Journal: Carcinogenesis

Article Title: Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer

doi: 10.1093/carcin/bgw088

Figure Lengend Snippet: CPI-7c efficiently degrades MDM2 but leads to stabilization of p53 and induces global ubiquitination response. In ( A ), MCF-7 cells were treated with 5, 10, 15 and 20 µM CPI-7c and Nutlin-3, and expression levels of MDM2, ubiquitin and p53 were assessed by using immunoblotting and shown in the image. In panel ( B ), MCF-7 cells were grown in coverslips and treated with either vehicle or 10 and 20 µM of CPI-7c and Nutlin-3, respectively, for 24h and subjected to immunofluorescence staining and analyzed by confocal microscope. Merged confocal photographs represent the superimposition of green (p53) and red (MDM2) images, and the magnified area of the box was shown in inset pictures. Scale bar, 20 µm. Representative of three independent experiments. In panel ( C ), RT–PCR analysis shows the mRNA expression MDM2 and GAPDH genes in response to 10 and 20 µM doses of CPI-7c and Nutlin-3 treatment.

Article Snippet: Nutlin-3 and ChIP grade antibody for ubiquitin, and HDM2 (catalytic RING domain) peptide were purchased from Enzo Life Sciences.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Microscopy, Reverse Transcription Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer

doi: 10.1093/carcin/bgw088

Figure Lengend Snippet: CPI-7c physically binds to RING and N-terminal domains of MDM2 and selectively induces ubiquitination of MDM2 as well as interferes at p53–MDM2 interaction. MCF-7 cells were treated with 10 µM dose of CPI-7c and Nutlin-3 for 24h, and protein lysates were prepared for immunoprecipitation using human anti-ubiquitin. Ubiqutinated MDM2 level was measured using western blotting of anti-MDM2 antibody with proper input control and shown in panel ( A ). Similarly, ( B ) represents the level of associated p53 when lysate was immunoprecipitated using anti-MDM2 antibody. Panel ( C ) represents standard scatchard exponential hyperbolic binding curve of direct physical binding of CPI-7c and Nutlin-3 with N-terminal peptide and RING domain peptide of MDM2 in left and right panels, respectively, along with respective dissociation equilibrium constant ( Kd ) values.

Article Snippet: Nutlin-3 and ChIP grade antibody for ubiquitin, and HDM2 (catalytic RING domain) peptide were purchased from Enzo Life Sciences.

Techniques: Immunoprecipitation, Western Blot, Binding Assay

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: Primer sequences for RT-qPCR.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques:

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: E2F7 is overexpressed in colon cancer tissues and negatively correlates with the survival rate of patients. (A) E2F7 expression in cancer tissues and adjacent normal tissues collected from 30 patients with colon cancer determined by RT-qPCR. The Y axis indicates the relative expression of E2F7 mRNA. (B) Immunohistochemical staining analysis of E2F7 protein in three pairs of colon cancer tissues and adjacent normal tissues (×200). (C) The expression of E2F7 in colon cancer tissues of the GEPIA2 database, where red indicates tumor samples and gray indicates normal tissues. The Y axis indicates the relative expression of E2F7 mRNA. (D) Correlation of survival rate of patients with E2F7 expression analyzed by a survival curve. X axis indicates time, and Y axis indicates survival rate. * p < 0.05. Data between colon cancer tissues and adjacent normal tissues were compared using paired t -test. The experiment was repeated three times independently.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: E2F7 silencing suppresses the stemness of colon cancer stem cells. (A) Flow cytometry and quantitative analysis of the proportion of ALDH1 + cells in SW403 cells transfected with shE2F7. (B) Flow cytometry and quantitative analysis of the changes in the proportion of ALDH1 + cells in SW620 cells transfected with shE2F7. (C) Flow cytometry and quantitative analysis of the proportion of CD133+ cells in SW403 cells transfected with shE2F7. (D) Flow cytometry and quantitative analysis of the proportion of CD133+ cells in SW620 cells transfected with shE2F7. (E) SW403 cell stemness determined by sphere formation test upon transfection with shE2F7. (F) SW620 cell stemness determined by sphere formation test upon transfection with shE2F7. Data between two groups were compared using unpaired t -test. * p < 0.05. The experiment was repeated three times independently.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques: Flow Cytometry, Transfection

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: E2F7 silencing represses antagonistic effects of ALDH1 + cells on 5-FU treatment and oxidative stress. (A) In SW403, flow cytometry and quantitative analysis of the effect of E2F7 silencing on antagonism of ALDH1 + cell to effects of 5-FU treatment. (B) In SW403 cells, flow cytometry and quantitative analysis of the effect of E2F7 silencing on antagonism of ALDH1 + cells to H 2 O 2 treatment. (C) In SW620 cells, flow cytometry and quantitative analysis of the effect of E2F7 on ALDH1 + cell antagonist 5-FU. (D) In SW620 cells, flow cytometry and quantitative analysis of the effect of E2F7 on ALDH1 + cells antagonizing H 2 O 2 treatment. Data between two groups were compared using unpaired t -test. * p < 0.05. The experiment was repeated three times independently.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques: Flow Cytometry

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: E2F7 reduces miR-199b expression by binding to the miR-199b promoter. (A) Analysis of Motif of E2F7 through the cistromeDB database. (B) Visualization of ChIP-seq results of miR-199b targeting E2F7 in the hTFtarget database. (C) Expression of miR-199b determined by RT-qPCR in SW403 cells transfected with shE2F7 or miR-199b inhibitor. (D) miR-199b expression in colon cancer tissues and adjacent normal tissues determined by RT-qPCR (n = 30). (E) Binding of E2F7 to the miR-199 promoter region confirmed by luciferase reporter gene assay. (F) Binding of E2F7 to the miR-199 promoter region analyzed by ChIP assay. Data between colon cancer tissues and adjacent normal tissues were compared using paired t- test, while data between other two groups were compared using unpaired t -test. Comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05. The experiment was repeated three times independently.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques: Expressing, Binding Assay, ChIP-sequencing, Quantitative RT-PCR, Transfection, Luciferase, Reporter Gene Assay

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: USP47 is a target gene of miR-199b in SW403 cells. (A) Prediction of lowly expressed target gene set of miR-199b by starBase, miDIP, and DianaTools. (B) The expression of USP47 in colon cancer tissues in the GEPIA2 database, where red indicates tumor samples and gray indicates normal tissues. (C) Putative miR-199b binding sites in the 3′UTR of UPS47 mRNA. (D) Binding of miR-199b to USP47 confirmed by dual luciferase reporter gene assay. (E) RNA pull-down analysis of the interaction between miR-199b and GST-USP47 in vitro . (F) USP47 expression determined by RT-qPCR and Western blot analysis in cells transfected with miR-199b or miR-199b inhibitor. (G) USP47 expression determined by RT-qPCR and Western blot analysis in cells transfected with shE2F7. Comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. Data between two groups were compared using unpaired t- test. * p < 0.05. The experiment was repeated three times independently.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Gene Assay, In Vitro, Quantitative RT-PCR, Western Blot, Transfection

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: USP47 upregulation by E2F7 deubiquitin-modifies MAPK K48 ubiquitin chain to stabilize MAPK. (A) Protein expression of MAPK in three pairs of colon cancer tissues (T) and adjacent normal tissues (N) determined by Western blot analysis. (B) Detection of the interaction between USP47 and MAPK in 293T cells and SW403 cells. (C) GST pull-down analysis of the interaction between USP47 and MAPK in vitro . (D) In 293T cells, detection of the type of MAPK deubiquitin by USP47. (E) In SW403 cells, detection of MAPK deubiquitination. (F) MAPK expression determined by Western blot analysis in SW403 and SW620 cells transfected with shE2F7. The experiment was repeated three times independently.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques: Expressing, Western Blot, In Vitro, Transfection

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: E2F7 promotes in vitro proliferation, invasion, and migration, and in vivo tumorigenesis of colon cancer cells by upregulating MAPK expression. (A) Detection of SW403 cell proliferation by EdU assay (×200). (B) Detection of the migration of SW403 cells by scratch test. (C) Detection of SW403 cell invasion by Transwell assay. (D) Tumor size and weight of mice (n = 10). Comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05. The experiment was repeated three times independently.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques: In Vitro, Migration, In Vivo, Expressing, EdU Assay, Transwell Assay

Journal: Frontiers in Oncology

Article Title: E2F7 Transcriptionally Inhibits MicroRNA-199b Expression to Promote USP47, Thereby Enhancing Colon Cancer Tumor Stem Cell Activity and Promoting the Occurrence of Colon Cancer

doi: 10.3389/fonc.2020.565449

Figure Lengend Snippet: The mechanism of E2F7 in colon cancer. E2F7 inhibited miR-199b expression and consequently upregulated the miR-199b target USP47, thus stabilizing MAPK protein and inducing the increase of the cancer stem cell activity, which would then be responsible for the aggravation of colon cancer.

Article Snippet: The membrane was blocked with 5% skimmed milk powder for 1 h, added with primary rabbit antibodies diluted with Tris-buffered saline Tween-20 (TBST) (E2F7, ab56022, 1:1,000; USP47, ab72143, 1:1,000; GAPDH, EPR16891, 1:3,000; K48 ubiquitin, EP8589, 1:1,000; FLAG, ab205606; HA, ab187915, Abcam Inc., Cambridge, UK; MAPK, and p44/42 MAPK [Erk1/2; #9102, Cell Signaling Technologies, Beverly, MA, USA]), and incubated overnight at 4°C.

Techniques: Expressing, Activity Assay

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: Neuron

Article Title: ARNT2 tunes activity-dependent gene expression through NCoR2-mediated repression and NPAS4-mediated activation

doi: 10.1016/j.neuron.2019.02.007

Figure Lengend Snippet: A. ChIP-seq signal for ARNT2 at presumptive NPAS4 sites in unstimulated cortical neuron cultures (left) and CA1 hippocampal tissue derived from standard housed mice (right) sorted by descending binding strength. Each ARNT2 binding site is represented as a single horizontal line (orange) centered at the peak summit with flanking 2kb. Color denotes ChIP-seq signal intensity (displayed in log2(counts per million + 1)). Peaks are split between distal enhancers bound by NPAS4 (n= 6,352) and TSS-proximal promoters bound by NPAS4 (n = 2,614).

Article Snippet: To prepare antibody bead complexes, Protein A Dynabeads (Life Technologies) were incubated with antibodies [NPAS4 (20 μg/45–50 million cells), ARNT2 (20 μg/35–50 million cells), rabbit IgG (Santa Cruz H270 (sc-66931); 20 μg/45–50 million cells)] for 30 min at room temperature followed by washing in NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X100, 1 mM DTT, 250 mM NaCl) to remove unbound antibody.

Techniques: ChIP-sequencing, Derivative Assay, Binding Assay

Journal: Neuron

Article Title: ARNT2 tunes activity-dependent gene expression through NCoR2-mediated repression and NPAS4-mediated activation

doi: 10.1016/j.neuron.2019.02.007

Figure Lengend Snippet: A. Heatmaps of normalized ChIP-seq signal for unstimulated (0 hr KCl) and depolarized (2 hr KCl) cortical neuron cultures (left eight columns) at presumptive NPAS4 enhancers. Rightmost two columns show ARNT2 binding in adult hippocampal tissue in standard housing (Strd) or following neuronal stimulation via the injection of kainate (KA). Each binding site is represented as a single horizontal line centered at the peak summit and intensity of color correlates with sequencing signal for the indicated factor. Peaks are ordered by decreasing NPAS4 peak intensity.

Article Snippet: To prepare antibody bead complexes, Protein A Dynabeads (Life Technologies) were incubated with antibodies [NPAS4 (20 μg/45–50 million cells), ARNT2 (20 μg/35–50 million cells), rabbit IgG (Santa Cruz H270 (sc-66931); 20 μg/45–50 million cells)] for 30 min at room temperature followed by washing in NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X100, 1 mM DTT, 250 mM NaCl) to remove unbound antibody.

Techniques: ChIP-sequencing, Binding Assay, Injection, Sequencing

Journal: Neuron

Article Title: ARNT2 tunes activity-dependent gene expression through NCoR2-mediated repression and NPAS4-mediated activation

doi: 10.1016/j.neuron.2019.02.007

Figure Lengend Snippet: A. −log10(FDR) versus log2 fold change in peptides obtained by immunoprecipitation followed by mass spectrometry (IP-MS) with an anti-ARNT2 antibody in stimulated primary cortical neurons relative to peptides immunoprecipitated with an IgG control. Blue points indicate proteins with FDR <0.1 and log2FC > 0. Purple points indicate members of the NCoR2 co-repressor complex.

Article Snippet: To prepare antibody bead complexes, Protein A Dynabeads (Life Technologies) were incubated with antibodies [NPAS4 (20 μg/45–50 million cells), ARNT2 (20 μg/35–50 million cells), rabbit IgG (Santa Cruz H270 (sc-66931); 20 μg/45–50 million cells)] for 30 min at room temperature followed by washing in NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X100, 1 mM DTT, 250 mM NaCl) to remove unbound antibody.

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Control

Journal: Neuron

Article Title: ARNT2 tunes activity-dependent gene expression through NCoR2-mediated repression and NPAS4-mediated activation

doi: 10.1016/j.neuron.2019.02.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To prepare antibody bead complexes, Protein A Dynabeads (Life Technologies) were incubated with antibodies [NPAS4 (20 μg/45–50 million cells), ARNT2 (20 μg/35–50 million cells), rabbit IgG (Santa Cruz H270 (sc-66931); 20 μg/45–50 million cells)] for 30 min at room temperature followed by washing in NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X100, 1 mM DTT, 250 mM NaCl) to remove unbound antibody.

Techniques: Virus, shRNA, Recombinant, Reporter Assay, Luciferase, Binding Assay, Modification, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 1. Stimulation of epithelial cells, RELA activation, DNA binding, and gene expression. (A) Time course of RELA activation. Detroit 562 cells were stimulated for 2 h with LPS, TNF-a, Pam2CSK4, or poly I:C, and RELA activation was determined with the NF-kB p65 TF assay at 30-min interval for 2 h. Results are average luminescence readouts of two independent experiments. (B) Differential gene expression in response to different stimuli. After stimulation with LPS for 100 min, TNF-a for 70 min, Pam2CSK4 for 80 min, or poly I:C for 110 min, RNAs were isolated from Detroit 562 cells for RNA- seq analysis. Average Log2FC of biological duplicates were used for hierarchical clustering of genes DE in at least one condition. Sets of genes UP in poly I:C only or all conditions are highlighted. (C) Annotation of RELA binding sites. ChIP-seq experiment was performed in duplicates after treatment of Detroit 562 cells with LPS for 80 min, TNF-a for 50 min, Pam2CSK4 for 60 min, or poly I:C for 90 min. The number of peaks identified for each stimulus is reported on the right. Peaks were annotated; the bar chart shows the number of peaks in the different genomic features indicated. (D) UP, DOWN, or nonregulated (NO) genes after treatment with the four stimuli were assigned RELA ChIP-seq peaks located within 50 kb (Figure legend continues)

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: Activation Assay, Binding Assay, Gene Expression, Transcription Factor Assay, Isolation, RNA Sequencing, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 2. Comparison of RELA binding sites across stimuli. (A) Comparison of RELA binding sites location. Genomic coordinates of RELA ChIP-seq peaks called upon each stimulus were compared and considered overlapping when the maximum distance between two peak centers was ,500 bp. The Venn diagram shows the number of peaks overlapping across the four conditions. (B) Differential binding analysis. The heatmap shows K-means clustering of the ChIP-seq signal (pooled biological duplicates) across the four stimulations. DB regions were separated into six groups with the number of peaks in each group shown on the right. (C) Annotation of DB and non-DB peaks. The percentage of peaks in each genomic feature for both sets of peaks is reported. Pearson p values from x2 test between the two sets are reported. ***p # 0.001, ****p # 0.0001.

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: Comparison, Binding Assay, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 3. Poly I:C–increased RELA binding. (A) GO analysis of Group 5 peaks. Using Genomic Regions Enrichment of Annotation Tools, genomic regions from Group 5 peaks were associated to the single nearest gene and GO analysis was performed. The top five biological process terms are reported in this figure together with their p value. (B) Gene expression of genes associated with Group 5 peaks. DE genes in at least one condition were assigned DB RELA peaks located within 50 kb upstream to 50 kb downstream of the gene. The boxplot shows Log2FC in each condition (average across biological duplicates) of the genes assigned to Group 5 peaks. The p values from a paired Wilcoxon test against the gene expression under poly I:C stimulation are indicated. (C) Motif analysis of Group 5 peaks. Known motifs enrichment was investigated in the set of Group 5 peaks against all RELA peaks identified across stimuli. Log10 (p values) of significantly enriched motifs are reported in the bar graph. (D) De novo motif analysis on Group 5 peaks. Top unknown motif enriched in Group 5 peaks together with the best match from JASPAR database are represented. (E) Overlap with IRF ChIP-seq data. Binding sites for IRFs were extracted from the ENCODE data and overlapped with the RELA peaks from Group 5 (yellow), DB peaks from the other groups (dark gray), or non-DB peaks (light gray). The fraction of RELA peaks overlapping IRF binding sites are reported; the Pearson p values from a x2 test against the results for Group 5 peaks are indicated. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001.

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: Binding Assay, Gene Expression, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 4. OASL locus as an example of stimulus-specific RELA target. (A) Genome browser view of RELA ChIP-seq signal under different stimuli. A RELA binding site with higher signal under poly I:C stimulation is detected ∼20 kb upstream of the OASL gene. (B) Validation of the poly I:C–increased peak by ChIP qPCR. Primers were designed to amplify the region highlighted in (A) (yellow). The bar chart shows the enrichment of RELA binding at this location as percentage of input recovered. (C) OASL expression upon five stimuli. Expression was determined by RT-qPCR and expressed as fold changes in OASL expression after stimulation as compared with untreated cells. (D) Inhibition of RELA binding. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control. Binding of RELA at the region highlighted in yellow in (A) was investigated by ChIP qPCR and shown as the average input percentage recovered from the immunoprecipitation. (E) NF-kB regulation of OASL. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control, and OASL expression was measured by RT-qPCR and represented as fold change of expression over the control. Data in (B)–(E) represents the average of two independent experiments and SD as error bars.

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: ChIP-sequencing, Binding Assay, Biomarker Discovery, ChIP-qPCR, Expressing, Quantitative RT-PCR, Inhibition, Control, Immunoprecipitation