khdrbs1 expression plasmid  (GenScript corporation)

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: 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: Scramble control, KHDRBS1 (Sam68) and CREBBP (CBP) targeting shRNA expression plasmids were purchased at Millipore Sigma ( ) and co-transfected with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) vectors in 293-FT cells using Lipofectamine 2000 (Thermo Fisher).

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: Scramble control, KHDRBS1 (Sam68) and CREBBP (CBP) targeting shRNA expression plasmids were purchased at Millipore Sigma ( ) and co-transfected with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) vectors in 293-FT cells using Lipofectamine 2000 (Thermo Fisher).

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: Scramble control, KHDRBS1 (Sam68) and CREBBP (CBP) targeting shRNA expression plasmids were purchased at Millipore Sigma ( ) and co-transfected with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) vectors in 293-FT cells using Lipofectamine 2000 (Thermo Fisher).

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: Scramble control, KHDRBS1 (Sam68) and CREBBP (CBP) targeting shRNA expression plasmids were purchased at Millipore Sigma ( ) and co-transfected with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) vectors in 293-FT cells using Lipofectamine 2000 (Thermo Fisher).

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