Journal: bioRxiv

Article Title: Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

doi: 10.1101/435776

Figure Lengend Snippet: ( A ) Schematic of S1PR1 modulator screening system Four lentiviral vectors were transduced into U2OS cell line to enable gene activation by SAM and monitoring S1PR1 activation by TANGO system. The cells introduced with SAM sgRNA library were starved with 0.5% charcoal treated FBS, then the Venus-positive population was sorted and next-gen sequence (NGS) analysis was carried out to identify the enriched SAM sgRNA sequences. ( B ) Scatter plot showing enrichment of sgRNAs after sorting. Most sgRNAs are equally distributed in the pre-sort sample (closed gray circles) while after sorting a small fraction of sgRNAs (2,770 out of 70,290 sgRNAs) were enriched and others were not detected (open blue circles). The y-axis shows the NGS reads of sgRNAs. ( C ) Identification of top candidate genes using the MAGeCK method . The names of top ten candidate genes are indicated.

Article Snippet: The single clones were isolated from antibiotics resistant cells by limiting dilution, then introduced with the SAM sgRNA library (a gift from Feng Zhang, Addgene #1000000057) at a low multiplicity of infection.

Techniques: Activation Assay, Sequencing

Journal: International journal of cancer

Article Title: The impact of SAMHD1 expression and mutation status in mantle cell lymphoma: An analysis of the MCL Younger and Elderly trial.

doi: 10.1002/ijc.33202

Figure Lengend Snippet: Figure 2. The impact of SAMHD1 expression on the failure-free survival of

Article Snippet: After heat-induced epitope retrieval at pH 9.0 the rabbit polyclonal SAMHD1 antibody (1:200; Proteintech) was incubated for 30 min, followed by washing and detection according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Nature medicine

Article Title: SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

doi: 10.1038/nm.4163

Figure Lengend Snippet: SAMHD1-mediated HIV-1 restriction in cells does not involve RNase activity. (a–e) Both D137N and Q548A mutants of SAMHD1 restrict HIV-1 infection in PMA-differentiated U937 cells by decreasing viral cDNA synthesis, but not viral genomic RNA (gRNA). U937 cell lines stably expressing WT and mutant SAMHD1 were differentiated with PMA (30 ng/ml) for 20 h. The reverse transcriptase inhibitor nevirapine (NVP) was used as a control. (a) Lysates from PMA-differentiated cells were collected for immunoblotting to confirm SAMHD1 expression and T592-phosphorylated SAMHD1 (phospho-T592). GAPDH was used as a loading control. Blot is representative of three independent experiments. (b) PMA-differentiated cells were infected with single-cycle HIV-1-Luc/VSV-G at a multiplicity of infection (MOI) of 1. At 24 h postinfection (h.p.i.), luciferase assay was used to measure HIV-1 infectivity. Graph depicts relative percentage of luciferase per 10 μg of protein, with the vector set as 100%. Data are presented as mean ± s.e.m. of six independent experiments with two biological replicates per experiment. Statistical analysis was performed using the one-way ANOVA with Dunnett’s correction, ***P < 0.001. (c) Decreased dNTP levels of PMA-differentiated U937 cell lines expressing WT or mutant SAMHD1. Data are presented as means ± s.e.m. of two independent experiments with two biological replicates per experiment. The average concentrations of dNTPs (left) and dCTP (right) in different cell lines are shown. Statistical analysis was performed using the Kruskal–Wallis one-way ANOVA. (d) SAMHD1 does not degrade HIV-1 gRNA in PMA-differentiated U937 cells. The levels of HIV-1 gRNA were measured at 1, 3 and 6 h.p.i., as described at an MOI of 1. Data are presented as mean ± s.e.m. of three independent experiments with two biological replicates per experiment and depicted as relative to HIV-1 gRNA, with the time point 1 h.p.i. set as 1. RNA samples without reverse transcription were used as negative controls and showed no detection of HIV-1 gRNA (data not shown). (e) The expression of WT or mutant SAMHD1 reduces HIV-1 late reverse transcription (RT) products in PMA-differentiated U937 cells at 12 and 24 h.p.i. The levels of HIV-1 late RT products were measured by qPCR. Data are presented as means ± s.e.m. (n = 4 experiments for 12 h.p.i. and n = 6 experiments for 24 h.p.i.). Statistical analysis was performed using the one-way ANOVA with Dunnett’s correction, ***P < 0.001, **P < 0.01, *P < 0.05. (f–h) Characterization of SAMHD1 enzymatic activities in vitro. (f) The purity of recombinant WT SAMHD1 from different batches of preparations (sample 1 and sample 2), as demonstrated by analytical size-exclusion chromatography and SDS–PAGE. SAMHD1 (150 μl) at 2 mg/ml was applied to a Superdex 200 10/300 GL column. Fraction numbers on the SDS–PAGE indicate elution volumes. (g) Moderate exonuclease activities on both ssDNA and ssRNA were observed for sample 1; only background-level activities were detected for sample 2. Ctrl indicates a control without protein. The nuclease-activity assays were performed at 37 °C for 1 h, with 1 μM of SAMHD1 and 1 μM of ssDNA or ssRNA in the presence of 5 mM Mg2+. (h) The dNTPase activity of SAMHD1 proteins (0.5 μM) was assayed with 1 mM dGTP from 5–15 min. The amount of dG products generated in the reactions was quantified by HPLC. Error bars represent s.e.m. from triplicate experiments.

Article Snippet: Rabbit antibodies specific for T592 phospho-SAMHD1 (ProSci, #8005) were used at a dilution of 1:1,000, as previously described 22 .

Techniques: Activity Assay, Infection, cDNA Synthesis, Stable Transfection, Expressing, Mutagenesis, Reverse Transcription, Control, Western Blot, Luciferase, Plasmid Preparation, In Vitro, Recombinant, Size-exclusion Chromatography, SDS Page, Generated

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Workflow showing the selection strategy for KHDRBS1 among DNA-damage response genes transcriptionally activated by Myc and significantly associated to breast cancer prognosis. Venn diagram showing the overlap between Myc-transcriptionally activated genes, DNA-damage response genes and genes associated to breast cancer. Specifically, genes were retrieved from: (i) microarray data of Myc-overexpressing mammospheres (M2) (GSE86407); (ii) published dataset (MD Anderson Human-DNA Repair Genes, https://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repair-genes.html ), BioRad DNA-damage signaling pathway (SAB Target List H96) and recently published DNA-damage-associated genes (Supplementary Table ); and (iii) breast cancer versus normal breast tissues TCGA BRCA and GTeX gene expression data (Supplementary Table ). Genes were further selected for association to the worse relapse-free survival probability in breast cancer (Supplementary Table ) and novelty in the field, excluding known genes associated with BRCAness . B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA ( n = 1212) and GTeX ( n = 179) gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. C Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients stratified by high or low KHDRBS1 expression levels. D GSEA of DNA-repair gene signatures in IMEC-WT versus M2 ( n =3). E Scheme showing MYC and H3K4me3 PCR amplicons localization (red box) on IMEC-WT and M2 cells and layered H3K27ac signals on KHDRBS1 ( SAM68 ) promoter from ENCODE. Chromatin state was assessed by ChromHMM from ENCODE. MYC-MAX binding on multiple cell lines was assessed by ChIP-seq from ENCODE. F ChIP-qPCR estimating MYC binding at SAM68 promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). G qRT-PCR analysis of SAM68 gene expression in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). H ChIP-qPCR of H3K4me3 deposition at KHDRBS1 ( SAM68 ) promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3).

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Selection, Microarray, Gene Expression, Expressing, Binding Assay, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Kaplan–Meier plots of distant relapse-free survival (DRFS) of BC patients stratified by high or low Sam68 protein expression levels. Patients were categorized according to all molecular subtypes ( n = 211) and Luminal-A ( n = 91), Luminal-B ( n = 61), HER2 + ( n = 27), TNBC ( n = 32), HER2 + + TNBC ( n = 59) BCs. B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. The indicated statistics refer to each molecular subtype versus basal subtypes. * p value ≤ 0.05; ** p value ≤ 0.01; **** p value ≤ 0.0001. C ChIP-qPCR estimating MYC and MAX binding at SAM68 promoter in BCSphCs (#4 and #15). Data are mean ± SEM of two independent experiment for each BCSphCs. D Expression of Myc (green color) and Sam68 (red color) on paraffin-embedded sections on parental BC and corresponding PDX tissue. Nuclei were counterstained with Toto-3 (blue color). Scale bar represents 40 µm. E Relative mRNA expression levels of MYC and KHDRBS1 on BCSphCs (#4, #13, and #21) expressing a MycER fusion protein induced by 50 nM of OHT. Data are represented as fold mRNA level changes of OHT-treated cells over vehicle. Data are represented as mean ± SD of three independent experiments. * p value ≤ 0.05; ** p value ≤ 0.01. F Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, and #21) transduced with doxycyclin-inducible non-targeting (nt) and short hairpin Sam68 (shSam68). Data are represented as fold variation of shSam68 over scr. ns not significant; ** p value ≤ 0.01. G Size of tumors generated by orthotopic injection of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4, #13) in immunocompromised mice (NOD/SCID) at the indicated time points. Data are expressed as mean ± SD ( n = 5 mice per group). ns not significant, *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Expressing, Gene Expression, ChIP-qPCR, Binding Assay, Transduction, Generated, Injection

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A MYC binding on DNA-damage related genes transcription start sites (TSS) on IMEC-WT and M2 breast cells. B Representative immunofluorescence analysis of Rad51 foci formation in ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC established cell lines and BCSphCs (#4) untreated (UT) and after 6 h of 8 Gy single dose γ-irradiation (IR). Nuclei were counterstained by Toto-3 (blue). Scale bar represents 10 µm. C Waterfall plot analysis of doxorubicin (DOX, 200 nM, left panel ), paclitaxel (PTX, 10 nM, middle panel ) and carboplatin (CARB, 100 µM, left panel ) response at 72 h in ER+ and TNBC BC established cell lines and BCSphCs. D Response rate distribution to chemotherapy for ER+ and TNBC BC established cell lines and BCSphCs treated as in ( C ). Middle line shows the median value of response per group, while single points represent the average value of BC cell response to DOX, PTX and CARB. Data are mean of three independent experiments. Statistical analysis was performed by using Kruskal–Wallis test. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01. E Immunoblot analysis of PARP and Sam68 (input) and after immunoprecipitation (IP) with Sam68 antibody in BCSphCs (#15) treated for 4 h with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB). Lamin-B was used as loading control. F Immunoblot analysis of nuclear PAR, PARP, and Sam68 in scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 4 h. H3 was used as loading control. G Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, #21) transduced with scramble and short hairpin Sam68 (shSam68) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 72 h. Data are represented as fold variation of shSam68 over scramble. Data are mean ± SD of three independent experiments. ns not significant; * p value ≤ 0.05; ** p value ≤ 0.01. H , I Relative mRNA expression levels of RAD51 (H) and MYC (I) on scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#12 and #13) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX), and carboplatin (CARB) for 24 h. Data are represented as fold mRNA level changes of treated scr and shSam68 cells over vehicle. Data are represented as mean ± SD of three independent experiments. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Binding Assay, Immunofluorescence, Irradiation, Western Blot, Immunoprecipitation, Control, Transduction, Expressing

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Schematic model of DNA-repair signaling pathways mediating the resistance of BC stem-like cells to chemotherapy. B Workflow of purification of sphere cells from serially transplanted BC PDX and their use for in vitro and in vivo drug toxicity testing. C Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and BO2. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). D Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #21) treated with vehicle, olaparib, BO2, cisplatin and olaparib plus BO2 and olaparib plus cisplatin and BO2. Arrows indicate the beginning and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. E Immunoblot analysis of Rad51 in BCSphCs (#15) treated with dinaciclib for 24 h at the indicated concentration. Β-actin was used as loading control. F Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #15, and #21) treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 2). * p value ≤ 0.05; *** p value ≤ 0.001. G Representative images ( left panel ) and quantification of area ( right panel ) of BC sphere cells (#21), transduced with scramble (scr) and short hairpin Sam68 (shSam68) lentiviral vectors, treated with vehicle and dinaciclib for 6 days. Data are represented as mean ± SEM ( n = 3). Ns not significant, ** p value ≤ 0.01; *** p value ≤ 0.001. Scale bar represents 100 µm. H Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and dinaciclib (din). Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. I Cell viability percentage of BCSphCs (#4, #13, #14, #15, #21) treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). J Synergy plot representing the combination index (CI), computed in CompuSyn by using Chou-Talalay method, for each olaparib and dinaciclib dose pair, calculated from cell viability data of BCSphCs (#13). K Size of tumors generated by orthotopic injection of BCSphCs treated with vehicle, olaparib, dinaciclib and olaparib plus dinaciclib. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated with BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Protein-Protein interactions, Purification, In Vitro, In Vivo, Generated, Injection, Western Blot, Concentration Assay, Control, Transduction

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cell line treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 4). * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001. B Relative mRNA expression levels of RAD51 and MYC on scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cells treated with vehicle and dinaciclib for 6 days. Data are represented as fold mRNA level changes of treated scr and shSam68 over vehicle ( n = 3). C Cell viability percentage in ER+ R (MCF7) BC cells treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). D Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients of all molecular subtypes stratified by high or low MYC , KHDRBS1 , and RAD51 expression levels. E Schematic model showing the persistence of a BC stem-like population, characterized by high expression levels of MYC, SAM68 , and RAD51 , following standard anticancer therapies.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Expressing

Journal: The Journal of biological chemistry

Article Title: SH2-kinase linker mutations release Hck tyrosine kinase and transforming activities in Rat-2 fibroblasts.

doi: 10.1074/jbc.274.37.26579

Figure Lengend Snippet: FIG. 1. Co-expression of Nef and Hck stimulates Hck tyrosine kinase activity in Rat-2 fibroblasts. Rat-2 fibroblasts expressing wild-type Nef, a Nef mutant with Ala substitutions for prolines 72 and 75 in the SH3-binding motif (Nef-PA), wild-type Hck, or combinations of wild-type Hck and Nef or Nef-PA were extracted with RIPA buffer, and clarified lysates were incubated with anti-Hck polyclonal antibodies. Lysates from fibroblasts infected with a retrovirus carrying only the neo selection marker were included as a negative control (Con). Hck-anti- body complexes were precipitated with protein G-Sepharose, washed, and resuspended in 20 ml of kinase buffer containing [g-32P]ATP and a GST-Sam 68 fusion protein of 50 kDa (p50) as substrate. Following incubation, Hck and p50 were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and phosphorylated p50 was vi- sualized by storage phosphor imaging (upper panel). The positions of p50 and autophosphorylated Hck are indicated by the arrows. The membrane was probed with anti-Hck monoclonal antibodies to ensure equivalent recovery of Hck in the immunoprecipitates (middle panel). Cell lysates were immunoblotted with anti-Nef monoclonal antibodies to verify expression of Nef and Nef-PA (lower panel).

Article Snippet: Kinase buffer (20 ml) containing 1 mg of the tyrosine kinase substrate p50 (50-kDa GST fusion protein containing residues 331–443 of the Src substrate protein Sam 68 (27, 28); Santa Cruz Biotechnology) and 5 mCi of [g-32P]ATP (3,000 Ci/mmol; NEN Life Science Products) were added, and the reactions were incubated for 15 min at 30 °C.

Techniques: Expressing, Activity Assay, Mutagenesis, Binding Assay, Incubation, Infection, Selection, Marker, Negative Control, SDS Page, Imaging, Membrane, Bioprocessing

Journal: The Journal of biological chemistry

Article Title: SH2-kinase linker mutations release Hck tyrosine kinase and transforming activities in Rat-2 fibroblasts.

doi: 10.1074/jbc.274.37.26579

Figure Lengend Snippet: FIG. 3. Release of Hck protein-tyrosine kinase activity by SH2- kinase linker mutations. Rat-2 fibroblasts expressing Hck wild-type (WT), KE, 2PA, YF, or 2PAYF were extracted with Hck lysis buffer. Extracts from fibroblasts infected with a retrovirus carrying only the neo selection marker were included as a negative control. Clarified lysates were incubated with anti-Hck polyclonal antibodies, and Hck- antibody complexes were precipitated with protein G-Sepharose. The immunoprecipitates were washed with Hck lysis buffer followed by kinase buffer and then incubated with [g-32P]ATP and a GST-Sam 68 fusion protein of 50 kDa (p50). Following incubation, Hck and p50 were resolved by SDS-PAGE, transferred to polyvinylidene difluoride mem- branes, and phosphorylated p50 was visualized by storage phosphor imaging (upper panel). The membrane was probed with anti-Hck mono- clonal antibodies to ensure equivalent recovery of Hck in the immuno- precipitates (lower panel). The positions of p50 and autophosphorylated Hck are indicated by the arrows.

Article Snippet: Kinase buffer (20 ml) containing 1 mg of the tyrosine kinase substrate p50 (50-kDa GST fusion protein containing residues 331–443 of the Src substrate protein Sam 68 (27, 28); Santa Cruz Biotechnology) and 5 mCi of [g-32P]ATP (3,000 Ci/mmol; NEN Life Science Products) were added, and the reactions were incubated for 15 min at 30 °C.

Techniques: Activity Assay, Expressing, Lysis, Infection, Selection, Marker, Negative Control, Incubation, SDS Page, Imaging, Membrane