗∗∗: p < 0.0001, two-tailed t test). Data are represented as mean ± SEM (error bars). Scale bar: 100 μm. (E) Quantification of nuclear Sam68 in HT29 cells treated with increasing doses of YB-0158, in the presence (n = 4) or absence (n = 3) of a PRMT1 inhibitor (Furamidine, 10 μM). Cells were treated for 48 h and nuclear Sam68 immunostaining was quantified by high-content imaging. Data are represented as mean ± SEM (error bars). (F) Western blot analysis of Sam68 levels in normal human intestinal progenitor cells HIEC; human colorectal cancer SW480, HT29, and HCT116 lines; mouse colon adenocarcinoma MC38 cells; t-hESCs; as well as patient-derived CSC-enriched spheroids and 3D organoids from colorectal tumor samples (n ≥ 3). Relative OD signal quantification for Sam68 versus loading control (GAPDH) is presented. (G) Dose-response experiment monitoring growth of normal intestinal cells HIEC, as well as HT29, SW480, and HCT116 colorectal cancer lines treated with YB-0158 (n ≥ 3, 48 h). A significant correlation was established between calculated EC 50 and Sam68 expression (R 2 = 0.8510, p < 0.0001, simple linear regression). (H) Cell growth experiment in HCT116 cells transduced with control/empty-mGFP (pLenti Control) or KHDRBS1 -mGFP (pLenti Sam68) overexpression vectors and treated with YB-0158 (0.3 μM, 48 h) or vehicle control (DMSO). GFP-positive cell counts upon treatments are presented versus their corresponding DMSO-treated group (n = 7, ∗∗: p = 0.003, two-tailed t test). Data are represented as mean ± SEM (error bars). (I) Cell growth experiment using HCT116 cells overexpressing wild-type Sam68 ( KHDRBS1 ) (wt Sam68) or with a mutated G305 motif (G305N Sam68) and subjected to increasing doses of YB-0158 (0.08–10 μM versus DMSO control) for 48 h. Residual transduced cells (GFP reporter) were counted for each dose and presented versus DMSO control (n ≤ 5, ∗∗∗: p < 0.001, two-tailed t test). Data are represented as mean ± SEM (error bars).

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Inhibition, Immunoprecipitation, Two Tailed Test, Negative Control, Immunofluorescence, Staining, Imaging, Immunostaining, Western Blot, Derivative Assay, Expressing, Transduction, Over Expression

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet:

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Recombinant, Derivative Assay, Immunoprecipitation, Staining, Chromatin Immunoprecipitation, DNA Purification, SYBR Green Assay, Purification, Western Blot, RNA Sequencing Assay, Sequencing, Expressing, Transformation Assay, shRNA, Software

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: Reverse/β-turn peptidomimetic compounds are direct interactors of Sam68 (A) Chemical structure of HAT inhibitor C646 and bromodomain ligand I-CBP112, as well as β-turn peptidomimetics ICG-001 and CWP232228. (B) Western blot analysis of CBP-catalyzed H3K14ac and H3K18ac histone acetylation marks in C646 (0.25 μM), I-CBP112 (0.25 μM), and CWP232228 (0.1 μM) t-hESCs versus control DMSO. Total histone H3 and GAPDH were used as loading control. Relative OD signal quantification versus H3 intensity is presented (C646: n = 3, I-CBP112: n = 5, CWP232228: n ≥ 3, ∗: p = 0.0183, ∗∗: p = 0.0078, ∗∗∗: p ≤ 0.00033, two-tailed t test). Data are represented as mean ± SEM (error bars). (C) Dose-response experiment assessing the impact of bromodomain ligand-based (C646 and I-CBP112), and peptidomimetic (CWP232228) inhibition of CBP on t-hESC growth (C646, I-CPB112: n = 4; CWP232228: n = 3). (D) Early endoderm differentiation assay performed in t-hESCs in the presence of CWP232228 (0.1 μM, n = 6), I-CBP112 (0.25 μM, n = 3), or C646 (0.25 μM, n = 3) versus control DMSO (n = 6) and basal culture media (n = 3). Bar graph represents relative counts of FOXA2-positive (early endoderm marker)/OCT4-negative cells in DMSO, CWP232228, I-CBP112, and C646-treated t-hESCs versus basal culture media (one-way ANOVA, ∗∗∗: p < 0.0001). Data are represented as mean ± SEM (error bars). Scale bar: 100 μm. (E) Pro-drug CWP232228 is converted into its active form CWP231904 via hydrolysis of the phosphate group by serum/cellular alkaline phosphatase. (F) Affinity pull-down experiments using CWP231904-conjugated magnetic beads performed on whole hESC lysates. Physical interaction between CBP, Sam68, beta-Catenin, ETS, MYB, GATA2, and PTMA with immobilized CWP231904 was assessed by immunoblotting. Each protein was previously shown as a member of the CBP interactome showing selective enrichment in human primary AML versus healthy blood ( Benoit et al., 2017 ). Excess of soluble compounds (CWP and ICG001, 100 μM) were used to compete with immobilized CWP231904. Whole-cell lysate was used as input, and amine-functionalized beads were used as negative control (n = 2). The heatmap presents mean background-corrected OD signal for each putative interactor tested (gray: not tested).

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Western Blot, Two Tailed Test, Inhibition, Differentiation Assay, Marker, Magnetic Beads, Negative Control

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: In silico screening of peptidomimetics with enhanced binding affinity for Sam68 (A) Representation of Sam68 interacting with SH3 domain in Src kinase family proteins via proline-rich motifs located in N-terminal P1-P2 and between residues 275 and 374 (P3, P4, P5). Small molecule UCS15A is known to disrupt SH3-mediated interaction of Src with Sam68 P3-5 domains ( Sharma et al., 2001 ). (B) 2D representation of UCS15A and the peptidomimetic ICG-001 in silico predicted binding pocket in Sam68 275-374 peptide (red, oxygen; blue, nitrogen). Common residues involved in both small-molecule-binding pockets are highlighted in red. Predicted hydrogen bond length is represented by dashed lines (Å). (C) Schematic representation of the in silico structure-activity relationship analysis pipeline (PyRx) used to identify β-turn peptidomimetic molecules with enhanced binding affinity for Sam68 275-374 domain. “A” and “B” represent the positions of distinct substituents added to reverse-turn mimetic cores. (D) Dose-response curves assessing selective toxicity of peptidomimetics ICG-001, CWP232228, and PRI-724 in HT29 human colorectal cancer cell line versus normal intestinal progenitor cells HIEC (n ≥ 4, 48-h treatments). (E) Compound ranking based on predicted Keq for each β-turn analog (black dots). Only molecules presenting a standard deviation below 0.1 for a minimum of three analysis runs, with an exhaustiveness (“E”) level of “8” were plotted. Dots corresponding to ICG-001, CWP231904, PRI-724-OH, and YB-0159 were highlighted in red. Random structure ranking is represented by green dots. See also . (F) Structure of YB-0158, a phosphate-stabilized prodrug of YB-0159. (G) Docked poses of CWP231904 (left) and YB-0159 (right) in human Sam68 257-374 fragment (red, oxygen; blue, nitrogen). Glycine 305 is highlighted in red, where distinct hydrogen bond (gray dashed line) was predicted between YB-0159 and Sam68. The inset in the right pose represents a higher magnification view of the predicted hydrogen bond formation between YB-0158 and Gly305. See also .

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: In Silico, Binding Assay, Activity Assay, Standard Deviation

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: YB-0158 alters Sam68 biology in human cancer cells (A) Dose-response experiment assessing growth inhibition caused by peptidomimetics analogs CWP232228 and YB-0158 in t-hESCs (n = 3, 48-h treatments). Calculated EC 50 for each small molecule is presented in the inset table. See also . (B) Dose-response experiment assessing growth inhibition caused by peptidomimetic analogs CWP232228 and YB-0158 in HT29 colorectal cancer cells (n = 2, 48-h treatments). Calculated EC 50 for each small molecule is presented in the inset table. (C) Co-immunoprecipitation (IP) assessing changes in interaction levels between Src and Sam68 in response to CWP232228 (1.5 μM) and YB-0158 (0.3 μM) in HT29 cells (48 h) (n = 3, ∗: p = 0.021, ∗∗: p = 0.0088, two-tailed t test). Data are represented as mean ± SEM (error bars). Mouse IgGs were used as negative control for pull down. (D) Immunofluorescence staining of Sam68 in DMSO, CWP232228, and YB-0158-treated t-hESCs (48 h, n = 9). Quantification of nuclear Sam68 was performed by high-content imaging and presented as relative levels versus DMSO (∗∗: p < 0.01, ∗∗∗: p < 0.0001, two-tailed t test). Data are represented as mean ± SEM (error bars). Scale bar: 100 μm. (E) Quantification of nuclear Sam68 in HT29 cells treated with increasing doses of YB-0158, in the presence (n = 4) or absence (n = 3) of a PRMT1 inhibitor (Furamidine, 10 μM). Cells were treated for 48 h and nuclear Sam68 immunostaining was quantified by high-content imaging. Data are represented as mean ± SEM (error bars). (F) Western blot analysis of Sam68 levels in normal human intestinal progenitor cells HIEC; human colorectal cancer SW480, HT29, and HCT116 lines; mouse colon adenocarcinoma MC38 cells; t-hESCs; as well as patient-derived CSC-enriched spheroids and 3D organoids from colorectal tumor samples (n ≥ 3). Relative OD signal quantification for Sam68 versus loading control (GAPDH) is presented. (G) Dose-response experiment monitoring growth of normal intestinal cells HIEC, as well as HT29, SW480, and HCT116 colorectal cancer lines treated with YB-0158 (n ≥ 3, 48 h). A significant correlation was established between calculated EC 50 and Sam68 expression (R 2 = 0.8510, p < 0.0001, simple linear regression). (H) Cell growth experiment in HCT116 cells transduced with control/empty-mGFP (pLenti Control) or KHDRBS1 -mGFP (pLenti Sam68) overexpression vectors and treated with YB-0158 (0.3 μM, 48 h) or vehicle control (DMSO). GFP-positive cell counts upon treatments are presented versus their corresponding DMSO-treated group (n = 7, ∗∗: p = 0.003, two-tailed t test). Data are represented as mean ± SEM (error bars). (I) Cell growth experiment using HCT116 cells overexpressing wild-type Sam68 ( KHDRBS1 ) (wt Sam68) or with a mutated G305 motif (G305N Sam68) and subjected to increasing doses of YB-0158 (0.08–10 μM versus DMSO control) for 48 h. Residual transduced cells (GFP reporter) were counted for each dose and presented versus DMSO control (n ≤ 5, ∗∗∗: p < 0.001, two-tailed t test). Data are represented as mean ± SEM (error bars).

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Inhibition, Immunoprecipitation, Two Tailed Test, Negative Control, Immunofluorescence, Staining, Imaging, Immunostaining, Western Blot, Derivative Assay, Expressing, Transduction, Over Expression

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet:

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Recombinant, Derivative Assay, Immunoprecipitation, Staining, Chromatin Immunoprecipitation, DNA Purification, SYBR Green Assay, Purification, Western Blot, RNA Sequencing Assay, Sequencing, Expressing, Transformation Assay, shRNA, Software