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

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: 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: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 4. Expression of the gene encoding KHDRBS3 is up-regulated in glioblastoma-derived neurospheres. (A) The differential expression levels for proteins signif icantly up-regulated in spheroids over monolayer culture and for proteins significantly down-regulated in butyrate treatment over monolayer culture (p < 0.05). (B and C) KHDRBS3 and BAG3 mRNA expression in neurospheres of four glioblastoma cell lines including U251, LN229, U87 and M059J. The numbers indicate mean of fold-change. ACTB was used as internal control. The relative value was expressed as the mean ± SD (n = 4). The statistical analyses were tested by One-way ANOVA. ** p < 0.01; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Control

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 5. Depletion of KHDRBS3 expression decreased proliferation of U251 and LN229 cells. (A) Realtime PCR analysis of KHDRBS3 in U251 and LN229 cells which were transfected with 50 nM of negative control siRNA (si-CTL) or KHDRBS3-specific siRNA (si-KHDRBS3) for 48 h. ACTB was used as internal control. The relative expression value was expressed as the mean ± SD from three independent experiments. (B) Western blot analysis of KHDRBS3. Band intensities were analyzed and compared using imageJ software. (C) si-CTL and si-KHDRBS3 treated U251 and LN229 were grown under monolayer culture up to 6 days. Cell number was counted at the indicated time points. (D) Cell proliferation was determined by MTT assay at the indicated time points. Data is represented as the mean ± SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. ** p < 0.01; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Expressing, Transfection, Negative Control, Control, Western Blot, Software, MTT Assay

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 6. KHDRBS3 facilitates neurosphere formation of U251 and LN229 cells. Sphere formation assay was performed in U251 (top) and LN229 (bottom) cells transfected with control (si-CTL) or KHDRBS3 siRNA (si-KHDRBS3). Spheres were photographed by light microscopy at days 3, 5 and 7 (magnification: 10×, scale bar: 200 μm). The number of spheres was counted in and represented as the mean ± SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Tube Formation Assay, Transfection, Control, Light Microscopy

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 7. KHDRBS3 promotes cell migration of U251 and LN229. Wound healing assay of U251 (left) and LN229 (right) transfected with si-CTL or si-KHDRBS3 was performed. Cells were seeded at density of 2.5 × 105 cells/well and incubated in normal culture media for 24 h prior wound scratching. The scratch-wounds were photographed at 0 and 24 h of incubation. The percentage of cell migration was calculated according to the reduction of wound area at 24 h compared with the area at 0 h. Error bars represent SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. * p < 0.05; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Migration, Wound Healing Assay, Transfection, Incubation

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 8. KHDRBS3 facilitates expression of genes involved in cancer stem cells of the glioblastoma cell lines U251 and LN229. Expression of the genes encoding SOX2, PAX6, CD133 and CD44 was determined by realtime PCR in neurospheres derived from U251 and LN229 cells treated with si-CTL or si-KHDRBS3. ACTB was used as internal control. The relative value was expressed as the mean ± SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Expressing, Derivative Assay, Control

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 9. Silencing of KHDRBS3 promotes TMZ sensitivity in glioblastoma. (A) U251 and LN229 cells transfected with si-CTL or si-KHDRBS3 were cultured in 96-well plates and treated with the indicated concentrations of TMZ (0–400 μM) for 48 h. The viability of cells treated with TMZ was determined by MTT assay. (B) Left panel: representative FACS analyses of apoptotic cell death of si-CTL and si-KHDRBS3 treated U251 cells in response to 100 and 200 μM TMZ. FITC-conjugated Annexin V and PI were used as the early-stage and late-stage apoptotic markers. Right panel: the histogram displayed percentages of apoptotic cells, including early and late apoptotic cells. Error bars represent SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. ** p < 0.01; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Transfection, Cell Culture, MTT Assay