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 immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

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 immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

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 immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

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 immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

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 immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Expressing

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 1. TGFβ pathway signature increases in TNBC cells upon anchorage-independent culture and androgen receptor and SMAD3 are predicted up- stream regulators. (A) Metacore pathway analysis of BT549 microarray data showing SMAD3 connecting with other genes altered under anchorage- independent culture for 24 hours. Original data set from Barton et al (2015) (GEO record GSE95472). (B) GSEA pathway enrichment analysis showing changes in the TGFβ pathway in BT549 cells cultured in attached or under anchorage-independent culture for 24 hours. (C) Heatmap of significantly altered genes associated with the TGFβ pathway in BT549 cells grown in attached versus suspended culture conditions for 24 hours (n = 4). Gene list from KEGG database on TGFβ Signaling Pathway. Red asterisk: genes associated with the canonical TGFβ signaling pathway. (D) qRT-PCR for ca- nonical TGFβ signaling pathway gene expression in attached versus under anchorage-independent culture conditions at 48 hours in SUM159PT and BT549 cell lines (n = 6). Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Microarray, Cell Culture, Quantitative RT-PCR, Gene Expression

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 2. Anchorage-independent conditions increase TGFβ1 expression and secretion and nuclear pSmad3 localization and activity. (A) RPPA data showing changes in protein in BT549 cells grown in attached versus under anchorage-independent culture conditions for 24, 48, and 72 hours with biological triplicates at each condition and timepoint shown. Red, high relative expression levels; black, intermediate relative expression levels; green, low relative expression levels. Full panel of RPPA data in (30). (B) ELISA measuring changes in TGFβ1 in conditioned medium from three TNBC lines cultured in attached versus suspended conditions for 24 and 48 hours normalized to total live cell count as determined by trypan blue. (C) IHC and quantification of percentage of pixels corresponding to nuclear DAB staining for pSmad3 in MDA-MB-453, SUM159PT, and BT549 cells in at- tached versus suspended culture conditions for 24 hours. (D) 3TP-lux TGFβ reporter-linked luciferase activity normalized to renilla in SUM159PT cells grown in attached versus suspended culture. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Cell Counting, Staining, Luciferase

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 3. Exogenous TGFβ increases AR protein levels and TGFβ inhibition decreases AR and cell viability in anchorage-independent conditions. (A) Western blot for pSmad2, Smad2, Smad3, ID1, AR, GR, and GAPDH in the SUM159PT cell line treated with recombinant TGFβ1 (10 ng/mL) over a time course of 24 hours. (B) qRT-PCR for AR in SUM159 and BT549 cells grown in attached conditions for 48 hours ± LY2109761 (10 µM). (C) Western blot for AR was performed on MDA-MB-453, SUM159PT, and BT549 cell lines grown in attached or suspended conditions for 48 hours ± LY2109761 (10 µM). (D) Western Blot to analyze the apoptosis marker cleaved-PARP in SUM159PT and BT549 cells in attached or suspended conditions for 48 hours ± LY2109761 or LY2157299 (10 µM). Densitometry values calculated as (cleaved-PARP/PARP)/GAPDH. (E) Trypan blue assay for percentage of dead cells in SUM159PT and BT549 cells in attached or suspended conditions for 48 hours ± LY2109761 or LY2157299 (10 µM). Western blot quantifica- tions performed by ratio of protein of interest to loading control and normalized to control. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Inhibition, Western Blot, Recombinant, Quantitative RT-PCR, Marker, Control

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 4. Clinical data suggests a high AR and TGFB3 is associated with worse outcome. (A) Correlation analysis on the co-expression of AR and the TGFβ ligand TGFB3 in TNBC patients in the TCGA-BRCA (n = 123) and SCAN-B (n = 143) cohorts. (B) GSEA analysis on TGFB3 relating to the androgen response gene set from the HCI-009 PDX. (C) KMplotter was used to stratify basal BC patients stratified into ARhigh (top) or ARlow (bottom) populations. TGFB3 expression was then inter- rogated based on overall survival (OS, n = 241) and distant metastasis free survival (DMFS, n = 232)

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 5. AR ChIP-seq peaks are identified near SMAD3 and TGFB1. (A) Heatmaps show that differential AR genome binding in attached and sus- pended conditions with vehicle, DHT, and DHT+Enza treatments. (B) Heatmaps display the signal at differential DHT-induced AR binding sites that show a 2-fold or greater enrichment in the suspended condition. (C) Gene ontology biological processes found nearby AR bound sites that are more than 2-fold enriched in suspended cells using GREAT (63). (D) Genes involved in focal adhesion assembly that were identified as being 2-fold en- riched in under anchorage-independent culture by AR ChIP-seq. (E) AR ChIP-seq peaks identified near SMAD3 are shown. The scale bar at the top is labeled for each loci to indicate the relative distance from the canonical SMAD3 TSS along with the respective gene annotations. For each region, the AR ChIP-seq signal for each treatment condition in attached (black) and suspended (red) are shown. The peaks at each loci are scaled to the same height for accurate comparison but are not directly comparable between loci due to differences in ChIP-seq signal intensity. The chromosomal coord- inates of the area covered by each peak are shown below each region. (F) Browser tracks show AR binding near the TGFβ1 promoter. AR ChIP-seq signal within a 2000 bp window covering 500 bp upstream of the TGFβ1 TSS and 1500 bp into the 5′ UTR and first exon is shown. ChIP-seq signal is scaled to the same height for comparison and represents each treatment condition in attached (black) and suspended (red), with the chromosomal coordinates included at the bottom for reference.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: ChIP-sequencing, Binding Assay, Labeling, Comparison

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 6. SUM159PT cells are more sensitive to a combined treatment with Enza and TGFβ inhibitor than either drug alone, particularly after anchorage-independent culture. (A) SUM159PT cells were grown in triplicate in 6-well plates in attached conditions for 72 hours with either vehicle, LY1 (10 μM), Enza (40 μM), or both, followed by crystal violet assay. (B) SUM159PT cells were grown in triplicate in 6-well plates in attached conditions for 72 hours with the same treatments, followed by washout of drug and an additional 72 hours in culture, then crystal violet assay was performed. (C) SUM159PT cells were grown in under anchorage-independent culture on poly-hema coated plates for 48 hours with the treatments mentioned above, followed by replating of cells in attached conditions for an additional 48 hours. Crystal violet assay was performed after. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Crystal Violet Assay

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 7. Proposed model of a positive feedback loop whereby TGFβ signaling increases AR expression, and AR increases core TGFβ pathway components.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: (A) Graph shows mRNA expression of highest induced lymphocyte-specific transcription factors in splenic Ly49H+ NK cells sorted from uninfected and MCMV-infected animals on day 2 PI, as assessed by microarray [data provided by the Immunological Genome Consortium (41)]. Data are shown as fold change in microarray signal intensity for the infected versus uninfected samples (n = 3 biological replicates per group and representative of three separate experiments). Solid black bars denote significant up-regulation or down-regulation as compared with uninfected controls (P < 0.05, two-tailed unpaired Student’s t test). (B) Normalized counts of Runx1, Runx2, Runx3, and Cbfb in splenic Ly49H+ NK cells sorted from MCMV-infected mice on day 2 PI and uninfected mice (top) or in unstimulated (US) or IL-12 plus IL-18-treated (12 + 18; 16-hour stimulation) splenic NK cells (bottom), as assessed by RNA-seq (n = 2 to 3 biological replicates per group). (C) RNA-seq was performed on purified Ly49H+ WT NK cells and Stat4−/− NK cells from uninfected and MCMV-infected mixed BMC mice (day 2 PI). Normalized counts of Runx family members are shown (n = 2 to 3 biological replicates per group). (D) RNA-seq was performed on purified Ly49H+ and Ly49H− WT NK cells from uninfected and MCMV-infected mice (day 2 PI). Normalized counts of Runx1 and Runx3 are shown (n = 2 to 3 biological replicates per group). Data are presented as means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Expressing, Infection, Microarray, Two Tailed Test, RNA Sequencing, Purification

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: Splenic NK cells (TCRβ−CD19−CD3ε−Ly6G−TER119−TCRγδ−NK1.1+) were sorted from WT mice and stimulated with IL-12 and IL-18 or media alone as a control (unstimulated). STAT4 ChIP was performed, followed by high-throughput DNA sequencing. (A) Proportions of STAT4 genome-wide occupancy at promoter (2 kb upstream and 0.5 kb downstream from TSS), intronic, exonic, or distal intergenic regions in cytokine-stimulated NK cells are shown. (B) RNA-seq was performed on splenic Ly49H+ WT NK cells and Stat4−/− NK cells sorted from mixed chimeras 2 days after MCMV infection. Venn diagram of overlap between differentially expressed genes (top; Padj < 0.05) identified through RNA-seq and reproducible STAT4-bound regions identified through ChIP-seq (bottom; IDR < 0.05). RNA-seq data were performed on n = 3 per condition. (C) Bar graphs depict the top 20 genes with greatest fold enrichment of STAT4 binding over input calculated by MACS2 in transcription factors that show differential expression in RNA-seq data. (D) Representative gene tracks for indicated core-binding factors from STAT4 ChIP-seq. ChIP-seq data are representative of three independent experiments with n = 15 to 20 pooled mice per group per experiment.

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Control, High Throughput Screening Assay, DNA Sequencing, Genome Wide, RNA Sequencing, Infection, ChIP-sequencing, Binding Assay, Quantitative Proteomics

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: Splenic NK cells (TCRβ−CD19−CD3ε−Ly6G−TER119−TCRγδ−NK1.1+) were isolated from WT and Stat4−/− mice and stimulated with IL-12 and IL-18 or media alone as a control (unstimulated; unstim). H3K4me3 ChIP was performed, followed by high-throughput DNA sequencing. (A) Global proportions of H3K4me3 permissive marks at promoter, intronic, exonic, or distal inter-genic regions in cytokine-stimulated NK cells are shown. (B) Bar plots depict number of peaks that change on the basis of fold change (FC) of stimulated versus unstimulated NK cells. FC was calculated by taking the difference between log2-transformed normalized counts for each condition. Only peaks that showed a log2 FC greater than a magnitude of 1 were counted. (C) Meta-peak of all H3K4me3 promoter regions. Overlap of midpoints of ChIP fragments (defined as regions between properly paired sequence reads) for each TSS region was counted for each base pair ± 1 kb from the transcriptional start site. Line plot depicts average signal for all regions for each base pair. (D) Heat map of all H3K4me3 binding regions, with each row representing a single-peak region, row-clustered by normalized peak counts. Signal is displayed as normalized read counts over 5 kb centered at the peak region and is binned at 100-bp windows. (E) H3K4me3 signals from Cbfb, Runx1, Runx2, and Runx3 loci plotted as normalized fragment counts binned at 200 bp across a 10-kb window centered on the transcriptional start site. (F) Zoomed-in histograms of STAT4 ChIP and H3K4me3 ChIP reads mapped to Runx1 and Runx3 loci. Dashed box within boxed tracks indicate STAT4 ChIP called peak region. Data are representative of two independent experiments with n = 15 to 20 pooled mice per group per experiment.

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Isolation, Control, High Throughput Screening Assay, DNA Sequencing, Transformation Assay, Sequencing, Binding Assay

Journal: Biochemical and biophysical research communications

Article Title: Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor

doi: 10.1016/j.bbrc.2014.01.125

Figure Lengend Snippet: Validations of MR target genes by ChIP-qPCR and RT-qPCR of the candidate MR target genes. (A) Target sites of ChIP-qPCR (arrow) are shown. The value based on each read number of immunoprecipitated (IP) DNA is visualized as a proportional altitude of each peak. Significantly higher peaks are shown in red, whereas background signals are represented in green. The target site for ChIP-qPCR in the exon 14 of Sgk1 (A, arrowhead) is also shown. 5′ side of DNA is left for the genes shown above the upper number line. The positional relation is inverted for those below the number line. (B) We performed qPCR targeting the genomic DNA shown in A. The ratios of IP DNA and the input DNA are shown. Sgk1 exon14 is used as a negative control for MR-binding. (N = 3, *P < 0.05 each gene vs. control) (also in E). (C) Of 25 genes as shown in Table 1, 5 genes were upregulated by aldosterone (Aldo) for 3 h compared with the control group and inhibited by pretreatment with spironolactone (Spi) (N = 3, *P < 0.05: Aldo vs. control, #P < 0.05: Aldo + Spi vs. Aldo). The results of ChIP-seq (D) and ChIP-qPCR (E) for Ctgf and Serpine1 are also shown in the same manner. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Microarray mDCT cells with overexpression of 3xFLAG-hMR were incubated with 5 × 10 −6 M spironolactone or vehicle for 2 h, and then with 10 −7 M aldosterone or the same concentration of ethanol for 3 h. mRNA were extracted from the cells treated with ethanol/ethanol (group 1), ethanol/aldosterone (group 2) and spironolactone/aldosterone (group 3) and were analyzed by Affymetrix Mouse Genome 430 2.0 microarrays over 39000 transcripts (Affymetrix, Santa Clara, CA).

Techniques: ChIP-qPCR, Quantitative RT-PCR, Immunoprecipitation, Negative Control, Binding Assay, Control, ChIP-sequencing

Journal: Biochemical and biophysical research communications

Article Title: Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor

doi: 10.1016/j.bbrc.2014.01.125

Figure Lengend Snippet: Overexpression of 3xFLAG-hMR in mDCT cells. (A) A picture of mDCT cells are shown. (B) mRNA of Na-Cl cotransporter (NCC) was expressed in mDCT cells as well as in murine kidney, while not in differentiated mouse podocyte cell line (MPC). GAPDH was used for internal PCR control. (C) Total amounts of 3xFLAG-hMR overexpressed in mDCT cells were analyzed by Western blotting. (D) 3xFLAG-hMR overexpressed in mDCT cells accumulates to the nucleus in response to a physiological dose of aldosterone.

Article Snippet: Microarray mDCT cells with overexpression of 3xFLAG-hMR were incubated with 5 × 10 −6 M spironolactone or vehicle for 2 h, and then with 10 −7 M aldosterone or the same concentration of ethanol for 3 h. mRNA were extracted from the cells treated with ethanol/ethanol (group 1), ethanol/aldosterone (group 2) and spironolactone/aldosterone (group 3) and were analyzed by Affymetrix Mouse Genome 430 2.0 microarrays over 39000 transcripts (Affymetrix, Santa Clara, CA).

Techniques: Over Expression, Control, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor

doi: 10.1016/j.bbrc.2014.01.125

Figure Lengend Snippet: Identification of the candidate MR target genes in mDCT cells by the combination of ChIP-seq and microarray analyses. (A) We compared MR target gene candidates identified by ChIP-seq and microarray analyses. 1414 genes were identified as those determined by GREAT to be associated with MR ChIP-seq peaks, and 186 genes were identified as those upregulated by aldosterone in the microarray. The venn diagram intersection identified 25 genes as MR target gene candidates. (B) MRE/GRE was discovered as de novo motif in MBSs of mDCT cells with the sufficient significance (E-value

Article Snippet: Microarray mDCT cells with overexpression of 3xFLAG-hMR were incubated with 5 × 10 −6 M spironolactone or vehicle for 2 h, and then with 10 −7 M aldosterone or the same concentration of ethanol for 3 h. mRNA were extracted from the cells treated with ethanol/ethanol (group 1), ethanol/aldosterone (group 2) and spironolactone/aldosterone (group 3) and were analyzed by Affymetrix Mouse Genome 430 2.0 microarrays over 39000 transcripts (Affymetrix, Santa Clara, CA).

Techniques: ChIP-sequencing, Microarray