Journal: Virology

Article Title: The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

doi: 10.1016/j.virol.2011.12.019

Figure Lengend Snippet: Fig. 1. Sub-cellular distribution of Sam68 is altered by FMDV infection. A. LFBK cells were infected with FMDV or mock-infected (top panel) or BEV-1 (bottom panel) at a MOI of 10 and incubated at 37 °C for 1, 3, and 5 h. Virus-infected cells were probed with rabbit anti-Sam68 (N-terminal) followed by goat-anti-rabbit-AF488 (green). Nuclear material was stained with DAPI (blue). B. LFBK cells were transfected with a plasmid encoding GFP-tagged full-length Sam68, and 2 days post-transfection, the transfected cell lysates were examined by Western blot probing with anti-Sam68 (C-terminal). Arrowhead indicates endogenous Sam68. Arrow indicates ectopically expressed Sam68. The lane marked with an asterisk contains a purified preparation of GST-tagged Sam68 expected to have a similar approximate molecular weight as GFP-tagged Sam68. C. LFBK cells were transfected with pGFP-Sam68, subsequently either mock-infected or FMDV-infected for 4 h, and then were examined by IFM using a GFP filter.

Article Snippet: Polyclonal rabbit anti-Sam68 directed against the N-terminus of Sam68 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Infection, Incubation, Virus, Staining, Transfection, Plasmid Preparation, Western Blot, Molecular Weight

Journal: Virology

Article Title: The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

doi: 10.1016/j.virol.2011.12.019

Figure Lengend Snippet: Fig. 2. FMDV-induced cytoplasmic accumulation of Sam68 is not triggered by Nup degradation. A. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and the infected cells were harvested at 1, 3, and 5 hpi. Virus-infected cell lysates were examined by Western blot probing with anti-nucleoporin protein or anti-tubulin (loading control). B. Densitometry scans were conducted using ImageJ software (NIH), and Nup62 (gray bars) and Nup98 (black bars) values, normalized against tubulin, obtained over 3 experiments were plotted.

Article Snippet: Polyclonal rabbit anti-Sam68 directed against the N-terminus of Sam68 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Infection, Virus, Western Blot, Control, Software

Journal: Virology

Article Title: The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

doi: 10.1016/j.virol.2011.12.019

Figure Lengend Snippet: Fig. 3. Sam68 is degraded during FMDV infection. A. Schematic representation of Sam68 with specific domains and regions indicated. The region recognized by the N-terminal anti- Sam68 antibody (A) and C-terminal anti-Sam68 antibody (B) are shown. B. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and incubated at 37 °C for 1, 3, or 5 hpi. Lysates were examined on side-by-side Western blots probing with antibodies directed against the N-terminus (left panel) or C-terminus (right panel) of Sam68. Arrowheads indicate full-length Sam68. Arrow indicates a stable Sam68-reactive degradation product. Asterisk (*) indicates a protein cross-reacting with the N-terminal anti-Sam68 antibody that might correspond to SLM-1. Shaded circle indicates the tubulin loading control. C. LFBK cells were mock-infected (not shown) and infected with either FMDV or BEV-1 at a MOI of 10 and incubated at 37 °C for 2, 3, 4, or 5 h. Lysates were examined by Western blot probing with anti-Sam68 (C-terminal), anti-RHA, or anti-tubulin (loading control).

Article Snippet: Polyclonal rabbit anti-Sam68 directed against the N-terminus of Sam68 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Infection, Incubation, Western Blot, Control

Journal: Virology

Article Title: The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

doi: 10.1016/j.virol.2011.12.019

Figure Lengend Snippet: Fig. 4. Sam68 degradation is not due to the FMDV leader protease. A. Recombinant full-length Sam68 was mixed with increasing concentrations of recombinant wild-type or mutant Lpro. The in vitro reactions were incubated at 37 °C for 5 h, and subsequently analyzed by Western blot probing with the N-terminal Sam68 antibody. B. LFBK cells grown on coverslips were mock-infected or infected with wild-type (WT) FMDV A24-Cruzeiro or leaderless (LL) FMDV A24-Cruzeiro at a MOI 10 and incubated at 37 °C for 1, 3 or 5 hpi. Mock and virus-infected cells were probed successively with rabbit anti-Sam68 then goat-anti-rabbit-AF488 (green).

Article Snippet: Polyclonal rabbit anti-Sam68 directed against the N-terminus of Sam68 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Recombinant, Mutagenesis, In Vitro, Incubation, Western Blot, Infection, Virus

Journal: Virology

Article Title: The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

doi: 10.1016/j.virol.2011.12.019

Figure Lengend Snippet: Fig. 5. Sam68 is cleaved by the FMDV 3C protease. A. Diagram of Sam68 with the RGG box, KH domain, tyrosine rich region, nuclear localization sequence (NLS) and polyproline regions (P0, P1, P2, P3, P4, P5) indicated. The anti-Sam68 antibody targeted to the N-terminus reacts with the region labeled as “A”, and the C-terminal anti-Sam68 antibody reacts with the region labeled as “B”. Arrowheads mark potential FMDV 3C protease cut sites. B. An active WT 3C protease was expressed in bacteria from a plasmid encoding the FMDV 3ABC polypeptide. Transformed cultures were induced, lysed, and fractionated into soluble (S) and insoluble (P) portions, which were examined on a Western blot probing with a mouse monoclonal anti-3Cpro antibody followed by goat-anti-mouse-HRP. C. Recombinant purified Sam68 was incubated 5 h at 30 °C with increasing amounts of bacterial lysates contain- ing the active wild-type 3Cpro or an inactive mutant. The reactions were analyzed on a Western blot probing first with rabbit anti-Sam68, then stripped and probed with mouse anti-3Cpro. D. Partially purified WT and inactive 3Cpro were mixed with LFBK or BHK cellular lysates and incubated at 37 °C for 5 h. The solutions were then examined by Western blot probing with the C-terminal anti-Sam68. Afterwards, the blot was stripped and re-probed first with anti-histone H3 to confirm the enzyme activity and second with anti-tubulin to confirm equal load- ing between the lanes. Densitometry scanning was used to assign arbitrary values to relative changes in protein concentration on the Western blots.

Article Snippet: Polyclonal rabbit anti-Sam68 directed against the N-terminus of Sam68 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Sequencing, Labeling, Bacteria, Plasmid Preparation, Transformation Assay, Western Blot, Recombinant, Incubation, Mutagenesis, Activity Assay, Protein Concentration

Journal: Virology

Article Title: The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

doi: 10.1016/j.virol.2011.12.019

Figure Lengend Snippet: Fig. 6. Sam68 interacts with the FMDV IRES. A. Full-length Sam68 was mixed with biotinylated single-stranded RNA transcripts corresponding to the FMDV S-fragment, IRES, cre, or 3′NTR. Following the previously described RNA filter-binding assay (see Materials and methods), protein-RNA interaction was evaluated by probing the nitrocellulose membrane with SA-HRP. B. Three of the RNA filter binding assays were quantified by densitometry scanning using ImageJ software (NIH) and the values were plotted using Microsoft Excel. C. Biotinylated RNA transcripts of the FMDV S-fragment, IRES, cre, and 3′NTR were separately mixed with SA-coated magnetic beads. Next, they were incubated with full-length Sam68, washed, and the eluates examined by Western blot probing with anti-Sam68. NC, negative control; PC, positive control.

Article Snippet: Polyclonal rabbit anti-Sam68 directed against the N-terminus of Sam68 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Binding Assay, Membrane, Software, Magnetic Beads, Incubation, Western Blot, Negative Control, Positive Control

Journal: Virology

Article Title: The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

doi: 10.1016/j.virol.2011.12.019

Figure Lengend Snippet: Fig. 7. Reduced Sam68 expression negatively affects progression of FMDV infection. LFBK cells were untransfected (C), transfected with nonsense negative control siRNA (NC), or individual and pooled Sam68-targeted siRNA molecules (#6, 7, 8, and 9) and incubated 72 h at 37°C. Post-incubation, the transfected cells were analyzed by XTT cytotoxicity assay (A) and Western blot probing with anti-Sam68 antibodies (B). Equal loading was confirmed by re-probing with anti-tubulin antibodies. C–D. LFBK cells were transfected with non- sense or all 4 Sam68-specific siRNA molecules, and the cells incubated for 72 h at 37 °C. Post-incubation, the transfected cells were infected with FMDV at an MOI of 0.001 and in- cubated at 37 °C for 24 h. The resulting samples were evaluated for Sam68 concentration by Western blot (the blot in C represents one replicate experiment with values obtained from densitometry scanning beneath the Western blot bands) and virus growth by plaque assay as previously described (D). The assay was conducted in triplicate.

Article Snippet: Polyclonal rabbit anti-Sam68 directed against the N-terminus of Sam68 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Infection, Transfection, Negative Control, Incubation, Cytotoxicity Assay, Western Blot, Concentration Assay, Virus, Plaque Assay

Journal: Virology

Article Title: The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

doi: 10.1016/j.virol.2011.12.019

Figure Lengend Snippet: Fig. 8. The cellular concentration of Sam68 affects translation from the FMDV IRES. A. Schematic of the bicistronic reporter plasmid, pBIC with the chloramphenicol acetyltransferase (CAT) gene under control of CAP-dependent translation and the firefly luciferase (FFLuc) gene under control of CAP-independent translation driven by the FMDV IRES. B. LFBK cells were transfected with pBIC simultaneously with Sam68-targeted siRNAs (Sam68 siRNA), nonsense negative control siRNAs (NC siRNA) or with no siRNA (No siRNA) and incubated for 48 h. Harvested cells were evaluated for reduction in Sam68 by Western blot probing with anti-Sam68 followed by anti-tubulin (loading control). C. Harvested cell lysates were also evaluated for FFLuc and CAT activity. Values obtained from the FFLuc assay were normalized against those obtained from the CAT assay, and the adjusted relative light units were plotted using Microsoft Excel. The graph is representative of 1 of 3 independent experiments. D. Overview of the step-wise triple transfection protocol employed to overcome the competition between siRNAs and plasmid DNA during co-transfection. E. Western blot of LFBK cells following the step-wise triple transfection depicted in D, probing with anti- Sam68 followed by anti-tubulin (loading control). F. Harvested cell lysates from the step-wise triple transfection were also evaluated for FFLuc and CAT activity. The values obtained for the FFLuc assay were normalized against the CAT assay values, and the normalized RLUs were plotted. Values shown are the average of 3 separate experiments. Densitometry scanning was used to assign arbitrary values to relative changes in protein concentration on the Western blots (B and E).

Article Snippet: Polyclonal rabbit anti-Sam68 directed against the N-terminus of Sam68 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Concentration Assay, Plasmid Preparation, Control, Luciferase, Transfection, Negative Control, Incubation, Western Blot, Activity Assay, Cotransfection, Protein Concentration

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: Molecular and Cellular Biology

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/mcb.23.1.92-103.2003

Figure Lengend Snippet: FIG. 1. Sam68 enhances CTE function in 293 and COS cells. Five micrograms of the pCMVGag-Pol-CTE reporter plasmid (pHR1361) and 0.25 g of pCMVSEAP (pHR1831) were transfected into 100-mm-diameter dishes of either 293 cells (A) or COS cells (B) together with 1 g each of pcDFSam68 (pHR2208), pcDHSam68KH (pHR2212), pcDHSam68(G178E) (pHR2210), or pcDNA3 with no insert (pHR2214). Superna- tants were collected at 72 h posttransfection, and p24 and SEAP levels in each sample were determined. p24 values were then adjusted for variations in SEAP levels. The data represent the average of two independent transfections.

Article Snippet: For detection of Sam68, blots were probed with an anti-Sam68 monoclonal antibody (SC-333; Santa Cruz Biotechnology, Inc.).

Techniques: Plasmid Preparation, Transfection

Journal: Molecular and Cellular Biology

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/mcb.23.1.92-103.2003

Figure Lengend Snippet: FIG. 2. Comparison of endogenous Sam68 produced in 293T and COS cells. Sam68 was immunoprecipitated with anti-Sam68 monoclo- nal antibodies from lysates of 293T and COS cells metabolically la- beled with [35S]Met/Cys Tran35S-label for either 3 or 24 h. Protein was resolved by SDS-PAGE and visualized by PhosphorImager analysis. IgG1, immunoglobulin G1.

Article Snippet: For detection of Sam68, blots were probed with an anti-Sam68 monoclonal antibody (SC-333; Santa Cruz Biotechnology, Inc.).

Techniques: Comparison, Produced, Immunoprecipitation, Metabolic Labelling, SDS Page

Journal: Molecular and Cellular Biology

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/mcb.23.1.92-103.2003

Figure Lengend Snippet: FIG. 3. Sam68 enhances CTE function in a stable 293T packaging cell line and Qcl3 quail cells. (A) Dishes (100-mm diameter) of B2.10 cells were transfected with 1 g of pcDFSam68 (pHR2208) or pcDNA empty vector (pHR2214). B2.10 is a cell line derived from 293T cells by stable transfection of pCMVGag-Pol-CTE (pHR1361) and pCMVEnv-CTE (pHR1374). p24 levels in the supernatant were determined at 72 h posttransfection. The data represent the average of two independent transfections. (B) Dishes (100-mm diameter) of Qcl3 quail fibroblast cells were transfected with 5 g of the pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 g of pCMVSEAP (pHR1831) together with 2 g of pCMVTap (pHR2128), 1 g of pcDFSam68 (pHR2208), or both. At 72 h posttransfection, supernatants were collected and analyzed for p24 and SEAP expression. The data represent the average of two independent transfections.

Article Snippet: For detection of Sam68, blots were probed with an anti-Sam68 monoclonal antibody (SC-333; Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Plasmid Preparation, Derivative Assay, Stable Transfection, Expressing

Journal: Molecular and Cellular Biology

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/mcb.23.1.92-103.2003

Figure Lengend Snippet: FIG. 4. Coexpression of the constitutively active Sik(Y447F) mutant protein with Sam68 inhibits Sam68-mediated enhancement of CTE function. (A) Five micrograms of pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 g of pCMVSEAP (pHR1831) were transfected into 293T cells with 1 g of a plasmid that expresses either the wild-type (pcDNA3-Sikwt, pHR2531), the kinase-inactive [pcDNA3-Sik(K219M), pHR2533], or the constitutively active [pcDNA3Sik(Y447F), pHR2533] form of Sik. These plasmids were expressed either alone or together with 1 g of pcDSam68 (pHR2208). At 72 h posttransfection, supernatants were collected and analyzed for p24 and SEAP expression. The data represent the average of two independent transfections. (B) Increasing amounts of pcDFSam68 or pcDNA3-Sik(Y447F) were cotransfected with 5 g of pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 g of pCMVSEAP (pHR1831) plasmid into 293T cells. At 72 h posttransfection, supernatants were collected and analyzed for p24 and SEAP expression. The data represent the average of two independent transfections.

Article Snippet: For detection of Sam68, blots were probed with an anti-Sam68 monoclonal antibody (SC-333; Santa Cruz Biotechnology, Inc.).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing

Journal: Molecular and Cellular Biology

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/mcb.23.1.92-103.2003

Figure Lengend Snippet: FIG. 5. Expression of the Sik(Y447F) mutant increases phosphor- ylation of Sam68 at multiple residues. 293T cells were transfected with plasmids expressing Flag-Sam68 either alone or with a plasmid ex- pressing Sik(Y447F) and labeled with [32P]orthophosphate. (A, left side) 32P analysis of immunoprecipitated Sam68. Sam68 was immuno- precipitated from transfected cells by using an agarose bead-conju- gated anti-FLAG antibody. Proteins were resolved by SDS-PAGE, and 32P incorporation was visualized by PhosphorImager analysis. (A, right side) Western blot analysis of Sam68 in the absence or presence of Sik(Y447F). Proteins from whole-cell lysates were resolved by SDS- PAGE and transferred to Immobilon. The blot was probed with an anti-Sam68 monoclonal antibody, and Sam68 was visualized by using an alkaline phosphatase-conjugated anti-mouse secondary antibody. (B) Two-dimensional phosphopeptide analysis of Sam68 expressed in the presence or absence of Sik(Y447F). Tryptic peptide analysis was carried out on Sam68 immunoprecipitated from 32P-labeled 293T cells by using an agarose bead-conjugated anti-FLAG antibody. The pep- tides were resolved in two dimensions, first by electrophoresis in pH 1.9 buffer and second by chromatography in phosphochromatography buffer. Labeled peptides were visualized by PhosphorImager analysis. Arrows indicate peptides that appeared de novo or increased in inten- sity in the presence of Sik(Y447F).

Article Snippet: For detection of Sam68, blots were probed with an anti-Sam68 monoclonal antibody (SC-333; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Western Blot, Phospho-proteomics, Electrophoresis, Chromatography

Journal: Molecular and Cellular Biology

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/mcb.23.1.92-103.2003

Figure Lengend Snippet: FIG. 6. Analysis of p24 and Gag-Pol RNA in 293T cells. 293T cells were transfected with the reporter plasmids pCMVGag-Pol-RRE (pHR354) or pCMVGag-Pol-CTE (pHR1361) and pCMVSEAP (pHR1831) in the presence or absence of Rev (pHR30)- and/or Sam68 (pHR2208)-expressing plasmids essentially as described in the legend to Fig. 1. (A) Supernatants were harvested at 72 h posttransfection, and p24 levels were analyzed by ELISA. (B) Cells were harvested at 72 h and analyzed by Western blot assay. The Western blot analysis was performed on whole-cell lysates prepared from transfected cells with an anti-p24 primary monoclonal antibody, a rabbit anti-mouse secondary antibody, and 125I-coupled protein A for detection. Brackets show the fold difference in p24 levels between indicated lanes. (C) Northern blot analysis of cytoplasmic and total poly(A) RNA isolated from 293T cells 60 h after transfection. Gag-Pol- and SEAP- specific mRNAs were detected by using specific radiolabeled DNA probes. The blot was analyzed with a Molecular Dynamics Phosphor- Imager and ImageQuant software. Brackets show the fold differences in the levels of the Gag-Pol RNA bands between the indicated lanes after adjustment for variations in SEAP RNA levels.

Article Snippet: For detection of Sam68, blots were probed with an anti-Sam68 monoclonal antibody (SC-333; Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Northern Blot, Isolation, Software

Journal: Molecular and Cellular Biology

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/mcb.23.1.92-103.2003

Figure Lengend Snippet: FIG. 7. In situ hybridization of Gag-Pol-CTE RNA in cells with or without Sam68 coexpression. Cells were transfected with plasmids expressing Gag-Pol-RRE RNA (with or without Rev) or Gag-Pol-CTE RNA (with or without Sam68), as indicated. At 72 h after transfection, cells were fixed and hybridized to an antisense DIG-labeled oligonucleotide probe specific for a sequence within gag. Hybridization was detected with an anti-DIG antibody conjugated to FITC and visualized by LSM5 Pascal confocal microscopy (Zeiss).

Article Snippet: For detection of Sam68, blots were probed with an anti-Sam68 monoclonal antibody (SC-333; Santa Cruz Biotechnology, Inc.).

Techniques: In Situ Hybridization, Transfection, Expressing, Labeling, Sequencing, Hybridization, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/mcb.23.1.92-103.2003

Figure Lengend Snippet: FIG. 8. Effect of Sam68 on Rev/RRE function. (A) Five micrograms of pCMVGag-Pol-RRE (pHR354) and 0.25 g of pCMVSEAP (pHR1831) were transfected into 100-mm-diameter dishes of 293 cells with the indicated amounts of pCMVRev (pHR30) with or without 1 g of pcDFSam68 (pHR2208). p24 analysis was carried out as described in the legend to Fig. 1. (B) Dishes (150-mm diameter) of 293T cells were transfected with 20 g of pCMVGag-Pol-RRE and 5 g of pCMVSEAP in the presence or absence of 5 g of pCMVRev and/or pcDFSam68. Northern blot analysis was performed on cytoplasmic (Cyto) and total poly(A) mRNA isolated at 48 h after transfection and detected with a radiolabeled gag/pol-specific probe. Gag-Pol- and SEAP-specific mRNAs were detected by using specific radiolabeled DNA probes. The blot was analyzed with a Molecular Dynamics Phosphorimager and ImageQuant software. Brackets show the fold differences in the levels of the Gag-Pol RNA bands between the indicated lanes after adjustment for variations in SEAP RNA levels.

Article Snippet: For detection of Sam68, blots were probed with an anti-Sam68 monoclonal antibody (SC-333; Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Northern Blot, Isolation, Software

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: In vivo association of SAM68 with U1 snRNP. ( A ) Schematic representation of a portion of mTor pre-mRNA spanning from exon4 to exon6 (upper panel), with a close-up of the 5′ splice site and the subsequent SAM68-binding site (SB-1), as well as the cryptic polyadenylation signal that harbor SAM68-binding site (SB-A). ( B ) Co-immunoprecipitation of U1 snRNP with Flag-hSAM68. HEK-293T cells depleted of endogenous SAM68 (sh SAM68 HEK-293T) were transiently transfected with Flag-hSAM68 or Flag-YFP (yellow-fluorescent protein), the latter serving as negative control. Flag-tagged proteins were immunoprecipitated using anti-Flag M2 agarose beads and immunoprecipitated proteins were detected with antibodies specific to U1–70K, U1A and U1C. β-Actin was used as negative control. Portion of the Flag-immunoprecipitates was used for RNA isolation and RT-PCR using U1 snRNA specific primers. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) RNA was used as negative control of the RT-PCR made from the RNA immunoprecipitation. ( C ) Co-immunoprecipitation of endogenous hSAM68 with U1–70K. Immunoprecipitated proteins were detected with antibodies directed against SAM68 and U1–70K. β-Actin was used as negative control of immunoprecipitated proteins. ( D ) Co-immunoprecipitation of endogenous hSAM68 with U1A. Immunoprecipitated proteins were detected with antibodies directed against SAM68 and U1A. β-Actin was used as negative control of immunoprecipitated proteins. ( E ) Coomassie staining of purified human SAM68 and U1A. ( F ) RNA binding assay with purified SAM68 and labeled U1snRNA. Reactions contained 10 nM γ-p32 labeled U1snRNA in buffer with no protein (lane 1) or with purified SAM68 (lanes 2–5). Bottom panel: quantification from three independent binding experiments. Error bars represent the corresponding standard error. Unpaired two-tailed t -tests were used to compare the different concentrations of purified protein to the RNA only control. SAM68 P -values are 0.0014, 0.0005, <0.0001, <0.0001 in increasing order of SAM68 concentration. ( G ) RNA binding assay with purified U1A and labeled U1snRNA. Reactions contained 10 nM γ-p32 labeled U1snRNA in buffer with no protein (lane 1) or with purified U1A (lanes 2–5). Bottom panel: U1snRNA P -values = 0.0008, <0.0001, <0.0001, <0.0001 in increasing order of U1A concentration. ** P -value < 0.005, *** P -value < 0.001.

Article Snippet: Due to the high sequence homology between human and mouse SAM68 , both constructs were produced using EcoRI- SAM68 -F and NotI- SAM68 -R. Flag tag was then inserted at the C-terminus of SAM68 by annealing the oligos, SacI-Flag-F and SacI-Flag-R, and inserting the adaptor at SacI sites of the plasmid. pGEX-6P2-U1A-His was generated by inserting U1A cds, obtained by polymerase chain reaction (PCR) from HEK-293T total cDNA using EcoRI-U1A-F and SalI-U1A-R at EcoRI-SalI sites. cDNA was amplified from total RNA of HEK-293T using Superscript VILO Master mix (Invitrogen). pGEX-6P2-U1C-His was sub-cloned by PCR from pGEX-2TK-U1C using EcoRI-U1C-F and XhoI-U1C-R and inserted at EcoRI-XhoI sites. pGEX-6P2-U1–70K was sub-cloned by PCR from pINTO-NSA:hSNRNP70 using EcoRI-U1–70K-F and XhoI-U1–70K-R and inserted at EcoRI-XhoI sites. pcDNA-Flag- SAM68 and pcDNA-Flag- Sam68 were constructed by inserting corresponding cds, obtained by PCR from pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag using EcoRI- SAM68 -F and NotI- SAM68 -R at EcoRI-NotI sites. pcDNA-Flag- SAM68 I184N and pcDNA-Flag- SAM68 V229F were generated by swapping the 679 bp, AgeI-XbaI fragment from pcDNA mCherry- SAM68 I184N and pcDNA mCherry- SAM68 V229F , respectively, to pcDNA-Flag- SAM68 WT. pcDNA-Flag- SAM68 -Nter and pEGFP- Sam68 -Nter were generated by deletion PCR with SAM68 -F and SAM68- Nter-R primers using pcDNA-Flag- SAM68 and pEGFP- SAM68 as templates, respectively. pcDNA-Flag- SAM68 -Cter was generated from pcDNA-Flag- SAM68 by PCR using EcoRI- SAM68 -Cter-F and NotI- SAM68 Cter-R and cloning the amplicon at EcoRI-SalI sites of pEGFP-C1. pEGFP-C1- SAM68 -C1 was obtained by deletion PCR using SAM68 -F and SAM68 -C1-R, respectively. pEGFP- SAM68 -C2 to C5 were generated by cloning the PCR amplicons obtained using the reverse primer, SalI- SAM68 -R and forward primers namely EcoRI- SAM68- C2, C3, C4, C5 at EcoRI-SalI sites of pEGFP-C1. pEGFP- SAM68 -NLS was obtained by deletion PCR using EcoRI- SAM68 -NLS-F and EcoRI- SAM68 -NLS-R. pLKO-sh SAM68 was generated by annealing and inserting the oligos, sh SAM68 -F and sh SAM68 -R, at AgeI-EcoRI restriction sites of pLKO.1 (Addgene plasmid #8453).

Techniques: In Vivo, Binding Assay, Immunoprecipitation, Transfection, Negative Control, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining, Purification, RNA Binding Assay, Labeling, Two Tailed Test, Concentration Assay

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: In vitro Purified SAM68 associated with U1 snRNP in an RNA-independent manner. ( A ) In vitro purified hSAM68-Flag was added to sh SAM68 HEK-293T cell lysates for 1 h at 4°C, in the presence or absence of 50 μg/ml RNaseA. hSAM68-Flag and associated proteins were immunoprecipitated using Flag-M2 affinity beads and treated further with RNaseA at 37°C for 30 min. Bound proteins were eluted with Laemmli and immunoblotted with antibodies specific to U1–70K, U1A and U1C. To assess RNaseA treatment efficiency, total RNA from sh SAM68 HEK-293T was treated with either Mock or RNaseA for 30 min at 37°C, and the remaining total RNA was assessed on agarose gel. ( B ) RNA-binding defective mutant hSAM68 I184N interacts with U1 snRNP. sh SAM68 HEK-293T were transiently transfected with Flag-hSAM68, Flag-hSAM68 I184N and Flag-YFP (negative control). The Flag-tagged proteins were immunoprecipitated using anti-Flag M2 agarose beads and immunoblotted with antibodies directed against U1–70K, U1A and U1C. ( C ) Association of hSAM68-Flag with U1 snRNP withstands high salt washes. Purified in vitro produced hSAM68-Flag was added to cell lysates of sh SAM68 HEK-293T for 1 h at 4°C. Flag-M2 affinity beads were added to the reaction and left for 1 h at 4°C. The washes were done, by increasing salt concentration, from 150 to 500 mM of NaCl. Bound proteins were eluted with Laemmli and immunoblotted with antibodies directed against U1–70K, U1A and U1C. ( D ) SAM68 interacts with U1A in vitro . About 300 ng of purified hSAM68-Flag was incubated with 100 ng of glutathione-agarose bound GST-U170k-His, GST-U1A-His, GST-U1C-His and GST-His. Following washes, the beads were washed five times in binding buffer and the bound proteins eluted with Laemmli and immunoblotted using anti-Flag or anti-His antibodies.

Article Snippet: Due to the high sequence homology between human and mouse SAM68 , both constructs were produced using EcoRI- SAM68 -F and NotI- SAM68 -R. Flag tag was then inserted at the C-terminus of SAM68 by annealing the oligos, SacI-Flag-F and SacI-Flag-R, and inserting the adaptor at SacI sites of the plasmid. pGEX-6P2-U1A-His was generated by inserting U1A cds, obtained by polymerase chain reaction (PCR) from HEK-293T total cDNA using EcoRI-U1A-F and SalI-U1A-R at EcoRI-SalI sites. cDNA was amplified from total RNA of HEK-293T using Superscript VILO Master mix (Invitrogen). pGEX-6P2-U1C-His was sub-cloned by PCR from pGEX-2TK-U1C using EcoRI-U1C-F and XhoI-U1C-R and inserted at EcoRI-XhoI sites. pGEX-6P2-U1–70K was sub-cloned by PCR from pINTO-NSA:hSNRNP70 using EcoRI-U1–70K-F and XhoI-U1–70K-R and inserted at EcoRI-XhoI sites. pcDNA-Flag- SAM68 and pcDNA-Flag- Sam68 were constructed by inserting corresponding cds, obtained by PCR from pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag using EcoRI- SAM68 -F and NotI- SAM68 -R at EcoRI-NotI sites. pcDNA-Flag- SAM68 I184N and pcDNA-Flag- SAM68 V229F were generated by swapping the 679 bp, AgeI-XbaI fragment from pcDNA mCherry- SAM68 I184N and pcDNA mCherry- SAM68 V229F , respectively, to pcDNA-Flag- SAM68 WT. pcDNA-Flag- SAM68 -Nter and pEGFP- Sam68 -Nter were generated by deletion PCR with SAM68 -F and SAM68- Nter-R primers using pcDNA-Flag- SAM68 and pEGFP- SAM68 as templates, respectively. pcDNA-Flag- SAM68 -Cter was generated from pcDNA-Flag- SAM68 by PCR using EcoRI- SAM68 -Cter-F and NotI- SAM68 Cter-R and cloning the amplicon at EcoRI-SalI sites of pEGFP-C1. pEGFP-C1- SAM68 -C1 was obtained by deletion PCR using SAM68 -F and SAM68 -C1-R, respectively. pEGFP- SAM68 -C2 to C5 were generated by cloning the PCR amplicons obtained using the reverse primer, SalI- SAM68 -R and forward primers namely EcoRI- SAM68- C2, C3, C4, C5 at EcoRI-SalI sites of pEGFP-C1. pEGFP- SAM68 -NLS was obtained by deletion PCR using EcoRI- SAM68 -NLS-F and EcoRI- SAM68 -NLS-R. pLKO-sh SAM68 was generated by annealing and inserting the oligos, sh SAM68 -F and sh SAM68 -R, at AgeI-EcoRI restriction sites of pLKO.1 (Addgene plasmid #8453).

Techniques: In Vitro, Purification, Immunoprecipitation, Agarose Gel Electrophoresis, RNA Binding Assay, Mutagenesis, Transfection, Negative Control, Produced, Concentration Assay, Incubation, Binding Assay

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: SAM68 interaction with U1A is mediated through its C-terminal portion. ( A ) Schematic representation of C-terminus (aa. 1–280) and N-terminus (aa.281–443) deletion domains of hSAM68 fused to flag. ( B ) sh SAM68 HEK-293T cells were transiently transfected with Flag- SAM68 (N-term), Flag- SAM68 (C-term), Flag- SAM68 (FL) and flag-YFP (negative control). Forty-eight hours post transfection, the flag-tagged proteins were immunoprecipitated using anti-flag M2 agarose beads and immunoblotted with antibodies specific to U1–70K, U1A and U1C. ( C ) Schematic representation of full-length SAM68, C-terminus deleted SAM68 (NT, aa. 1–280), C-terminus truncated to proline rich C1 (aa. 269–364) and tyrosine rich C2 (aa. 365–443), C3 (aa. 370–443), C4 (aa. 385–443), C5 (aa. 340–443) and NLS (aa. 430–443). Fragments were fused to GFP tag at their N-terminus and all fragments had SAM68 NLS at their C-terminus. ( D ) GFP-Trap-A pulldown of GFP-tagged proteins. sh SAM68 HEK-293T cells were transiently transfected with GFP, GFP-SAM68 (FL), GFP - SAM68 (NT), GFP - SAM68 (C1) and GFP-SAM68 (C2). Forty-eight hours post transfection, cells were lysed and GFP-Trap-A beads were used to pull down GFP-tagged proteins, and their association with U1A was validated by western blot using specific antibodies. ( E ) Primary amino acid sequence of the various deletion constructs of SAM68 YY domain (GFP-hSAM68 C2 to C5). Underlined indicates YXXY motifs in the YY domain. Also highlighted is the minimal ARM-binding region. ( F ) GFP-Trap-A pulldown of GFP-tagged proteins. shSAM68 HEK-293T cells were transiently transfected with GFP, GFP-SAM68 (C2), GFP-SAM68 (C3), GFP-SAM68 (C4), GFP-SAM68 (C5) and GFP-SAM68 (NLS). Forty-eight hours post transfection, cells were lysed and GFP-Trap-A beads were used to pull down GFP-tagged proteins, and their association with U1A was validated by western blot using specific antibodies. ( G ) U1A binds preferentially to the minimal ARM motif (YEGYEGY) within the YY domain of SAM68. Flag-hSAM68(FL) and Flag-hSAM68(ΔARM) were transiently transfected in sh SAM68 HEK-293T cells. Forty-eight hours post transfection, cells were lysed and Flag-tagged proteins were immunoprecipitated using anti-flag M2 agarose beads, and U1A association was assessed using U1A antibody. ★: denotes an unspecific band.

Article Snippet: Due to the high sequence homology between human and mouse SAM68 , both constructs were produced using EcoRI- SAM68 -F and NotI- SAM68 -R. Flag tag was then inserted at the C-terminus of SAM68 by annealing the oligos, SacI-Flag-F and SacI-Flag-R, and inserting the adaptor at SacI sites of the plasmid. pGEX-6P2-U1A-His was generated by inserting U1A cds, obtained by polymerase chain reaction (PCR) from HEK-293T total cDNA using EcoRI-U1A-F and SalI-U1A-R at EcoRI-SalI sites. cDNA was amplified from total RNA of HEK-293T using Superscript VILO Master mix (Invitrogen). pGEX-6P2-U1C-His was sub-cloned by PCR from pGEX-2TK-U1C using EcoRI-U1C-F and XhoI-U1C-R and inserted at EcoRI-XhoI sites. pGEX-6P2-U1–70K was sub-cloned by PCR from pINTO-NSA:hSNRNP70 using EcoRI-U1–70K-F and XhoI-U1–70K-R and inserted at EcoRI-XhoI sites. pcDNA-Flag- SAM68 and pcDNA-Flag- Sam68 were constructed by inserting corresponding cds, obtained by PCR from pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag using EcoRI- SAM68 -F and NotI- SAM68 -R at EcoRI-NotI sites. pcDNA-Flag- SAM68 I184N and pcDNA-Flag- SAM68 V229F were generated by swapping the 679 bp, AgeI-XbaI fragment from pcDNA mCherry- SAM68 I184N and pcDNA mCherry- SAM68 V229F , respectively, to pcDNA-Flag- SAM68 WT. pcDNA-Flag- SAM68 -Nter and pEGFP- Sam68 -Nter were generated by deletion PCR with SAM68 -F and SAM68- Nter-R primers using pcDNA-Flag- SAM68 and pEGFP- SAM68 as templates, respectively. pcDNA-Flag- SAM68 -Cter was generated from pcDNA-Flag- SAM68 by PCR using EcoRI- SAM68 -Cter-F and NotI- SAM68 Cter-R and cloning the amplicon at EcoRI-SalI sites of pEGFP-C1. pEGFP-C1- SAM68 -C1 was obtained by deletion PCR using SAM68 -F and SAM68 -C1-R, respectively. pEGFP- SAM68 -C2 to C5 were generated by cloning the PCR amplicons obtained using the reverse primer, SalI- SAM68 -R and forward primers namely EcoRI- SAM68- C2, C3, C4, C5 at EcoRI-SalI sites of pEGFP-C1. pEGFP- SAM68 -NLS was obtained by deletion PCR using EcoRI- SAM68 -NLS-F and EcoRI- SAM68 -NLS-R. pLKO-sh SAM68 was generated by annealing and inserting the oligos, sh SAM68 -F and sh SAM68 -R, at AgeI-EcoRI restriction sites of pLKO.1 (Addgene plasmid #8453).

Techniques: Transfection, Negative Control, Immunoprecipitation, Western Blot, Sequencing, Construct, Binding Assay

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: Tyrosine-rich (YY) domain of SAM68 mediates the interaction with U1 snRNP via YXXY repeated motif. ( A ) Overlay of the 2D 15 N- 1 H HSQC spectra of GB1-hSAM68 (C2) recorded before and after the addition of unlabeled GB1-U1A. The spectra are colored according to the molar ratio hSAM68 (C2):U1A (1:0; 1:0.6 and 1:1.4 are colored in blue, red and black, respectively). Strongly perturbed signals are marked by red arrows and their assignment is indicated. ( B ) Overlay of the 2D 15 N- 1 H HSQC spectra of U1A RRM1 recorded before and after the addition of unlabeled GB1-hSAM68 (C2). The spectra are colored according to the molar ratio U1A RRM1:hSAM68 (C2) (1:0; 1:0.6 and 1:1.4 are colored in blue, red and black, respectively). ( C ) Plot of the normalized chemical shift perturbations observed in panel (B) in function of the sequence of U1A RRM1. The chemical shift perturbations are then plotted onto the surface representation of the structure of the free form of the RRM1 of U1A . Amino acids that experienced chemical shift perturbation between 0.03 and 0.05 are colored in orange while the CSP higher than 0.05 are colored in red.

Article Snippet: Due to the high sequence homology between human and mouse SAM68 , both constructs were produced using EcoRI- SAM68 -F and NotI- SAM68 -R. Flag tag was then inserted at the C-terminus of SAM68 by annealing the oligos, SacI-Flag-F and SacI-Flag-R, and inserting the adaptor at SacI sites of the plasmid. pGEX-6P2-U1A-His was generated by inserting U1A cds, obtained by polymerase chain reaction (PCR) from HEK-293T total cDNA using EcoRI-U1A-F and SalI-U1A-R at EcoRI-SalI sites. cDNA was amplified from total RNA of HEK-293T using Superscript VILO Master mix (Invitrogen). pGEX-6P2-U1C-His was sub-cloned by PCR from pGEX-2TK-U1C using EcoRI-U1C-F and XhoI-U1C-R and inserted at EcoRI-XhoI sites. pGEX-6P2-U1–70K was sub-cloned by PCR from pINTO-NSA:hSNRNP70 using EcoRI-U1–70K-F and XhoI-U1–70K-R and inserted at EcoRI-XhoI sites. pcDNA-Flag- SAM68 and pcDNA-Flag- Sam68 were constructed by inserting corresponding cds, obtained by PCR from pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag using EcoRI- SAM68 -F and NotI- SAM68 -R at EcoRI-NotI sites. pcDNA-Flag- SAM68 I184N and pcDNA-Flag- SAM68 V229F were generated by swapping the 679 bp, AgeI-XbaI fragment from pcDNA mCherry- SAM68 I184N and pcDNA mCherry- SAM68 V229F , respectively, to pcDNA-Flag- SAM68 WT. pcDNA-Flag- SAM68 -Nter and pEGFP- Sam68 -Nter were generated by deletion PCR with SAM68 -F and SAM68- Nter-R primers using pcDNA-Flag- SAM68 and pEGFP- SAM68 as templates, respectively. pcDNA-Flag- SAM68 -Cter was generated from pcDNA-Flag- SAM68 by PCR using EcoRI- SAM68 -Cter-F and NotI- SAM68 Cter-R and cloning the amplicon at EcoRI-SalI sites of pEGFP-C1. pEGFP-C1- SAM68 -C1 was obtained by deletion PCR using SAM68 -F and SAM68 -C1-R, respectively. pEGFP- SAM68 -C2 to C5 were generated by cloning the PCR amplicons obtained using the reverse primer, SalI- SAM68 -R and forward primers namely EcoRI- SAM68- C2, C3, C4, C5 at EcoRI-SalI sites of pEGFP-C1. pEGFP- SAM68 -NLS was obtained by deletion PCR using EcoRI- SAM68 -NLS-F and EcoRI- SAM68 -NLS-R. pLKO-sh SAM68 was generated by annealing and inserting the oligos, sh SAM68 -F and sh SAM68 -R, at AgeI-EcoRI restriction sites of pLKO.1 (Addgene plasmid #8453).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: Both SAM68 and intronic enhancer sequences in mTor intron 5 are required for U1A recruitment to 5′SS in vitro . ( A ) Schematic representation of the various in vitro transcribed mTor minigene baits with the 5′ splice site. As shown, the baits span from last 7 nucleotides of exon5 to the poly-adenylation signal in intron 5. WT refers to the wild-type intronic SAM68-binding sequences of SB-1 (UUUUAU) and SB-A (UAAAA), the latter is embedded in the cryptic poly-adenylation signal (AAUAAA). The ‘mut’ denotes the combined mutations of SB-1 (UUUUAU to UUUCAU) and SB-A (AAUAAAA to AAUAACC). ( B ) SAM68 recruits U1A to 5′ splice site in vitro . Recombinant in vitro purified hSAM68-Flag was tested for its ability to recruit U1A to mTor intron 5 baits with either WT or mutated SAM68-binding sites. GST-Flag was used as negative control. ( C ) Schematic representation of the various in vitro transcribed mTor minigene baits that are deleted for the 5′ splice site. As shown, the baits span 18 nucleotides downstream of the 5′ splice site to the poly-adenylation signal of intron 5. WT refers to the wild-type intronic SAM68-binding sequences, SB-1 (UUUUAU) and SB-A (UAAAA). The ‘mut’ denotes the combined mutations of SB-1 (UUUUAU to UUUCAU) and SB-A (AAUAAAA to AAUAACC). ( D ) SAM68 recruits U1A in the absence of 5′ splice site in vitro . Recombinant in vitro purified hSAM68-Flag was tested for its ability to recruit U1A to mTor intron 5 baits lacking 5′SSs with either WT or mutated SAM68-binding sites. GST-Flag was used as negative control. ( E ) Schematic representation of the in vitro transcribed mTor minigene bait and the primers used for the RppH/Xrn1 protection assays. ( F ) Assessment of the processivity of RppH and Xrn1 enzyme on the naked mRNA bait, showing that RppH treatment is necessary for Xrn1-mediated degradation of the mRNA bait. ( G ) RppH and Xrn1 protection assays in vitro produced mRNA bait incubated with either WT MEFs cell lysate (lane 1), Sam68 −/− MEFs cell lysate (lane 2), in vitro produced mSAM68(WT) + Sam68 −/− MEFs cell lysate (lane 3) or in vitro produced mSAM68(WT) + Sam68 −/− MEFs cell lysate + U1 nRNAs antisense oligo (lane 4). U1snRNP components (U1A, U1C) and mSAM68 levels were assessed by western blot, while U1 snRNA levels was assessed by RT-PCR. GAPDH served as loading control for the western blot. ( H ) SAM68 protects the mTor RNA bait from Xrn1 degradation. Biotinylated RNA baits were incubated with buffer (lane 1), 100 ng of GST-Flag (lane 2) or 100 ng of mSAM68-Flag (lane 3) for 30 min on ice. Sam68 levels were assessed by western blotting using anti-Flag, while baits levels were measured by semi-quantitative RT-PCR using FSS-RSB primers for the full-length RNA and FSB-RSB for the SAM68 protected fragment.

Article Snippet: Due to the high sequence homology between human and mouse SAM68 , both constructs were produced using EcoRI- SAM68 -F and NotI- SAM68 -R. Flag tag was then inserted at the C-terminus of SAM68 by annealing the oligos, SacI-Flag-F and SacI-Flag-R, and inserting the adaptor at SacI sites of the plasmid. pGEX-6P2-U1A-His was generated by inserting U1A cds, obtained by polymerase chain reaction (PCR) from HEK-293T total cDNA using EcoRI-U1A-F and SalI-U1A-R at EcoRI-SalI sites. cDNA was amplified from total RNA of HEK-293T using Superscript VILO Master mix (Invitrogen). pGEX-6P2-U1C-His was sub-cloned by PCR from pGEX-2TK-U1C using EcoRI-U1C-F and XhoI-U1C-R and inserted at EcoRI-XhoI sites. pGEX-6P2-U1–70K was sub-cloned by PCR from pINTO-NSA:hSNRNP70 using EcoRI-U1–70K-F and XhoI-U1–70K-R and inserted at EcoRI-XhoI sites. pcDNA-Flag- SAM68 and pcDNA-Flag- Sam68 were constructed by inserting corresponding cds, obtained by PCR from pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag using EcoRI- SAM68 -F and NotI- SAM68 -R at EcoRI-NotI sites. pcDNA-Flag- SAM68 I184N and pcDNA-Flag- SAM68 V229F were generated by swapping the 679 bp, AgeI-XbaI fragment from pcDNA mCherry- SAM68 I184N and pcDNA mCherry- SAM68 V229F , respectively, to pcDNA-Flag- SAM68 WT. pcDNA-Flag- SAM68 -Nter and pEGFP- Sam68 -Nter were generated by deletion PCR with SAM68 -F and SAM68- Nter-R primers using pcDNA-Flag- SAM68 and pEGFP- SAM68 as templates, respectively. pcDNA-Flag- SAM68 -Cter was generated from pcDNA-Flag- SAM68 by PCR using EcoRI- SAM68 -Cter-F and NotI- SAM68 Cter-R and cloning the amplicon at EcoRI-SalI sites of pEGFP-C1. pEGFP-C1- SAM68 -C1 was obtained by deletion PCR using SAM68 -F and SAM68 -C1-R, respectively. pEGFP- SAM68 -C2 to C5 were generated by cloning the PCR amplicons obtained using the reverse primer, SalI- SAM68 -R and forward primers namely EcoRI- SAM68- C2, C3, C4, C5 at EcoRI-SalI sites of pEGFP-C1. pEGFP- SAM68 -NLS was obtained by deletion PCR using EcoRI- SAM68 -NLS-F and EcoRI- SAM68 -NLS-R. pLKO-sh SAM68 was generated by annealing and inserting the oligos, sh SAM68 -F and sh SAM68 -R, at AgeI-EcoRI restriction sites of pLKO.1 (Addgene plasmid #8453).

Techniques: In Vitro, Binding Assay, Recombinant, Purification, Negative Control, Produced, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: U1snRNP is recruited in a SAM68-dependent manner at the exon5–intron 5 junction (ei5) in mTor pre-mRNA. ( A ) RNA immunoprecipitation (RIP) assay of mSAM68 on mTor pre-mRNA. (Top) Schematic representation of mTor pre-mRNA showing location of amplicon used to detect U1 snRNP binding by RIP (ei4 in red, ei5 in green and ei37 in black). (Below) U1A-RIP was done from WT MEFs or Sam68 −/− MEFs using anti-U1A or control IgG antibodies. Bound RNA was analyzed by RT-qPCR using the highlighted primers. Mean values are expressed as fold enrichment over input and normalized to WT signal. Error bars represent ± standard deviations of the means. ( B ) U1snRNP recruitment is restored at mTor EI5, in Sam68 −/− MEFs expressing mSam68(WT) but not with mSam68(ΔArm) . U1A-RIP was done using anti-U1A or control IgG antibodies in Sam68 −/- MEFs, Sam68 −/- MEFs rescued with mSam68(WT) or mSam68(ΔArm) . Bound RNA was analyzed in triplicates by RT-qPCR using the highlighted primers. Mean values are expressed as fold enrichment over input and normalized to WT signals. Error bars represent ± standard deviations of the means.

Article Snippet: Due to the high sequence homology between human and mouse SAM68 , both constructs were produced using EcoRI- SAM68 -F and NotI- SAM68 -R. Flag tag was then inserted at the C-terminus of SAM68 by annealing the oligos, SacI-Flag-F and SacI-Flag-R, and inserting the adaptor at SacI sites of the plasmid. pGEX-6P2-U1A-His was generated by inserting U1A cds, obtained by polymerase chain reaction (PCR) from HEK-293T total cDNA using EcoRI-U1A-F and SalI-U1A-R at EcoRI-SalI sites. cDNA was amplified from total RNA of HEK-293T using Superscript VILO Master mix (Invitrogen). pGEX-6P2-U1C-His was sub-cloned by PCR from pGEX-2TK-U1C using EcoRI-U1C-F and XhoI-U1C-R and inserted at EcoRI-XhoI sites. pGEX-6P2-U1–70K was sub-cloned by PCR from pINTO-NSA:hSNRNP70 using EcoRI-U1–70K-F and XhoI-U1–70K-R and inserted at EcoRI-XhoI sites. pcDNA-Flag- SAM68 and pcDNA-Flag- Sam68 were constructed by inserting corresponding cds, obtained by PCR from pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag using EcoRI- SAM68 -F and NotI- SAM68 -R at EcoRI-NotI sites. pcDNA-Flag- SAM68 I184N and pcDNA-Flag- SAM68 V229F were generated by swapping the 679 bp, AgeI-XbaI fragment from pcDNA mCherry- SAM68 I184N and pcDNA mCherry- SAM68 V229F , respectively, to pcDNA-Flag- SAM68 WT. pcDNA-Flag- SAM68 -Nter and pEGFP- Sam68 -Nter were generated by deletion PCR with SAM68 -F and SAM68- Nter-R primers using pcDNA-Flag- SAM68 and pEGFP- SAM68 as templates, respectively. pcDNA-Flag- SAM68 -Cter was generated from pcDNA-Flag- SAM68 by PCR using EcoRI- SAM68 -Cter-F and NotI- SAM68 Cter-R and cloning the amplicon at EcoRI-SalI sites of pEGFP-C1. pEGFP-C1- SAM68 -C1 was obtained by deletion PCR using SAM68 -F and SAM68 -C1-R, respectively. pEGFP- SAM68 -C2 to C5 were generated by cloning the PCR amplicons obtained using the reverse primer, SalI- SAM68 -R and forward primers namely EcoRI- SAM68- C2, C3, C4, C5 at EcoRI-SalI sites of pEGFP-C1. pEGFP- SAM68 -NLS was obtained by deletion PCR using EcoRI- SAM68 -NLS-F and EcoRI- SAM68 -NLS-R. pLKO-sh SAM68 was generated by annealing and inserting the oligos, sh SAM68 -F and sh SAM68 -R, at AgeI-EcoRI restriction sites of pLKO.1 (Addgene plasmid #8453).

Techniques: Immunoprecipitation, Amplification, Binding Assay, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: SAM68 deletion of ‘ARM binding region’ shows decrease in U1A binding. ( A ) Schematic of the pcDNA mTor 4–6 minigene, comprising the mTor genomic fragment from exon4 to exon6. ( B ) (Left panel) Sam68 −/- MEFs cells were infected with Sam68 (WT) or Sam68 (ΔARM) and compared to uninfected Sam68 −/- MEFs or WT MEFs. Total RNA was extracted in each cell lines and semi-quantitative RT-PCRs were performed using endogenous mTor specific primers. Forward (Fe4) and Reverse (Re6) were used to quantify mTor transcripts that were spliced normally (mTor 4–6 ), while Forward (Fe4) and Reverse (Ri5) were used to quantify intron 5 including mTor transcripts ( mTor i5 ). Gapdh was used to normalize the values obtained. Total protein was also extracted and run on 10% SDS-PAGE and blotted with SAM68, U1A and GAPDH antibodies. (Right panel) Quantification of intron 5-induced termination over normally spliced mRNA based on three independent experiments using endogenous mTor specific primers. ** P -value ≤ 0.05 and # = non-significant (two-tailed t -test).

Article Snippet: Due to the high sequence homology between human and mouse SAM68 , both constructs were produced using EcoRI- SAM68 -F and NotI- SAM68 -R. Flag tag was then inserted at the C-terminus of SAM68 by annealing the oligos, SacI-Flag-F and SacI-Flag-R, and inserting the adaptor at SacI sites of the plasmid. pGEX-6P2-U1A-His was generated by inserting U1A cds, obtained by polymerase chain reaction (PCR) from HEK-293T total cDNA using EcoRI-U1A-F and SalI-U1A-R at EcoRI-SalI sites. cDNA was amplified from total RNA of HEK-293T using Superscript VILO Master mix (Invitrogen). pGEX-6P2-U1C-His was sub-cloned by PCR from pGEX-2TK-U1C using EcoRI-U1C-F and XhoI-U1C-R and inserted at EcoRI-XhoI sites. pGEX-6P2-U1–70K was sub-cloned by PCR from pINTO-NSA:hSNRNP70 using EcoRI-U1–70K-F and XhoI-U1–70K-R and inserted at EcoRI-XhoI sites. pcDNA-Flag- SAM68 and pcDNA-Flag- Sam68 were constructed by inserting corresponding cds, obtained by PCR from pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag using EcoRI- SAM68 -F and NotI- SAM68 -R at EcoRI-NotI sites. pcDNA-Flag- SAM68 I184N and pcDNA-Flag- SAM68 V229F were generated by swapping the 679 bp, AgeI-XbaI fragment from pcDNA mCherry- SAM68 I184N and pcDNA mCherry- SAM68 V229F , respectively, to pcDNA-Flag- SAM68 WT. pcDNA-Flag- SAM68 -Nter and pEGFP- Sam68 -Nter were generated by deletion PCR with SAM68 -F and SAM68- Nter-R primers using pcDNA-Flag- SAM68 and pEGFP- SAM68 as templates, respectively. pcDNA-Flag- SAM68 -Cter was generated from pcDNA-Flag- SAM68 by PCR using EcoRI- SAM68 -Cter-F and NotI- SAM68 Cter-R and cloning the amplicon at EcoRI-SalI sites of pEGFP-C1. pEGFP-C1- SAM68 -C1 was obtained by deletion PCR using SAM68 -F and SAM68 -C1-R, respectively. pEGFP- SAM68 -C2 to C5 were generated by cloning the PCR amplicons obtained using the reverse primer, SalI- SAM68 -R and forward primers namely EcoRI- SAM68- C2, C3, C4, C5 at EcoRI-SalI sites of pEGFP-C1. pEGFP- SAM68 -NLS was obtained by deletion PCR using EcoRI- SAM68 -NLS-F and EcoRI- SAM68 -NLS-R. pLKO-sh SAM68 was generated by annealing and inserting the oligos, sh SAM68 -F and sh SAM68 -R, at AgeI-EcoRI restriction sites of pLKO.1 (Addgene plasmid #8453).

Techniques: Binding Assay, Infection, SDS Page, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: SAM68 is up-regulated in Lung adenocarcinoma (LUAD) and correlates with a poor prognosis for LUAD patients. (A~C) SAM68 mRNA levels was up-regulated in LUAD compared to normal lung tissue based on the Oncomine, GEO and TCGA database. (D~G) From TCGA LUAD specimen cohorts, compared with the patients with low expression level of SAM68 , the patients with high mRNA expression of SAM68 had higher recurrence rates and death rates, shorter RFS, and OS. (H~J) The SAM68 mRNA and protein levels were detected by qPCR, RT-PCR and Western blot in the LUAD tissues (T) and their corresponding adjacent non-tumoral tissues (N). (K) IHC analysis of the expression of SAM68 protein was represented in LUAD tissues and the corresponding adjacent normal lung tissues. (L) Differences in expression levels of SAM68 protein in LUAD tissues (n = 50) and adjacent normal lung tissues (n = 50). Two-tailed t-tests were used B, C, H and L. Pearson Chi-Square tests were used in D and F.Log rank tests were used in E and G.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Downregulation of SAM68 inhibited the malignant phenotypes of LUAD cells in vitro and tumorigenesis and progression in vivo . (A and B) NCI-H1975 and A549 cells were transfected with anti-SAM68 siRNAs, SAM68 mRNA (A) and protein levels (B) level were determined by qPCR and Western blot, respectively. (C) After SAM68 silencing, SAM68 was immuno-stained with anti-SAM68 antibody in NCI-H1975 cells. (D-F) The effects of SAM68 silencing on NCI-H1975 and A549 cells growth (D), colony formation (E), and migration and invasion (F) were detected. (G) CRISPR-Cas9 mediated knockout (KO) of SAM68 in NCI-H1975 cells as detected by immunofluorescence. (H) The in vivo growth of SAM68 KO (sgSAM68) NCI-H1975 cellswas detected (n = 6). (I)The in vivo lung metastasis of SAM68 KO NCI-H1975 cells was examined (n = 5). (J and K) The hematoxylin and eosin (HE) staining and Kaplan-Meier curves are shown for two cohorts of transplanted mice carrying SAM68 KO cells and control groups. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001. Two-tailed t-tests were used A, E, F and I. Two-way ANOVA was used in D. Log rank tests was used in L.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: In Vitro, In Vivo, Transfection, Western Blot, Staining, Migration, CRISPR, Knock-Out, Immunofluorescence, Control, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: The 351~443 motif of SAM68 binds to the Glycine residues in RGG box of hnRNP A1. (A) HEK293T cells were transfected with Flag-SAM68. After Co-IP, proteins that interacted with Flag-SAM68 were identified using silver staining combined mass spectrometry. (B) The SAM68-interacting proteins were performed by GO analysis. (C and D) The unique peptide of SAM68 (C) and hnRNP (D) A1 were identified by mass spectrometry. (E and F) Flag-SAM68 and HA-hnRNP A1 plasmid were transfected into NCI-H1975 cells, cellular lysates were treated with 10 mg/mL RNase A (Thermofisher, EN0531) for 1 h or no treatment, Flag-SAM68 complexes were co-immunoprecipitated by anti-Flag antibody, then hnRNP A1 was detected (E), and HA-hnRNP A1 complexes were co-immunoprecipitated by anti-HA antibody, then SAM68 was detected (F). (G) Confocal images of endogenous SAM68 and hnRNP A1 in NCI-H1975 cells. (H) Wild-type (WT) and indicated mutations with the different domain of hnRNP A1 were constructed. (I and J) The indicated HA-hnRNP A1 WT and mutation constructs and Flag-SAM68 were co-transfected into HEK293T cells, Flag-SAM68 and HA-hnRNP A1 complexes were co-immunoprecipitated by anti-Flag and HA antibody, respectively; HA-hnRNP A1 mutants were detected using anti-HA antibodies, and Flag-SAM68 were detected using anti-Flag antibodies. (K and L) The HA-hnRNP A1 WT or its RAA mutation with Flag-SAM68 were co-transfected into HEK293T cells, and the interactions of hnRNP A1 RAA mutant with Flag-SAM68 were detected as described in (I) and (J). (M and N) The Flag-SAM68 WT or its indicated mutation with HA-hnRNP A1 were co-transfected into HEK293T cells, the interactions of Flag-SAM68 indicated mutation with HA-hnRNP A1 were detected as described in (I) and (J).

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Transfection, Co-Immunoprecipitation Assay, Silver Staining, Mass Spectrometry, Plasmid Preparation, Immunoprecipitation, Construct, Mutagenesis

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Silencing hnRNP A1 antagonized the enhancement of malignant phenotypes induced by SAM68 overexpression. (A~D) The anti-hnRNP A1 siRNAs were transfected into NCI-H1975 and A549 cells, hnRNP A1 protein levels (A) level was detected by Western blot, and the effects of silencing hnRNP A1 on NCI-H1975 and A549 cells growth (B), colony formation (C), migration and invasion (D) were detected. (E~H) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975 and A549 cells, the indicated protein levels (E), cell growth (F), colony formation (G), migration and invasion (H) were detected. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001. Two-way ANOVA were used in B and F, two-tailed t-tests were used C, D, G and H.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Over Expression, Transfection, Western Blot, Migration, Plasmid Preparation, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: The 351~443 motif of SAM68 favors the binding of the RGG motif of hnRNP A1 to the intronic sequences flanking exon 9 (EI9) by binding to the Glycine residues in RGG box of hnRNP A1. (A) Quantification of SAM68-regulated AS events in each category was measured RNA sequencing. (A3SS/A5SS, alternative 3'/5' splice sites, MXE, mutually exclusive exons, RI, retained introns, SE, skipped exons) (B) Changes in PSI values of SAM68-regulated AS events were shown. (C) SAM68 regulated PKM pre-mRNA splicing and promoted PKM2 isoform formation. (D) CLIP assay of SAM68 and hnRNP A1 binding to the PKM pre-mRNA. NCI-H1975 cells were UV-crosslinked and immunoprecipitated with control IgGs or antibodies, as indicated. (E) RNA affinity purification followed by Western blot showed in vitro binding of the indicated biotin-labeled RNAs with endogenous hnRNP A1 or SAM68. (F) The Flag-SAM68 plasmid at the indicated doses was transfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68). (G) NCI-H1975 cells were co-transfected with Flag-SAM68 plasmid and the indicated hnRNP A1 mutations, and in vitro binding of EI9 (50-68) RNA probes with WT or the indicated hnRNP A1 mutations was detected. (H) The HA-hnRNP A1 WT or its RAA mutation was transfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68). (I) HA-hnRNP A1 WT or its RAA mutation with Flag-SAM68 plasmids were co-transfected into NCI-H1975 cells, and in vitro binding of EI9 (50-68) RNA probes with WT or its RAA mutation was detected. (J) The WT SAM68 or its indicated mutant with WT hnRNP A1-HA plasmids were cotransfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68).

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Binding Assay, RNA Sequencing, Immunoprecipitation, Control, Affinity Purification, Western Blot, In Vitro, Labeling, Plasmid Preparation, Transfection, Mutagenesis

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Sam68 and hnRNP A1 cooperated in PKM splicing to decrease PKM1 isoform formation and increase PKM2 isoform formation, then promoting the aerobic glycolysis of LUAD cells. (A and B) NCI-H1975 cells were transfected with the Flag-SAM68 plasmids (A) or anti-SAM68 siRNAs (B), followed by the PKM splicing assay. (C) PKM splicing was performed in SAM68 KO NCI-H1975 cells. (D) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975, followed by the PKM splicing assay. (E) PKM splicing was performed in the LUAD tissue samples with low (n = 6) and high (n = 6) SAM68 expression. (F) The ratio of PKM2/PKM1 is calculated in the LUAD tissue samples with low (n = 6) and high (n = 6) SAM68 expression (G and H) NCI-H1975 cells were transfected with the Flag-SAM68 plasmids, glucose uptake and lactate production were measured. (I and J) Glucose uptake and lactate production were measured in SAM68 KO NCI-H1975 cells. (K and L) NCI-H1975 cells were transfected with anti- hnRNP A1 siRNAs, then glucose uptake (K) and lactate production (L) were detected. (M and N) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975, and glucose uptake (M) and lactate production (N) were detected. (O and P) The Flag-SAM68 plasmid and anti-PKM2 siRNAs were co-transfected into NCI-H1975, and glucose uptake (O) and lactate production (P) were detected.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Transfection, Splicing Assay, Plasmid Preparation, Expressing

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: SAM68 increases PKM2 isoform formation, and promotes malignant phenotypes and aerobic glycolysis by cooperating to hnRNP A1 . The WT SAM68 or its indicated mutant was transfected into SAM68 KO NCI-H1975 cells; the indicated proteins were detected by Western blot (A), and cell growth (B), colony formation (C and D), migration and invasion (E), PKM splicing (F), glucose uptake (G), and lactate production (H) were detected. (I) A regulatory model of SAM68 on tumorigenesis proposed in this study. Data are represented as mean ± SEM. **p < 0.01 or ***p < 0.001. Two-way ANOVA was used in B; two-tailed t-tests were used in D~ H.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Mutagenesis, Transfection, Western Blot, Migration, Two Tailed Test

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: Arginine methylation of HNRNPUL2, KHDRBS1(SAM68) and SRSF1 is reduced in HD ISPNs ( A ) Total cell lysates from non-differentiated control (33CAG) and HD (180CAG) ISPNs were prepared as described in the Experimental Section. IPs with a mixture of mono-methyl (MMA) and asymmetric dimethyl (ADMA) antibodies were performed followed by western blot analysis with indicated protein-specific antibodies. Protein bands were visualized and quantified using the Licor System and Image Studio software. ( B ) – ( D ) Graphs (left) show the mean intensity values (±SEM) of the signal detected with antibodies to total HNRNPUL2, KHDRBS1(SAM68) and SRSF1 in the IPs normalized to the signal obtained from input lysates with the same antibodies. Total protein levels normalized to β-tubulin were also quantified (right graphs). n = 3 (biological replicates), Normality Test (Shapiro–Wilk): Passed, Equal Variance Test: Passed. T -test with equal variances was performed: (B) * 33CAG versus 180CAG, P 0.04. (C) * 33CAG versus 180CAG, P 0.0046. * * 33CAG versus 180CAG, P 0.0039.

Article Snippet: FLAG-tagged HNRNPUL2 and KHDRBS1 expression plasmids were obtained from GenScript.

Techniques: Methylation, Control, Western Blot, Software

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: RNA association of hypomethylated RBPs is decreased in HD cells. RNA-binding assay in ISPNs. RNA pull-downs were performed as described in the Experimental Section according to Castello et al . (2013) . ( A ) Validation of RNA-binding assay in ISPNs demonstrating detection of indicated RNA-bound proteins in cross-linked cells (but not in the eluates of nonirradiated control cells) with protein-specific antibodies. ( B ) The amounts of mRNA within RNA-protein complexes were estimated by absorbance at A260, and equal amounts were analyzed by western blotting with indicated protein-specific antibodies. Representative experiment is shown. ( C ) – ( G ) Quantitation of blots shown in (B). Graphs show the mean intensity values (±SD) of the signal with antibodies to indicated proteins detected in the RNA-bound fraction normalized to the signal obtained from input lysates with the same antibodies. n = 3 (biological replicates). Normality test (Shapiro–Wilk): Passed, equal variance test: Passed. One-way ANOVA was performed with pairwise multiple comparison procedures (Holm–Sidak method). (C) HNRNPUL2, * 33EE versus 180, P 0.002; * * 33DG versus 180, P 0.002. (D) KHDRBS1, * 33EE versus 180, P 0.004; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001. (E) FUS, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.008. (F) SRSF1, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.011. (G) TAF15, * 33EE versus 180, P < 0.001; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001.

Article Snippet: FLAG-tagged HNRNPUL2 and KHDRBS1 expression plasmids were obtained from GenScript.

Techniques: RNA Binding Assay, Biomarker Discovery, Control, Western Blot, Quantitation Assay, Comparison

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Western blot analysis of endogenous SAM68 expression in siRNA-transfected cells with densitometric quantification indicated below (N=3). Statistics: p-values: **** <0.0001. Figure 1—figure supplement 1—source data 1. Western blot uncropped membranes.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Western Blot, Expressing, Transfection

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) HUVECs plated on FN-coated coverslips for 20 min were stained for SAM68, F-actin, cortactin and phospho-tyrosine. Scale bars=10 μm. Dotted squares depict enlarged areas (10 μm wide) shown in the same panel. ( B ) Labelling of SAM68 and FAK was performed on HUVECs plated on FN-coated coverslips for the indicated times; dotted squares depict enlarged areas shown in the same panel.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Staining

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) Scheme of the experimental procedure used to induce and image artificial adhesion sites in contact with FN-coated beads. ( B ) Labelling of SAM68 and vinculin was performed 20 min after seeding cells on FN-coated beads. Dotted squares in top panels depict enlarged z-projections shown in the middle panel. Orthogonal views are shown in bottom panels. Scale bars=5 μm. ( C ) Immunolabeling of α5β1 and activated FAK (pFAK-Y397) were performed 20 min after deposition of FN-coated beads onto siCTRL- or siSAM68-transfected cells and pFAK-Y397 foci were quantified (n=at least 8 beads per condition, N=3). ( D ) siSAM68-transfected cells were transduced with lentiviral constructions encoding SAM68 WT and mutants shown in the left panel of the figure. Immunolabeling of activated FAK (pFAK-Y397) was performed 20 min after deposition of FN-coated beads onto cells and pFAK-Y397 foci were quantified (n=at least 5 beads per condition, N=4). Statistics: p-values: *<0.05 **<0.01. Student’s t-test (paired CTRL-siSAM68) was used for ( C, D ). Figure 3—source data 1. Quantification of pFAK397 foci from . Figure 3—source data 2. Quantification of pFAK397 foci from .

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Immunolabeling, Transfection, Transduction

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) smRNA FISH and SAM68 stainings were performed 20 min after deposition of FN-coated beads. Scale bars top image=10 μm, enlarged area 5 μm. Arrowheads point to overlapping signals between β-actin mRNA and SAM68 protein. ( B ) smRNA FISH of β-actin performed on cells 20 min after addition of FN-coated beads to cultures of siCTRL- or siSAM68-transfected cells (n=at least 12 beads per condition, N=3). Scale bars = left 10 μm, enlarged area 5 μm. ( C ) smiRNA FISH staining of β-actin performed on cells 20 min after deposition of FN-coated beads onto endothelial cells transfected with CTRL or blocking oligonucleotides (#SBE1 and #SBE2, as indicated) (n=at least 12 beads per condition, N=3). Statistics: p-values: *<0.05 **<0.01. Student’s t-test (paired CTRL-siSAM68) was used. Figure 4—source data 1. Quantification of pFAK397 foci from . Figure 4—source data 2. Quantification of pFAK397 foci from .

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Transfection, Staining, Blocking Assay

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) Immunofluorescence staining of α5β1 integrin was performed to identify and quantify fibrillar adhesion in siRNA transfected cells plated overnight on glass coverslips (n=at least 15; N=3). ( B ) Immunofluorescence staining of FN was performed on siRNA transfected cells plated on glass coverslips and quantification on whole-coverslip scans is expressed as the ratio of FN-stained area to the number of cells (N=8). Representative 40x field views. ( C ) Representative high magnification images of FN staining are shown with areas between the dotted lines selected for fluorescence intensity profiles. ( D ) Western blot analysis of cell-associated FN in siRNA transfected cells with densitometric quantification indicated below (N=3). ( E ) qPCR analysis of total FN (tFN) and Extra Domain-containing isoform expression in siRNA transfected cells using the indicated qPCR primer pairs (N=7). ( F ) Measurements of Luciferase activity driven by the FN1 promoter when SAM68 is overexpressed (N=5). ( G ) DNA fragments located in the FN1 promoter were quantified by qPCR in anti-SAM68 or IgG immunoprecipitated complexes (N=3). Statistics: p-values: *<0.05 **<0.01 ***<0.001 ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for ( A, B, D, E, F ). Statistical analysis of fold enrichment in ( G ) was performed with R using pairwise t-test with p-values adjusted using ‘Bonferroni correction’. Figure 5—source data 1. Quantification of endothelial fibrillar adhesions. Figure 5—source data 2. Western blot uncropped membranes. Figure 5—source data 3. Quantification of FN1 mRNA levels. Figure 5—source data 4. Quantification of FN1 promotor reporter activity. Figure 5—source data 5. Quantification of SAM68 protein recruitment onto the FN1 promotor.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Immunofluorescence, Staining, Transfection, Fluorescence, Western Blot, Expressing, Luciferase, Activity Assay, Immunoprecipitation

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Migration of individual siRNA-transfected cells was analyzed by time lapse microscopy. ( A ) Representation of individual tracks of the experiment presented in for the second siRNA directed against SAM68. ( B ) Additional quantification of average speed and total distance travelled are shown (N=3). Statistics: p-values: *<0.05. Student’s t-test (paired CTRL-siSAM68) was used.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Migration, Transfection, Time-lapse Microscopy

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Distribution of average sprout length per bead observed in the 3D angiogenesis assay presented in . Results from two SAM68-targeting siRNAs in four different experiments are shown (N1 to N4).

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Angiogenesis Assay

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Upon integrin activation, a cytoplasmic fraction of SAM68 participates transiently in adhesion complex stabilization, through regulation of integrin signaling and localization of β-actin mRNA. In the nucleus, SAM68 concomitantly regulates the expression of key subendothelial matrix genes, thereby promoting basement membrane assembly and conditioning. These coalescent functions of SAM68 enhance endothelial cell adaptation to their microenvironment and point to an important role for SAM68 during angiogenesis.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Activation Assay, Expressing, Membrane

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet:

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Transduction, Sequencing, Control, Blocking 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

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left ; and IBRS2, right ) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 ( red ). Nuclei were stained with DAPI ( blue )

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Infection, Staining

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: FMDV-induced cytoplasmic Sam68 co-localizes with TIA-1. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and were fixed at 3 and 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and goat polyclonal anti-TIA-1 ( a ) or mouse monoclonal anti-G3BP ( b ) followed by donkey-anti-goat-AF568 ( red , a ) or goat-anti-mouse-AF568 ( red ; b ). Nuclei were stained with DAPI ( blue )

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Infection, Staining

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 interacts with FMDV IRES 4. a Cartoon diagram in the upper panel describes the modular structure of FMDV A24 IRES and the location of two unpaired UAAA and a CAAA sequence motifs in domain 4 and 3, respectively. Sam68 potential binding sites are shown as a grey incomplete oval. Lower panel in Fig. 3a shows anti-Sam68 Western blot (rabbit anti-Sam68) of pull-down experiments conducted between Sam68 and selected IRES domains. IRES domains used in the experiment are shown. b Determination Sam68 binding to FMDV IRES RNAs by EMSA. WT probe in the left panel consists of 5′ biotin labeled 65 nt long synthetic RNA representing residues 435–499 of FMDV A24-Cru IRES that spanned at least 20 bases upstream and downstream of the two UAAA sequence motifs present in IRES domain 4. The binding of Sam68 to RNA probe was carried out in the presence of 100-fold excess of tRNA. The concentrations of Sam68 used are indicated in each lane. In the mutant probe in the right panel, the two UAAA motifs aremutated to UACG. c Upper panel depicts cartoon representation of the wild-type and KH-domain deleted Sam68 constructs. Lower panel shows EMSA results with the addition of Sam68-WT (left) and Sam68-delta KH (right). Probe and conditions used were the same as in section ( b ). d Determination of binding interference by various 5′ NTR RNA segments on the complexes formed between WT probe representing partial FMDV IRES domain 4 and Sam68. The binding of Sam68 to WT probe was performed under similar conditions as mentioned in section ( b ) but using a 2 μM Sam68 and 30 nM of probe. WT probe-Sam68 binding was competed with 10-fold molar excess of either full-length IRES (lane 3) or miscellaneous RNAs, including the FMDV cre (lane 4), S-fragment (lane 5), and IRES domains 2, 3, 4 (lanes 6, 7, 8, respectively). Lane 1 contains the binding mixture of Sam68 and domain 4 RNA in the absence of competitor RNAs, whereas lane 9 contains a probe alone control. Lane 2 was left blank

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Sequencing, Binding Assay, Western Blot, Labeling, Mutagenesis, Construct, Control

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Oligonucleotides used in this study

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Sequencing

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Effect of Sam68-depletion on FMDV protein and RNA synthesis using cell-free extracts. a Depletion of Sam68 from BHK-21 CFE. BHK-21 CFE was prepared as described in Materials and Methods. The depletion of Sam68 was confirmed by Western blot probing of non-depleted ( left lane ) and depleted ( right lane ) extract with anti-Sam68. b Determination of the effect of Sam68-6H addition on the translation of FMDV A 24 -Cru. FMDV A 24 -Cru RNA was translated using non-depleted or depleted BHK-21 CFE that were supplemented with 0 or 1 μM Sam68-6H as marked. The reaction was carried out at 32 ° C for 2 h and the products were resolved by SDS-PAGE and Western blot probed for FMDV 3D pol . c Determination of the effect of Sam68-6H addition on the synthesis of FMDV A 24 -Cru RNA. FMDV A 24 -Cru RNA was used for RNA synthesis using non-depleted or depleted BHK-21 CFE that were supplemented with 0–2.5 μM Sam68-6H as marked. The reaction was carried out at 37 ° C for 5 h and the products were SDS-PAGE resolved by dot blotting

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Western Blot, SDS Page

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 interacts with FMDV 3C pro and 3D pol . a Co-immunoprecipitation of FMDV 3D pol and Sam68 during FMDV infection. BHK-21 cells either mock-infected or infected with FMDV at a MOI of 10 were lysed and the lysates were immunoprecipitated using either anti-FMDV 3D pol or anti-Sam68 and the eluates examined by Western blot. One lane in each panel (as indicated below) was not subject to IP as a control. Equal amount of isotype control antibody served as an IP control. The eluates from the anti-3D pol IP reaction were probed with anti-Sam68 (left panel). Conversely, the Sam68 IP eluates were probed with an anti-3D pol (right panel). In the left panel, lane 1 corresponds to the isotype IP control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP and lane 4 is the anti-3D pol IP eluate from FMDV-infected cell lysates. Similarly, in the right panel, lane 1 corresponds to the isotype control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP, and lane 4 is the anti-Sam68 IP eluate from FMDV-infected cell lysates. b The fragments (frag) listed in the table correspond to the amino acid (aa) sequence of FMDV 3D pol , starting from the N-terminus: frag #1 aa 1–48, frag #2 aa 49–108, frag #3 aa 109–157, frag #4 aa 158–217, frag #5 aa 218–268, frag #6 aa 269–331, frag #7 aa 332–404, and frag #8 aa 405–470. A scrambled peptide was used as a negative control. c Computational prediction of the interaction between FMDV 3D pol and Sam68. (i) Electrostatic surface representation of FMDV 3D pol in the docking pose (PDB: 1U09); red color depicts the negatively charged surface, white shows the neutral surface, and blue color shows the positively charged surface. Color intensity is proportional to the surface charge. Areas under dashed lines indicate Sam68 binding interface of FMDV 3D pol . (ii) Electrostatic surface representation of Sam68 in the docking pose. Surface charge and color annotation are same as section (i). Surface marked with dashed lines indicates FMDV 3D pol binding interface of Sam68. (iii) Electrostatic representation of Sam68 docked to FMDV 3D pol . FMDV 3D pol green docked on Sam68 blue in cartoon representation. The 3D pol frag-4 residues 193–217 (orange), frag-5 residues 221, 222, 225, 226 (magenta) and frag-8 residues 453–470 (red) form the Sam68 binding interface of 3D pol ( d ) LFBK cells were uninfected or infected with FMDV at a MOI of 10, and cells were harvested at 1, 3, and 5 hpi by treatment with versine. Left panel: cell lysates were IP with mouse monoclonal anti-FMDV 3C pro , and examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Right panel: collected cells were lysed and separated into nuclear and cytoplasmic fractions, and the cytoplasmic fractions were examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Loading control is indicated confirming equivalent loading per lane

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Immunoprecipitation, Infection, Western Blot, Control, Sequencing, Negative Control, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Met Receptors Induce Sam68-dependent Cell Migration by Activation of Alternate Extracellular Signal-regulated Kinase Family Members

doi: 10.1074/jbc.m110.211409

Figure Lengend Snippet: FIGURE 2. Sam68 is required for HGF-induced MAPK-dependent CD44v5 up-regulation and keratinocyte cell migration. A, HaCaT cells were starved for 18 h and treated with either vehicle or 50 ng/ml HGF for 5, 15, and 60 min (top panel) or 15, 30, and 60 min (bottom panel). B, HaCaT cells were starved for 18 h, pretreated with either vehicle or 10 M of the MEK inhibitor U0126 for 30 min, and treated with 50 ng/ml HGF for 15 min. Cellular lysates (A and B) were immunoprecipitated (IP) for Sam68 and immunoblotted (IB) using Sam68-specific antibodies. Normal rabbit IgG was used as specificity control. ERK1/2 activation was evaluated using phospho-specific antibodies; total ERK1/2 served as a loading control. C, cells transiently expressing either control or Sam68 siRNA were starved for 18 h, induced with 50 ng/ml HGF for 4 h, and Western-blotted (IB) with antibodies specific to CD44v5, CD44v6, and Sam68. Total p38 served as a loading control. Densitometry analysis was performed and indicated below the proper lane as a ratio between CD44v5 and p38 intensity or CD44v6 and p38. D, control and Sam68 siRNA-expressing cells were assayed for their ability to migrate in the presence of HGF. * denotes significance (p 0.05) relative to vehicle and between indicated groups, as determined by unpaired Student’s t test.

Article Snippet: Antibodies and Reagents—Antibodies to p38, ERK1/2, Sam68, ERK5, human CD44, Myc-tag and phospho-ERK1/2 (Thr-202/Tyr-204) were purchased from Cell Signaling Technology (Danvers,MA) and used at 1:1000 in 1%milk, except for Sam68 (used at 1:5000).

Techniques: Migration, Immunoprecipitation, Control, Activation Assay, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Met Receptors Induce Sam68-dependent Cell Migration by Activation of Alternate Extracellular Signal-regulated Kinase Family Members

doi: 10.1074/jbc.m110.211409

Figure Lengend Snippet: FIGURE 3. HGF-induced migration requires Sam68 phosphorylation. A, top panel, schematic representation of MAPK consensus (PXX(S/T)P) sites on Myc-tagged WT-Sam68 and phospho-mutant m1 wherein all eight Thr or Ser residues are replaced by Ala (30). Bottom panel, cells were transiently transfected with 2.5 g of vector and 1 and 2.5 g of Myc-tagged wild-type Sam68 or Myc-tagged phospho-mutant m1. After 48 h, cells were assayed for HGF-induced migration in Boyden chambers. * denotes significance (p 0.05) relative to vehicle and between indicated groups, as determined by unpaired Student’s t test. B, Western blots showing Myc-tagged Sam68 and endogenous Sam68 expression; C, HGF-induced activation of ERK1/2. Total ERK1/2 served as a loading control.

Article Snippet: Antibodies and Reagents—Antibodies to p38, ERK1/2, Sam68, ERK5, human CD44, Myc-tag and phospho-ERK1/2 (Thr-202/Tyr-204) were purchased from Cell Signaling Technology (Danvers,MA) and used at 1:1000 in 1%milk, except for Sam68 (used at 1:5000).

Techniques: Migration, Phospho-proteomics, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Expressing, Activation Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Met Receptors Induce Sam68-dependent Cell Migration by Activation of Alternate Extracellular Signal-regulated Kinase Family Members

doi: 10.1074/jbc.m110.211409

Figure Lengend Snippet: FIGURE 4. MAPK signaling mediates Sam68 phosphorylation and migra- tion in MDA-MB-231 breast cancer cells. A, HaCaT and MDA-MB-231 cells were starved for 18 h and treated with either vehicle or 50 ng/ml HGF for 4 h. CD44v5 expression was determined with specific antibodies. Total p38 served as a loading control. B, MDA-MB-231 cells were starved for 18 h, pre- treated with either vehicle or 10 M MEK1/2 inhibitor (U0126) for 30 min, and subsequently treated with 50 ng/ml HGF for 30 min. Sam68 was immunopre- cipitated (IP) from whole cell lysates and then subjected to Western blotting (IB) using Sam68-specific antibodies. Normal rabbit IgG served as a specificity control. Upshifted Sam68 band indicates multisite phosphorylation. C, Boy- den chamber migration assays preformed with MDA-MB-231 stimulated with HGF in the presence or absence of MEK1/2 inhibitor, U0126. Inset shows HGF- induced ERK1/2 activation; ERK1/2 protein levels served as loading control. * denotes significance (p 0.05) relative to vehicle and between indicated groups, as determined by unpaired Student’s t test.

Article Snippet: Antibodies and Reagents—Antibodies to p38, ERK1/2, Sam68, ERK5, human CD44, Myc-tag and phospho-ERK1/2 (Thr-202/Tyr-204) were purchased from Cell Signaling Technology (Danvers,MA) and used at 1:1000 in 1%milk, except for Sam68 (used at 1:5000).

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Migration, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Met Receptors Induce Sam68-dependent Cell Migration by Activation of Alternate Extracellular Signal-regulated Kinase Family Members

doi: 10.1074/jbc.m110.211409

Figure Lengend Snippet: FIGURE 5. ERK5 mediates breast cancer cell migration through ERK5-Sam68 complex dissociation. MDA-MB-231 cells were pretreated with either 10 M U0126 for 30 min (A) or 10 nM or 10 M PD (B) and then treated with either vehicle or 50 ng/ml HGF for 15 min. ERK1/2 and ERK5 activation was evaluated using phospho-specific and total specific antibodies, respectively; total ERK1/2 served as a loading control. C, MDA-MB-231 cells were assayed for migration in response to HGF in the presence or absence of either low (10 nM) or high (10 M) concentrations of PD. D, Boyden migration assays were performed using cells transiently expressing control or ERK5 siRNA (inset shows ERK5 and a nonspecific protein loading control) and stimulated with HGF. * denotes significance (p 0.05) relative to vehicle and between indicated groups, as determined by unpaired Student’s t test. § denotes no statistical difference relative to vehicle. E, cells were treated with either vehicle or 50 ng/ml HGF for 15–30 min. ERK5 was immunoprecipitated (IP) from whole cell lysates using total ERK5-specific antibodies. ERK5 immunoprecipitates were then subjected to Western blotting (IB) with total ERK5 and Sam68-specific antibodies. F, cells were pretreated for 30 min with low (10 nM) or high (10 M) concentration of PD, followed by 15 min of HGF treatment. ERK5 was immunoprecipitated from whole cell lysates using total ERK5-specific antibodies and Western-blotted with ERK5 and Sam68-specific antibodies. ERK1/2 and ERK5 activation was evaluated using phospho-specific and total specific antibodies, respectively.

Article Snippet: Antibodies and Reagents—Antibodies to p38, ERK1/2, Sam68, ERK5, human CD44, Myc-tag and phospho-ERK1/2 (Thr-202/Tyr-204) were purchased from Cell Signaling Technology (Danvers,MA) and used at 1:1000 in 1%milk, except for Sam68 (used at 1:5000).

Techniques: Migration, Activation Assay, Control, Expressing, Immunoprecipitation, Western Blot, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Met Receptors Induce Sam68-dependent Cell Migration by Activation of Alternate Extracellular Signal-regulated Kinase Family Members

doi: 10.1074/jbc.m110.211409

Figure Lengend Snippet: FIGURE 6. Sam68 mediates HGF-induced migration through MAPK-directed phosphorylation sites in breast cancer cells. A, MDA-MB-231 cells express- ing either control or Sam68 siRNA were assayed for HGF-induced migration. Inset, Western blots showing Sam68 knockdown and p38 loading control. B, MDA-MB-231 breast cancer cells were transiently transfected with 2.5 g of vector or 1–2.5 g of Myc-tagged WT- or m1-Sam68 and assayed in Boyden chambers for HGF induced migration. C, expression of Myc-tagged and endogenous Sam68 protein and ERK1/2 activation were detected using specific antibodies; total ERK1/2 served as a loading control. D, MDA-MB-231 cells transiently transfected with 2.5 g of Myc-tagged WT-, m1-, and m4-Sam68 were subjected to HGF-induced Boyden chamber migration assays. Schematic representations of Myc-tagged WT- and m4-Sam68 are shown. Inset, transfected Sam68 protein levels were detected using Myc tag-specific antibodies. E, HaCaT cells were transiently transfected as in D and assayed for migration. * denotes significance (p 0.05) relative to vehicle and between indicated groups, as determined by unpaired Student’s t test. F, endogenous and Myc-tagged WT- and m4-Sam68 and total ERK5 were visualized by Western blotting using specific antibodies. Slower running (higher migrating) Sam68 bands indicate Myc-tagged proteins.

Article Snippet: Antibodies and Reagents—Antibodies to p38, ERK1/2, Sam68, ERK5, human CD44, Myc-tag and phospho-ERK1/2 (Thr-202/Tyr-204) were purchased from Cell Signaling Technology (Danvers,MA) and used at 1:1000 in 1%milk, except for Sam68 (used at 1:5000).

Techniques: Migration, Phospho-proteomics, Control, Western Blot, Knockdown, Transfection, Plasmid Preparation, Expressing, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Met Receptors Induce Sam68-dependent Cell Migration by Activation of Alternate Extracellular Signal-regulated Kinase Family Members

doi: 10.1074/jbc.m110.211409

Figure Lengend Snippet: FIGURE 7. Phospho-Sam68 mediates HGF-induced Met cancer cell migration. A, MDA-MB-435 cells were transiently transfected as described above with vector, WT-, or m1-Sam68 and assayed for HGF-induced migra- tion. B, Western blot showing transfected and endogenous Sam68 protein expression and MAPK activation. Endogenous and Myc-tagged WT- and m4-Sam68 were visualized by Western blotting using Sam68-specific anti- bodies. Slower running bands indicate Myc-tagged proteins. ERK1/2 activa- tion was evaluated using phospho-specific antibodies; total ERK1/2 served as a loading control. * denotes significance (p 0.05) relative to vehicle between indicated groups, as determined by unpaired Student’s t test. C, mechanism of action of Sam68 downstream of activated Met receptors. Sam68 is the common effector of HGF-induced migration in keratinocyte and breast cancer cells. Contrary to ERK1/2- and CD44v5-dependent keratinocyte migration, breast cancer cell migration occurs independently of CD44v5 expression via an ERK1/2- or ERK5-dependent mechanism, suggesting a role for phospho-Sam68 in the regulation of splicing of multiple gene targets important for HGF-induced cell migration.

Article Snippet: Antibodies and Reagents—Antibodies to p38, ERK1/2, Sam68, ERK5, human CD44, Myc-tag and phospho-ERK1/2 (Thr-202/Tyr-204) were purchased from Cell Signaling Technology (Danvers,MA) and used at 1:1000 in 1%milk, except for Sam68 (used at 1:5000).

Techniques: Migration, Transfection, Plasmid Preparation, Western Blot, Expressing, Activation Assay, Control