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: Molecular neurobiology

Article Title: Inhibition of tRF- 02514 in Extracellular Vesicles Preserves Microglia Pyroptosis and Protects Against Parkinson's Disease.

doi: 10.1007/s12035-025-04925-2

Figure Lengend Snippet: Fig. 6 tRF- 02514 siR inhibits the expression of NLRP3, GSDMD, and Caspase- 1 while promoting ATG5 (n = 6); all bar charts were created using GraphPad Prism software and error bars (SEM) were added. A IHC analysis of NLRP3, GSDMD, Caspase- 1, and ATG5 in SN (nucleus staining appears blue, while positive tis- sue staining appears brown-yel- low, × 200). B WB assessment of NLRP3, GSDMD, Caspase- 1 and ATG5 expression in brain tissue (*P < 0.05; **P < 0.01; ***P < 0.001)

Article Snippet: The sections were incubated with 5% bovine serum albumin V at 37 °C for 30 min. Primary antibodies, including BDNF (Affinity, DF6387, 1:50), NGF (HUABIO, ET1606 - 29, 1:100), TH (Bioss, bs- 0016R, 1:100), NLRP3 (Proteintech, 68,102–1-IG, 1:100), GSDMD (Abcam, Ab191860, 1:500), Caspase- 1 (Cell Signaling Technology, D57 A2, 1:100), and ATG5 (Huaan Biotech, ET1611 - 38, 1:100), were diluted with 2% bovine serum albumin V. The diluted primary antibodies were added to the slides and incubated overnight at 4 °C.

Techniques: Expressing, Software, Staining

Journal: bioRxiv

Article Title: Mechanical cues of extracellular matrix determine tumor innervation

doi: 10.1101/2024.03.25.586535

Figure Lengend Snippet: ( A ) Representative images of ND-L and ND-H samples, as displayed by PGP9.5 IF stain. Correlation analysis of tissue stiffness with ND (n = 22, right panel). Scale bar, 100 μm. ( B ) Representative images of NH-L and NH-H samples, as displayed by PGP9.5 IF stain. Correlation analysis of tissue stiffness with NH (n = 22, right panel). Scale bar, 100 μm. ( C ) Representative histology images of ND-L and ND-H samples, obtained by co-immunofluorescence (IF) analysis of S100β and NF-L, Trichrome stain, collagen I IF stain, LOX IF stain, TGM2 IF stain, α-SMA IF stain, and picrosirius red (including polarized images and collagen fiber individualization analysis). Scale bar, 50 and 200 μm as indicated. ( D ) Quantification of Trichrome (collagen content), collagen I area, LOX area, TGM2 area, and α-SMA area between ND-L and ND-H samples (n = 37 per group). ( E ) Collagen fiber width, length, and straightness in ND-L and ND-H samples (n = 37 per group). ( F ) The association between ND and tumor size in PDAC (n = 37 per group). ( G ) The association between ND and tumor location in PDAC (n = 37 per group). ( H ) Stromal score and matrisome score of ND-L and ND-H cases in the TCGA cohort (n = 150). The arrow heads indicate nerves. Scale bar, 200 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001. Values were compared by the Spearman’s rank correlation methods ( A , B ), Student’s t test ( D , E , H ), and the chi-square test ( F , G ). ND, neural density; NH, neural hypertrophy.

Article Snippet: Primary antibodies were S100β (66616-1-Ig, Proteintech, 1:200), NF-L (ab223343, Abcam, 1:100), Collagen I (#72026, Cell Signaling Technology, 1:200), LOX (17958-1-AP, Proteintech, 1:200), TGM2 (15100-1-AP, Proteintech, 1:200), α-SMA (A5228, Sigma-Aldrich, 1:400), PGP9.5 (14730-1-AP, Proteintech, 1:200), PGP9.5 (66230-1-Ig, Proteintech, 1:500; for co-IF analysis), CK19 (1:200, Proteintech, 10712-1-AP), and AMY2A (15845-1-AP, Proteintech, 1:100).

Techniques: Staining, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: The Motor Protein Kinesin-1 Links Neurofibromin and Merlin in a Common Cellular Pathway of Neurofibromatosis

doi: 10.1074/jbc.c200434200

Figure Lengend Snippet: FIG. 1. NF1-containing complexes derived from the soluble or particulate fractions. a, particulate and soluble fractions from the HeLa cell line were fractionated by chromatography as described under “Materials and Methods.” Western blot analysis of P11 fractions using NF1 (sc-68) antibodies. b, Western blot analysis of the soluble eluate fractionated by Superose 6 gel filtration using NF1 (sc-68) antibodies. c, Western analysis of the particulate eluate fractionated by Superose 6 gel filtration using NF1 (sc-68) antibodies.

Article Snippet: Anti-NF1 antibodies (500 g, COOH-terminal, Santa Cruz Biotechnology, sc-68) were cross-linked to protein A-Sepharose (1 ml, Repligen) using standard techniques for affinity purification.

Techniques: Derivative Assay, Chromatography, Western Blot, Filtration

Journal: Journal of Biological Chemistry

Article Title: The Motor Protein Kinesin-1 Links Neurofibromin and Merlin in a Common Cellular Pathway of Neurofibromatosis

doi: 10.1074/jbc.c200434200

Figure Lengend Snippet: FIG. 2. Purification of NF1-containing complexes derived from the particulate fraction of HeLa and calf brain. a, HeLa particu- late extract was fractionated by chromatography as described under “Materials and Methods.” The affinity-purified -NF1 (sc-68) complex was separated in an SDS-polyacrylamide gel (4–12%), and proteins were visualized by colloidal blue staining and Western blot analysis using anti-NF1 and anti-kinesin-1 antibodies. Molecular masses of marker proteins are indicated on the left and the proteins analyzed by ion trap mass spectrometry on the right. b, calf brain particulate extract was fractionated by chromatography and analyzed as described above.

Article Snippet: Anti-NF1 antibodies (500 g, COOH-terminal, Santa Cruz Biotechnology, sc-68) were cross-linked to protein A-Sepharose (1 ml, Repligen) using standard techniques for affinity purification.

Techniques: Purification, Derivative Assay, Chromatography, Affinity Purification, Staining, Western Blot, Marker, Mass Spectrometry

Journal: Journal of Biological Chemistry

Article Title: The Motor Protein Kinesin-1 Links Neurofibromin and Merlin in a Common Cellular Pathway of Neurofibromatosis

doi: 10.1074/jbc.c200434200

Figure Lengend Snippet: FIG. 3. Kinesin-1 is a component of NF1 complexes derived from both the soluble and the particulate fractions. a, immuno- precipitation using three affinity-purified antibodies for kinesin-1 (one polyclonal -KIF5B and two monoclonals -H1 and -H2) and -TRAP220 (control) followed by Western analysis using -NF1 (sc-68) and -KIF5B antibodies. Calf brain particulate extract was used as the input. b, Western analysis using -KIF5B antibodies following immu- noprecipitation using the affinity-purified -H2, -NF1 (sc-68), and -TRAP220 from HeLa soluble fraction. c, after transfection of HeLa cells with either FLAG-KIF5B or pFLAG-CMV2, anti-FLAG antibodies were used to immunoprecipitate complexes associated with KIF5B. The eluate was analyzed by Western blot using the antibodies shown to the right of the figure. Antibodies against KIF5B also detected a heterodimeric complex formed by the endogenous KIF5B and FLAG-KIF5B.

Article Snippet: Anti-NF1 antibodies (500 g, COOH-terminal, Santa Cruz Biotechnology, sc-68) were cross-linked to protein A-Sepharose (1 ml, Repligen) using standard techniques for affinity purification.

Techniques: Derivative Assay, Immunoprecipitation, Affinity Purification, Control, Western Blot, Transfection

Journal: Journal of Biological Chemistry

Article Title: The Motor Protein Kinesin-1 Links Neurofibromin and Merlin in a Common Cellular Pathway of Neurofibromatosis

doi: 10.1074/jbc.c200434200

Figure Lengend Snippet: FIG. 4. NF2 is a component of a distinct kinesin-1-containing complex. a, Western analysis using -NF1 (sc-68) and -NF2(A19) antibodies. Immunoprecipitation was performed using affinity-puri- fied -NF1 (sc-68), -NF2(C18), and -TRAP220 antibodies from the HeLa-soluble fraction. b, immunoprecipitation using -NF2(C18), -NF2(A19), and -TRAP220 from the HeLa-soluble fraction was ana- lyzed by Western blotting using antibodies shown to the left of the panel. c, after transfection of HeLa cells with either FLAG-KIF5B or pFLAG-CMV2, anti-FLAG antibodies were used to immunoprecipitate complexes associated with KIF5B. Western blot analysis using -KIF5B and -NF2(C18). d, Western blot analysis of Superose 6 col- umn fractions using antibodies shown to the left of the figure.

Article Snippet: Anti-NF1 antibodies (500 g, COOH-terminal, Santa Cruz Biotechnology, sc-68) were cross-linked to protein A-Sepharose (1 ml, Repligen) using standard techniques for affinity purification.

Techniques: Western Blot, Immunoprecipitation, Transfection