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: Cell chemical biology

Article Title: Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

doi: 10.1016/j.chembiol.2017.05.026

Figure Lengend Snippet: Figure 3. Sam68 Is an Essential Co-factor Mediating CWP Response in Human AML (A) mRNA expression profile plot of CBP (CREBBP) in AML (red dots, n = 202) versus healthy (blue dots, n = 69) samples. Expression profile is from Mills et al. (2009). (B) STRING analysis interaction network of gene products found to be (1) CBP direct interactors and (2) differentially expressed between AML and healthy donor samples with a p value %1.0 3 1010. CBP interactors with the strongest level of confidence are illustrated. See also Key Resources Table. (C) mRNA expression profile plot of Sam68 (KHDRBS1) in AML (red dots, n = 202) versus healthy (blue dots, n = 69) samples (p = 2.45 3 1014). (D) Western blot analyses of Sam68 protein expression in normal hematopoietic MNCs (from adult and cord blood) (n = 9) and AML patient blasts (n = 7). Samples used: #6, #7, and #8 (Table S2). Dotted lines indicate membrane cropping from original images presented in Figure S3A: Tracks #1 (MPB), #3 (CB MNCs), and #5 (CB Lin) were selected from Sam68 5-s exposure. Densitometry analysis of Sam68 signal versus GAPDH is expressed in the bar graph (***p < 0.0001). (E) Effect of CWP and ICG-001 on siRNA knockdowns of CBP and Sam68 in AML cells. Cell viability assessments were performed in knockdowns and controls 48 hr post transfection and 24 hr post drug treatment. Viability values are presented as relative to vehicle-treated control siRNA (vehicle + siCTRL, n = 11; CWP + siCTRL, n = 11; ICG-001 + siCTRL, n = 3; vehicle + siCBP, n = 6; CWP + siCBP, n = 6; ICG-001 + siCBP, n = 3; vehicle + siSam68, n = 8; CWP + siSam68, n = 8; ICG-001 + siSam68, n = 3; *p = 0.0346, ***p < 0.001). Error bars represent the SEM.

Article Snippet: SUMOylated recombinant Sam68 (Origene TP300263) used as positive control was in vitro modified using a SUMOylation Kit (ENZO Life Sciences).

Techniques: Expressing, Western Blot, Membrane, Transfection, Control

Journal: Cell chemical biology

Article Title: Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

doi: 10.1016/j.chembiol.2017.05.026

Figure Lengend Snippet: Figure 4. Sam68 Is Associated with Selective Response to CWP in Human Breast and Colon CSC-like Models (A) CBP and Sam68 protein expression levels in normal versus neoplastic breast (normal adult dermal fibroblasts [HDF-A], MCF-7, and MDA-MB-231: n = 3), and colon (normal intestinal progenitors [HIEC cells], SW480, and HT29: n = 3). Densitometry analyses were performed from the series of blots presented in Fig- ure S3B and expressed as relative amounts versus GAPDH loading control (*p % 0.019). See also Figures S4A and S4B. (B) Cell count-based selective toxicity assay on non-CSC MCF-7 and SW480 cells versus CSC-like MDA-MB-231 and HT29 lines comparing CWP (500 nM, 48 hr; breast n = 6, **p = 0.0062; colon n = 3, **p = 0.0019) and ICG-001 (3 mM, 48 hr; breast n = 4, **p = 0.0022; colon n = 3, *p = 0.0317). See also Figure S4C. (C) Ki67 expression profiling from vehicle and CWP-treated (500 nM, 48 hr) CSC-like cell lines (DMSO n = 4, CWP n = 8; ***p < 0.001). (D) Lentiviral-mediated overexpression of 6x-histidine-tagged full-length Sam68 in MCF-7 cells. Ratios of Hoechst-positive nuclei counts from vehicle versus CWP-treated (500 nM, 48 hr) cells are presented (n = 3, *p = 0.0345). EGFP-transduced MCF-7 cells were used as control. Error bars represent the SEM. See also Figures S4D and S4E.

Article Snippet: SUMOylated recombinant Sam68 (Origene TP300263) used as positive control was in vitro modified using a SUMOylation Kit (ENZO Life Sciences).

Techniques: Expressing, Control, Cell Counting, Over Expression

Journal: Cell chemical biology

Article Title: Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

doi: 10.1016/j.chembiol.2017.05.026

Figure Lengend Snippet: Figure 5. CWP Treatment Selectively Affects Sam68 Cellular Distribution and CBP/b-Catenin Partnership in CSCs (A) CBP and Sam68 protein expression levels in normal versus neoplastic human pluripotent stem cells (PSCs and t-PSCs). GAPDH was used as loading control (p % 0.019). (B) Cell count-based selective toxicity assay for CWP and ICG-001 on human normal (n = 8) versus transformed (CWP, n = 3; ICG-001, n = 4) pluripotent stem cells (***p < 0.0001). (C) Sam68 immunostaining in vehicle control and CWP-treated PSCs (n = 8) and t-PSCs (n = 9). Scale bar, 50 mm. See also Figures S5H and S5J. (D) Quantification of Sam68 nuclear protein levels upon CWP (100 nM, 48 hr) or ICG-001 (3 mM, 48 hr) treatment was performed by high-content imaging (***p % 0.0003). (E) Interaction assessment between Sam68, b-catenin, and CBP using co-immunoprecipitation (IP) upon CWP treatment in t-PSC (Sam68, n = 7; b-catenin, n = 5) and PSC (Sam68, n = 6; b-catenin, n = 3). See also Figure S5K. (F) Changes in CBP/Sam68 and CBP/b-catenin interaction levels were quantified in t-PSC and PSC models (**p % 0.01; n.s, not significant). (G) Representative western blots of total H3K14ac (n = 3), H3K18ac (n = 6), and H3K4me3 (n = 3) levels in control and CWP-treated t-PSCs. Histone H3 and GAPDH were used as loading controls. (H) Quantification of protein and histone modification levels from western blots of control versus CWP-treated cells are represented in a bar chart (*p = 0.049; **p = 0.0098; ***p = 0.00012). Histone H3 and GAPDH were used as loading controls. (I) Co-immunoprecipitation (IP) kinetics assessing changes in interaction levels between CBP, b-catenin, and Sam68 in response to CWP (100 nM) in t-PSC. Error bars represent the SEM.

Article Snippet: SUMOylated recombinant Sam68 (Origene TP300263) used as positive control was in vitro modified using a SUMOylation Kit (ENZO Life Sciences).

Techniques: Expressing, Control, Cell Counting, Transformation Assay, Immunostaining, Imaging, Immunoprecipitation, Western Blot

Journal: Cell chemical biology

Article Title: Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

doi: 10.1016/j.chembiol.2017.05.026

Figure Lengend Snippet: Figure 6. SUMOylation of Sam68 Participates in CWP Response to CSCs (A) Representative immunofluorescence images of Sam68 (red) and SUMO1 (green) distribution in control and CWP-treated (100 nM, 48 hr) t-PSC (n = 8). Hoechst (blue) was used as nuclear counterstaining. Magnification: 320. (B) Dot-blot estimation of global SUMOylation levels (total SUMO1) in human PSCs versus t-PSCs, and cord blood MNCs versus primary AML samples (n = 9). Samples used: #9, #10, and #11 (Table S2). (C) Immunodetection of SUMOylated Sam68 species in vehicle, CWP, and ICG-001 treated t-PSCs (88 kDa). Purified 6xHis-Sam68 in vitro SUMOylated was used as a positive control. GAPDH performed on input lysates was used as loading control. Quantitative analysis of t-PSCs (Sam68 immunoprecipitation [IP] + western blot [WB]: n = 3) and AML cells (total Sam68 WB: n = 3) showed increased levels of SUMOylated Sam68 in response to CWP (*p = 0.0214, #p = 0.0611). See also Figures S6A–S6C. (D) Schematic illustration of wild-type (WT) and semi-SUMOylable mutant (K96R) Sam68 constructs used for overexpression experiments in t-PSCs. (E) HCI assessment of Sam68 nuclear levels from GFP-positive populations for control vector (EGFP), as well as Sam68 WT and K96R mutant transduced t-PSCs treated with CWP (100 nM) or ICG-001 (3 mM). Data are expressed as relative amounts versus control EGFP for each treatment (n = 6, **p % 0.0094). Repre- sentative micrographs for CWP versus vehicle controls are also presented. Magnification: 320. (F) Cell counts of GFP-positive populations from t-PSCs transduced with control (EGFP), WT, and K96R mutant Sam68 vectors in response to CWP (100 nM) or ICG-001 (3 mM) 48-hr treatments. Data are expressed as relative counts versus vehicle control treatment (n = 6; *p = 0.0377, ***p % 0.0001). Error bars represent the SEM. See also Figures S6D and S6E.

Article Snippet: SUMOylated recombinant Sam68 (Origene TP300263) used as positive control was in vitro modified using a SUMOylation Kit (ENZO Life Sciences).

Techniques: Control, Dot Blot, Immunodetection, In Vitro, Positive Control, Immunoprecipitation, Western Blot, Mutagenesis, Construct, Over Expression, Plasmid Preparation, Transduction

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: pLenti- KHDRBS1 -C-mGFP-P2A-Puro , Origene Tech. , Cat#RC200263L4.

Techniques: Western Blot, Control, 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: pLenti- KHDRBS1 -C-mGFP-P2A-Puro , Origene Tech. , Cat#RC200263L4.

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: pLenti- KHDRBS1 -C-mGFP-P2A-Puro , Origene Tech. , Cat#RC200263L4.

Techniques: Inhibition, Immunoprecipitation, Two Tailed Test, Negative Control, Immunofluorescence, Staining, Imaging, Immunostaining, Western Blot, Derivative Assay, Control, 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: pLenti- KHDRBS1 -C-mGFP-P2A-Puro , Origene Tech. , Cat#RC200263L4.

Techniques: Recombinant, Derivative Assay, Membrane, Immunoprecipitation, Staining, Chromatin Immunoprecipitation, DNA Purification, SYBR Green Assay, cDNA Synthesis, Purification, Western Blot, RNA Sequencing, Sequencing, Gene Expression, Transformation Assay, shRNA, Control, Software