Journal: Cell Reports Medicine

Article Title: LTA4H improves the tumor microenvironment and prevents HCC progression via targeting the HNRNPA1/LTBP1/TGF-β axis

doi: 10.1016/j.xcrm.2025.102000

Figure Lengend Snippet:

Article Snippet: Anti-Histone H3 , Bioss , BSM-33042M.

Techniques: Microarray, Recombinant, Lysis, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Reverse Transcription, Activity Assay, Immunoprecipitation, Extraction, Purification, In Vitro, Transfection, Sequencing, Mass Cytometry, ChIP-sequencing, Transgenic Assay, Software

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Top panel: cDCs were rested 16 h and stimulated as indicated for 15 min. pSTAT3 was evaluated by flow cytometry. cDCs were gated as CD11c hi cells. Bottom panel: cDCs were rested 1 h, treated with 20 ng ml −1 IL-21, IL-6, IL-10 or Flt3L for 4 h, and intracellular pro-IL-1β analysed by flow cytometry. Shown are data representative of three experiments. ( b ) Summary of three experiments from lower panel of a . ** P values of the untreated sample compared with IL-21, IL-6 and IL-10 treated samples are 0.0002, 0.0017 and 0.0049, respectively; NS, P =0.4; error bars are means±s.e.m. ( c – e ) cDCs were stimulated with 100 ng ml −1 IL-21 or IL-10 for 1 h, then stimulated with 100 ng ml −1 LPS for 4 h, and the expression of Il1b ( c ), Il6 ( d ), and Tnf ( e ) mRNA were determined. Shown are combined results of 3 independent experiments; error bars are means±s.e.m. ( f ) Top panel: BMMs were rested without M-CSF for 16 h, treated with IL-21 or LPS for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: BMMs (gated as CD11c + F4/80 + cells) were rested and treated with IL-21 or LPS for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of two experiments (total of 6 individual samples). ( g ) Top panel: CD4 + T cells were pre-activated with 5 μg ml −1 plate-bound anti-CD3+2 μg ml −1 soluble anti-CD28 for 3 days, washed, rested 16 h, treated with IL-21 for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: Rested CD4 + T cells were treated with IL-21 for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of three experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a – g ), cDCs were rested 1 h, treated with IL-21 for 1 h; pre-activated CD4 + T cells were washed, rested 16 h, treated with IL-21 for 1 h, and ChIP-Seq performed for STAT3, H3K4me1, and H3K27ac. ( a ) Venn diagram showing overlapping and distinctive STAT3 binding sites in cDCs and CD4 + T cells. ( b – g ) For IL-21-induced-STAT3 binding sites that differentially exist in cDCs or CD4 + T cells, there were cell type-specific binding profiles of STAT3 ( b versus c ) and H3K4me1 ( d versus e ) and H3K27ac ( f versus g ) enhancer marks. Shown are normalized read densities near peak summits for cDC- or CD4 + T-cell specific STAT3 binding sites. ‘Dips' at the plot centres ( d – g ) represent open chromatin corresponding to nucleosome depletion. Data are representative of two experiments.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: ChIP-sequencing, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Freshly isolated cDCs were rested 1 h, then stimulated with IL-21 for 4 h; CD4 + T cells were pre-activated for 3 days, then washed and rested 16 h, and stimulated with IL-21 for 4 h. Shown are genes differentially regulated by IL-21 in cDCs versus pre-activated CD4 + T cells. For cDCs, gene expression profiling was performed by microarray analysis, where cDCs were pooled from three independent experiments, as described in ref. . For pre-activated CD4 + T cells, gene expression profiling data were generated by RNA-Seq analysis. Shown are data from one of two similar experiments. ( b ) Il1b and Il21 expression in cDCs and CD4 + T cells not treated or stimulated with IL-21 as in a . Data are representative of 3 experiments. Error bars are technical duplicates of the representative experiment. ( c , d ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3, and H3K27me3 marks at the Il1b locus in cDCs ( c ) and CD4 + T cells ( d ). Arrows in c indicate STAT3 binding sites at GAS1, GAS2 and GAS3 regions (GAS1: TTAgggGAA (−155 bp), TACcctGAA (−175 bp), TCCctgGAA (−195 bp); GAS2: TTTgggGAA (−2,452 bp), TTCctcCAA (−2,525 bp), TTCttcAAA (−2,549 bp); GAS3: TTGtgtGAA (−9,761 bp)). Arrows in d indicate the STAT3 binding sites identified in cDCs, but no STAT3 binding was seen at these sites in CD4 + T cells. ( e , f ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3 and H3K27me3 marks at the Il21 gene locus in cDCs ( e ) and CD4 + T cells ( f ). Arrow in f indicates the STAT3 binding site at the GAS motif in the Il21 promoter region. Arrow in e indicates this same site, but no STAT3 binding was seen at this site in cDCs. Data are representative of two experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Isolation, Expressing, Microarray, Generated, RNA Sequencing Assay, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) cDCs were rested 1 h, treated with 100 ng ml −1 IL-21 or LPS at the indicated time points, and intracellular pro-IL-1β expression was determined. β-actin was used as control. Shown is one of two similar experiments. ( b ) cDCs were treated as in a , with 5 mM ATP added 1 h prior indicated time points in LPS-stimulated samples, and the secretion of IL-1β was determined by enzyme-linked immunosorbent assay (ELISA). Shown are combined results of two independent experiments; error bars are means±s.e.m. ( c ) CD4 + T cells from WT or Il1r −/− mice were cultured in Th17 cell differentiation conditions for 2 days, then supernatant from a 24 h, IL-21-treated cDC culture was added to the Th17 cells and incubated for 2 days, with or without addition of 10 μg ml −1 of anti-IL-1β. Expression of IL-2Rα (MFI) was determined by flow cytometry. The amount of biologically active IL-1β was determined using a standard curve constructed by assaying recombinant IL-1β. Shown are the combined results of two independent experiments with total of six samples. ( d , e ) WT, Casp1 −/− , Nlrp3 −/− and Pycard −/− cDCs were rested 1 h. In d , cDCs were then treated with 100 ng ml −1 LPS for 20–24 h with 5 mM ATP added in the final 1 h, and IL-1β assessed. Data are from two experiments; error bars are means±s.e.m. In e , cDCs were then treated with IL-21 for 20–24 h and IL-1β protein determined. Data are from five experiments. P values of IL-21-treated WT samples as compared with Casp1 −/− , Nlrp3 −/− and Pycard −/− samples are 0.99, 0.22 and 0.96, respectively; error bars are means±s.e.m. ( f ) cDCs from Ripk3 −/− , Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− mice were treated as in e , and IL-1β assessed. Data shown are from three experiments. P values of IL-21-treated Ripk3 −/− sample compared with Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− samples are 0.57 and 0.93, respectively. In b and d – f , IL-1β production in the culture supernatant was determined by ELISA. Pro-IL-1β induced by IL-21 in the culture supernatant was minimal, based on a pro-IL-1β-specific ELISA. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Cell Differentiation, Incubation, Flow Cytometry, Construct, Recombinant

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 Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Cyclin D1-Dependent and Independent Function in Estrogen-Regulated Development in Vivo. A, Schematic depicting experimental procedure for ovariectomy and estrogen pellet implantation (n = 16 female mice). Mice were implanted with an estrogen pellet or placebo pellet 14 days after ovariectomy. Tissues were harvested at day 21. B, The representative images of uterus from cyclin D1+/+ and cyclin D1−/− mice with or without estrogen treatment. Graph depicts uterus weights as a percentage of body weight in cyclin D1+/+ and cyclin D1−/− mice with or without estrogen treatment. C, Mouse mammary gland whole mounts stained with Carmine dye.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: In Vivo, Staining

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Genome-Wide Profiling of Cyclin D1-Dependent Estrogen-Regulated Genes in Vivo. A, Venn diagram displays the number of genes that were differentially regulated by estrogen in cyclin D1+/+ mouse mammary glands compared with cyclin D1−/− mouse mammary glands. The directionality of change is depicted by up and down arrows. B, Cyclin D1-dependent estrogen-regulated genes were grouped by hierarchical clustering via complete linkage (Cluster 3.0) and visually depicted using Treeview (left). The up-regulated genes are in red and down-regulated genes are in green (P < .05). Chart to the right of heat map depicts Log2 fold change of E2-induced and -repressed genes comparing cyclin D1+/+ with cyclin D1−/− mouse mammary glands. C–E, DAVID analysis was used to classify the pathways differentially regulated by cyclin D1 in E2-treated mice. Pathways depicted represent member genes from microarray analysis of estrogen-treated cyclin D1+/+ mouse mammary glands vs cyclin D1−/− mouse mammary glands, growth factors (C), growth factor receptors (D), and peptidases (E). V, vehicle; Veh., vehicle.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Genome Wide, In Vivo, Microarray

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Genes Regulated by E2 in Vivo Are Bound by Cyclin D1 by ChIP-Seq. A, Comparison of genes occupied by cyclin D1 in ChIP-Seq to those regulated by E2 in cyclin D1+/+ mouse mammary gland and (B) those E2-responsive genes that are regulated in a cyclin D1-dependant manner. C, Location of and (D) representative tag density profiles for, cyclin D1-occupied genes that are regulated by E2. Vertical axis shows average peak height and horizontal axis depicts chromosomal location of cyclin D1-associated interval sequence. TSS, transcription start site.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: In Vivo, ChIP-sequencing, Comparison, Sequencing

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: ERα Induces AREG Gene Expression in a Cyclin D1-Dependent Manner. A and B, MCF7 cells were transfected with siRNAs targeting cyclin D1 and treated with vehicle or E2 (10−8 M) for 24 hours. CCND1 and AREG mRNA abundance was determined by quantitative RT-PCR. C, Western blot was performed to determine the cellular levels of AREG expression in cyclin D1 knockdown cells compared with scramble siRNA control. β-tubulin was included as loading control for protein abundance. D, The concentration of AREG in cell culture medium was measured by ELISA. Concentration of AREG in the conditioned media was normalized to total protein. Data are mean ± SEM. E, AREG promoter luciferase reporter plasmids were transfected into MCF7 cells with a cyclin D1 expression vector. Relative luciferase activity is shown as mean ± SEM normalized to β-galactosidase activity of a cotransfected vector and as (F) fold-induction by cyclin D1. G, Promoter sequence alignment of mouse and human amphiregulin promoter. Homologous nucleotides (:) and regions of discontinuity (−) are indicated. Predicted BRCA1 sites are highlighted for human (− strand), and mouse (2 sites on + strand) with predicted confidence values of 86%, 87%, and 94% respectively. Ctrl., control.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Gene Expression, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Control, Quantitative Proteomics, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Luciferase, Plasmid Preparation, Activity Assay, Sequencing

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Cyclin D1 Is Recruited to a BRCA1 Binding Site. A, ChIP assay in MCF7 cells treated with E2 (10 nM) for ERα at the AREG gene promoter. B. The pS2 gene was included as a positive control. C, Western blot shows FLAG-cyclin D1 expression in transduced MCF7 cells. D, ChIP assay to determine cyclin D1 occupancy at the AREG gene promoter. Using either MCF7 cells transduced with FLAG-cyclin D1 or (E) MCF7 cells ChIP analysis with antibodies directed to endogenous cyclin D1, BRCA1 or ERα (E2 10 nM 24 hours). F and G, GST pulldown was performed to determine the minimal region of BRCA1 required for cyclin D1 binding. H and I, GST-cyclin D1 or mutants were incubated with in vitro translated BRCA1. The N terminus (1–100 amino acids) of cyclin D1 was required for BRCA1 binding. IB, immunoblot; IP, immunoprecipitation; IVT, in vitro translation.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Binding Assay, Positive Control, Western Blot, Expressing, Transduction, Incubation, In Vitro, Immunoprecipitation

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: E2 Induces Cyclin D1 Distribution within a LMW Complex with AIB1. A,. Western blot analysis of Superose 6 chromatography from asynchronously cycling MCF7 cell lysates using antibodies as indicated to the left of the figure. The molecular weight of the fractions is indicated at the bottom of the figure. Cells were treated with (+) E2 (10−8 M). The coeluting fractions > 4 mDa (HMC) and 670 kDa (LMC) are indicated by the boxes. B, Western blot analysis of MCF7 cell extracts after Superose 6 chromatographic fractionation. The antibodies are as indicated. Extracts were treated with E2 (10−8M) for 30 minutes. Molecular weight markers are shown below panel B, indicating the HMC (4 MDa) or LMC (670 kDa). C, Hormone-deprived MCF7 cells infected with shCCND1 or shControl were treated for 1 hour with 10 nM E2 or vehicle control; ERα was immunoprecipitated followed by Western blotting for AIB1, cyclin D1, and ERα. IP, immunoprecipitation; V, vehicle.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Western Blot, Chromatography, Molecular Weight, Fractionation, Infection, Control, Immunoprecipitation

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: ERα Recruitment to the LMW Complex Requires Cyclin D1. A, Western blot analysis of superose 6 chromatographic fractions from female cyclin D1−/− or cyclin D1+/+ mice cell lysates (liver) using antibodies as indicated to the left of the figure. The molecular weight markers are shown below. B, The relative abundance of ERα in the HMC or LMC (670 kDa) is shown graphically indicating increased ERα in the HMC in cyclin D1−/− mice. C, Schematic presentation of cyclin D1 regulation of ERα activation proposes a model in which cyclin D1 participates in ERα signaling by binding to BRCA1 and in the presence of E2 facilitates an LMC that includes ERα and AIB1. Cyclin D1 binding to BRCA1 antagonized BRCA1 action, including BRCA1 repression of Areg expression.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Western Blot, Molecular Weight, Activation Assay, Binding Assay, Expressing

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Global analyses of IKZF1 and c‐FOS binding to the myeloma genome. (A) Upper panel: Average plot (middle) and heatmap (left) of IKZF1 chromatin immunoprecipitation (ChIP)‐seq reads over all transcription start sites (TSSs) ± 5000 bp. The pie chart shows the gene section breakdown (right). Lower panel: Average plot (middle) and heatmap (left) of c‐FOS ChIP‐seq reads over all TSSs ± 5000 bp. The pie chart shows the gene section breakdown (right). Genes (rows) were ordered in the same way in heatmaps. (B) Left panel: Overlap of IKZF1‐ ( n = 13 932) and c‐FOS‐binding ( n = 10 173) sites in MM.1S cells. Right panel: Status of IKZF1 and c‐FOS binding at promoter/enhancer regions of representative genes based on ChIP‐seq data. (C) Nucleotide sequences of IKZF1‐ and c‐FOS‐binding sites deduced from ChIP‐seq analyses of MM.1S cells. The ChIP‐seq data were analyzed using a Partek Flow genomic analysis software v10.0 (Partek Inc.).

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing, Software

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: c‐FOS‐binding sites are present in nearly one‐half of IKZF1‐target genes in multiple myeloma (MM). (A) Using chromatin immunoprecipitation (ChIP)‐seq data of MM.1S cells, we visualized IKZF1 binding near transcription start sites (TSSs) (red triangles) of the indicated genes in the UCSC genome browser. The genes possessing the activator protein‐1 (AP‐1)‐binding motif based on a TRANSFAC search ( https://genexplain.com/transfac/ ) are marked in red, and those without the conventional AP‐1‐binding motif are marked in blue. (B) The peak values of IKZF1 binding and the presence of AP‐1‐binding sites in selected genes. (C) An example of the co‐occupancy of IKZF1 and c‐FOS in MM.1S cells.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Co‐occupancy of IKZF1 and c‐FOS at promoter/enhancer regions of actively transcribed genes in multiple myeloma (MM). Upper panel: chromatin immunoprecipitation (ChIP)‐seq data of IKZF1 and c‐FOS binding in MM.1S cells were aligned with acetylated histone H3K27 marks in the UCSC genome browser. The transcription start site (TSS) of each gene is shown with red triangles. Lower panel: Gene expression was assayed using Affymetrix U133 plus 2.0 microarrays. The data are unpaired GCRMA‐normalized expression signals for each gene in CD138‐positive cells from patients with (1) monoclonal gammopathy of undetermined significance, (2) newly‐diagnosed MM and (3) plasma cell leukaemia ( n = 8 each). ⁵³ (A) The results of three representative genes that are highly expressed in plasma cell disorders. (B) The results of three representative genes not expressed in plasma cell disorders.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Chromatin Immunoprecipitation, ChIP-sequencing, Binding Assay, Gene Expression, Expressing, Clinical Proteomics

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Biological functions of activator protein‐1 (AP‐1) family proteins in multiple myeloma (MM). (A) We determined the correlation of expression levels between IKZF1‐target genes (y‐axis) and the indicated genes (x‐axis) using the GenomicScape tool ( http://www.genomicscape.com ). ³² The data were extracted from DNA microarray analyses of gene expression in newly‐diagnosed MM patients. ⁵³ (B) Cell lysates were prepared from the indicated MM cell lines and analyzed by immunoblotting using antibodies against c‐FOS, FOSB, c‐JUN and GAPDH (loading control). (C) We examined the expression of FOS , FOSB and JUN transcripts in normal plasma cells ( n = 22), CD138‐positive cells derived from patients with monoclonal gammopathy of undetermined significance (MGUS; n = 44) and MM cells from newly‐diagnosed patients ( n = 12). The data were extracted from DNA microarray analyses deposited in the GenomicScape database by the Arkansas group. ⁵³ (D) Left panel: We transduced the indicated MM cell lines with shRNA against FOS or scrambled sequences (sh‐control) and evaluated the expression of candidate IKZF1‐target genes ( IRF4 , SLAMF7 , BSG and CD48 ) along with GAPDH (Control) and FOS (for the evaluation of knockdown efficiency) using quantitative real‐time reverse transcription‐PCR after 24 h. The data were quantified by the 2 –∆∆Ct method using GAPDH as a reference and are shown as the fold changes against the values of sh‐control‐transfected cells. * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. Right panel: Correlation between the expression levels of FOS mRNA and those of IRF4 or CD48 mRNA in MM cell lines.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Expressing, Microarray, Gene Expression, Western Blot, Control, Clinical Proteomics, Derivative Assay, shRNA, Knockdown, Reverse Transcription, Transfection, Comparison

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Direct interaction of IKZF1 and c‐FOS in multiple myeloma (MM) cells. (A) Left panel: Nuclear extracts from MM.1S and KMS12‐BM cells were immunoprecipitated with rabbit anti‐IKZF1 antibody or isotype‐matched immunoglobulin (IgG). The immunoprecipitates were analyzed by immunoblotting with specific antibodies against IKZF1, IKZF3, c‐FOS, c‐JUN, IRF4 and rabbit IgG. Input: Immunoblotting of nuclear extracts fractioned before immunoprecipitation. Right panel: The same experiments were carried out with c‐FOS immunoprecipitates. (B) The binding of the IKZF1 complex to oligonucleotides containing an IKZF consensus motif was measured by sandwich immunoassay and is shown as the relative activity against the data obtained in the absence of blocking antibodies. Antibody perturbation was carried out with isotype‐matched immunoglobulin (Control), an anti‐IKZF1 antibody, an anti‐c‐FOS antibody and a combination of the two antibodies. p < .05 by Student's t ‐test against Control ( n = 5). (C) HEK293T cells were transfected with an empty vector (Mock) or expression vectors carrying HA‐tagged full‐length IKZF1 protein, the exon 1‐exon 4 fragment, the exon 5‐exon 6 fragment, or the exon 7 fragment of IKZF1 together with a FLAG‐tagged c‐FOS expression vector. Nuclear extracts were isolated 24 h after transfection and immunoprecipitated with an anti‐HA antibody, followed by immunoblotting with antibodies against HA tag (IKZF1), FLAG tag (c‐FOS) or rabbit immunoglobulin (IgG). Red arrows denote the positions of the transfected IKZF1 fragments. (D) Structure‐based prediction of IKZF1‐c‐FOS interactions using the AlphaFold2 program.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Activity Assay, Blocking Assay, Control, Transfection, Plasmid Preparation, Expressing, Isolation, FLAG-tag

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: c‐FOS mediates lenalidomide resistance in multiple myeloma (MM) cells. (A) Left panel: MM.1S and KMS12‐BM cells were transfected with c‐FOS expression vector or empty vector (Mock) and treated with the vehicle alone (None) or 2.5 μM lenalidomide for 24 h, followed by immunoblot analysis for the expression of the indicated molecules. Right panel: MM.1S and KMS12‐BM cells were transfected with a c‐FOS expression vector or an empty vector (Mock) and treated with various concentrations of lenalidomide for 72 hours. Cell viability was determined by MTT reduction assay with a Cell Counting Kit (Fujifilm Wako Biochemicals). The graphs show the means of triplicate samples; the S.D. was less than 10% and thus omitted. * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. (B) Upper panel: Schematic representation of the IRF4 promoter region from the chromatin immunoprecipitation (ChIP)‐Atlas data. The relative positions of the putative binding sites of transcription factors are approximated by the symbols shown in the box. TSS: transcription start site. Bidirectional red arrows indicate regions that were PCR amplified in ChIP assays. Lower panel: Chromatin suspensions were prepared from KMS12‐BM cells cultured with vehicle alone (DMSO) or 2.5 μM lenalidomide for 24 h and immunoprecipitated with anti‐IKZF1 (grey bars) and c‐FOS (pink bars) antibodies or IgG (back bars). The resulting precipitates were subjected to PCR to amplify the regions shown in the upper panel. * p < .05 against IgG by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. (C) The expression of IRF4 protein and mRNA in DMSO‐ or lenalidomide‐treated KMS12‐BM cells. (D) MM.1S and KMS12‐BM cells were transfected with sh‐FOS expression vector or empty vector (Control) and treated with vehicle alone (DMSO) or 10 μM lenalidomide. (E) MM.1S and KMS12‐BM cells were treated with vehicle alone (DMSO), 10 μM lenalidomide, 20 μM T‐5224, or the combination of lenalidomide and T‐5224. Upper panels: The expressions of IRF4 and GAPDH transcripts were examined by quantitative real‐time reverse transcription‐PCR after 24 h. The results were normalized to the values of DMSO‐treated cells. Lower panels: Cell viability was determined by MTT reduction assay after 72 h and is shown as the percentage of untreated cells (%Control). The data are presented as the means of three biological replicates with S.D. (bars). * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, MTT Reduction Assay, Cell Counting, Comparison, Chromatin Immunoprecipitation, Binding Assay, Amplification, Cell Culture, Immunoprecipitation, Control, Reverse Transcription

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Graphical abstract. c‐FOS, a subunit of the activator protein‐1 (AP‐1) transactivator, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in multiple myeloma (MM) cells. Left panel: The IKZF complex binds to the enhancer/promoter regions of the genes involved in the growth and survival of MM cells such as IRF4 and SLAMF7 through the canonical IKZF‐binding motif CTTCC with c‐FOS/c‐JUN. Middle panel: Lenalidomide induces ubiquitin‐dependent degradation of IKZF1/IKZF3; however, residual c‐FOS, the level of which is often increased by lenalidomide treatment, binds to the AP‐1 consensus sequences, which present in the vicinity of IKZF‐binding sites of certain genes including IRF4 and SLAMF7 , leading to sustained expression of these genes and lenalidomide resistance. Right panel: A selective AP‐1 inhibitor, T‐5224, binds to the DNA‐binding domain of c‐FOS and mitigates the residual activity of the MM‐specific activator complex, resulting in complete IRF4 down‐regulation and augmentation of the anti‐MM effects of lenalidomide.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Ubiquitin Proteomics, Expressing, Activity Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Pathway analysis of microarray data on genes differentially expressed between control and SH-SY5Y-APP cells. b ChIP-seq screening results in SH-SY5Y cells showed that the region of AICD interaction is in the fourth intron of the JNK3 locus in chromosome 4. TSS, transcription starting site. c Validation of the ChIP-seq results. SH-SY5Y cells were transfected with either pcDNA4/V5-His or pcDNA4/V5-His-hAICD59 plasmid, and subjected to ChIP assay, using normal mouse IgG as control. Input, 5% of the sonicated chromatin. Left panel, real-time PCR results show V5, but not IgG control interacts with the JNK3 intron region ( n = 3). Right panel, representative image shows the real-time PCR products resolved by agarose gel. The molecular weight of the DNA ladder shown in bp on the left. d Real-time PCR results showing the JNK3 mRNA level in pcDNA4/V5-His (control) or pcDNA4/V5-His-hAICD59 (AICD) transfected SH-SY5Y cells ( n = 3). Student’s t- test: ** p < 0.01. Error bars represent SEM. e Luciferase assay showing the enhancer activity of the AICD-interacting site. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Microarray, Control, ChIP-sequencing, Biomarker Discovery, Transfection, Plasmid Preparation, Sonication, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Luciferase, Activity Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Intact retinae from wild-type mice were fixed, paraffin-embedded and 5 μm thick longitudinal sections were cut. The sections were stained with anti-JNK3, anti-APP and anti-Tuj1 antibodies after antigen retrieval. The inserts show lower magnification photomicrographs illustrating the immunofluorescence signal distribution across all the cell layers of the retina. APP and JNK3 were co-expressed in Tuj1 + RGCs. b Intact retinae from wild-type, APP-null or JNK3-null mice were stained with anti-APP or anti-JNK3 antibodies after antigen retrieval. The inserts show lower magnification photomicrographs illustrating the immunofluorescence signal distribution across all the cell layers of the retina. The anti-APP and anti-JNK3 antibodies were specific as no immunofluorescence signal is seen in the APP-null and JNK3-null retinal sections, respectively. c Adult, wild-type mouse retinae were retrogradely labeled with OHSt, fixed, and 5 μm thick sections were cut. The sections were stained with anti-APP and anti-JNK3 antibodies without antigen retrieval as antigen retrieval destroys the OHSt signal. Arrow heads indicate several OHSt-labeled RGCs, which co-express both APP and JNK3. Scale bar, 20 μm

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Staining, Immunofluorescence, Labeling

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Quantitative analysis of relative mRNA levels of Jnk s in intact retinae or retinae 1 day (1 d), 1 week (1 w) or 2 weeks (2 w) after ONA ( n = 3 for each time point). b Representative western blots from at least three biological repeats showing JNK expression after ONA. Intact retina or retinae 1 d, 1 w, 2 w after ONA were harvested, lysed and immunoblotted using antibodies against JNK1, JNK2, JNK3, pJNK or GAPDH. The location of molecular weight markers is shown in kDa on the right. c Representative fluorescence microscope images show immunostaining of mouse retina flat-mounts from wild-type or JNK1, JNK2, or JNK3-null mice ( n = 6 for each genotype) 14 days after ONA using an anti-Tuj1 antibody. d The bar chart shows quantitative analysis of Tuj1 + RGC numbers 14 days after ONA. Scale bar, 50 μm. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Molecular Weight, Fluorescence, Microscopy, Immunostaining

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: Representative western blots show JNK3 expression in a pcDNA3-FLAG-APP695 transfected or pcDNA3 transfected SH-SY5Y cells, e retinae from wild-type or APP-null mice, or i pcDNA4/V5-His-AICD59 or pcDNA4/V5-His transfected SH-SY5Y cells. The samples were lysed, resolved in SDS-page gel, and immunoblotted using antibodies against JNK3 or GAPDH. b , f , j The bar charts show quantitative analysis of relative JNK3 expression ( n = 3 for each experiment). Representative western blots show phosphorylative activation of JNK3 in c APP695-overexpressing or normal SH-SY5Y cells, g retinae from wild-type or APP-null mice, or k pcDNA4/V5-His-AICD59 or pcDNA4/V5-His transfected SH-SY5Y cells. JNK3 was immunoprecipitated with anti-JNK3 antibody and then detected for kinase activity with anti-phospho-JNK (pJNK) or anti-JNK3 antibody. Whole-cell lysate western blots show the downstream substrate pc-Jun73 or c-Jun level. d , h , l The bar charts show quantitative analysis of activated pJNK/mg lyate proteins or pc-Jun73/c-Jun ratio ( n = 3 for each experiment). Student’s t -test: * p < 0.05; ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Transfection, SDS Page, Activation Assay, Immunoprecipitation, Activity Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Representative western blots show JNK3 expression and overall phosphorylative activation of JNK in retinae from wild-type or APP-null mice 1 day (1 d), 1 week (1 w) and 2 weeks (2 w) after ONA ( n = 3 for each time point). Anti-JNK3, anti-pJNK and anti-GAPDH antibodies were used to detect the respective protein expression. b The bar charts show quantitative analysis of relative JNK3 expression or overall phosphorylative activation of JNK in the ipsilateral-injured retinae vs. the contralateral-uninjured retina. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Activation Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a – d Representative western blots show JNK3 expression and overall phosphorylative activation of JNK in SH-SY5Y-APP cells treated with vehicle or γ-secretase inhibitors a L-685,458 (5 μM) or c BMS 299897 (1 μM). b , d The bar charts show quantitative analysis of relative JNK3 expression and overall phosphorylative activation of JNK normalized to the GAPDH ( n = 3 for each treatment). e Representative western blots show phosphorylative activation of JNK3 in retinae from vehicle-treated or γ-secretase inhibitor BMS 299897 treated mice 2 weeks after ONA. f Representative fluorescence microscope images show immunostaining of mouse retina flat-mounts from vehicle or γ-secretase inhibitor BMS 299897 treated mice 2 weeks after ONA using anti-Tuj1 antibody. Scale bar, 50 μm. g The bar chart shows quantitative analysis of the surviving Tuj1 + RGC number in injured mouse retinae 14 days after ONA ( n = 4 for each treatment). Student’s t -test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Activation Assay, Fluorescence, Microscopy, Immunostaining

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: ONA induces upregulation of APP expression. APP is sequentially cleaved by α- or β-secretase and γ-secretase, producing several fragments and AICD, which is inhibited by APP deficiency or BMS 299897. AICD is translocated to the nucleus and upregulates JNK3 expression (probably functioning with other partners). Phosphorylated JNK3 then contributes to RGC death

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Expressing

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 3. HDAC3 is required for extracellular matrix homeostasis and remodeling of semilunar valves. A, Movat’s pentachrome staining of remodeling aortic valve shows an increase in proteoglycans (arrows, blue) from E13.5 to E18.5 in Hdac3Isl1KO aortic valves with a reduction and remodeling of proteoglycans in control valves. B, Movat’s pentachrome staining of remodeling pulmonic valve shows an increase in proteoglycans (arrows, blue) from E13.5 to E18.5 in Hdac3Isl1KO pulmonic valves with a reduction and remodeling of proteoglycans in control valves. C, Masson’s trichrome staining demonstrates disorganized collagen expression (arrow, blue) in Hdac3Isl1KO E18.5 aortic valve. D, Masson’s trichrome staining demonstrates disorganized collagen expression (arrow, blue) in Hdac3Isl1KO E18.5 pulmonic valve. E, Verhoeff-Van Gieson (VVG) staining shows disorganized collagen expression (arrow, red) in Hdac3Isl1KO E18.5 aortic valve. F, Verhoeff-Van Gieson staining shows disorganized collagen expression (arrow, red) in Hdac3Isl1KO E18.5 aortic valve. G, Movat’s pentachrome-stained sections showincreasedproteoglycans(blue)inaorticvalvecuspsofE18.5Hdac3Mef2CKOhearts.H,Movat’spentachromestainingrevealsincreasedproteoglycans(blue) in E18.5 Hdac3Mef2CKO pulmonic valve. I, representative images of cleaved caspase-3 immunostaining in E13.5 semilunar valves. Arrows, positive staining. J, quantification of cleaved caspase-3-positive cells in E13.5 control and Hdac3Isl1KO aortic valves. K, quantification of cleaved caspase-3-positive cells in E13.5 control and Hdac3Isl1KO pulmonic valves. L, schematic model depicting disorganized extracellular matrix and reduced apoptosis in Hdac3Isl1KO E13.5 semilunar valves. M, schematic model showing hyperplastic, enlarged, and disorganized Hdac3Isl1KO E18.5 semilunar valves.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Staining, Control, Expressing, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 4. HDAC3 is a critical regulator of TGF- signaling pathway. A–E, IPA of microarray data from E9.5 Hdac3Isl1KO hearts. A, cardiovascular development and function subcategories significantly dysregulated in E9.5 Hdac3Isl1KO hearts, sorted by significance, from IPA diseases and function analysis. B, classes of congenital heart anomalies affected in E9.5 Hdac3Isl1KO hearts, sorted by significance, from IPA diseases and function analysis. C, 30 most significant upstream regulatorsfromIPAupstreamanalysisofE9.5Hdac3Isl1KOmicroarraydata.D,clusteredheatmapofdifferentiallyexpressedgenesrelatedtoupstreamregulator TGF-1 from IPA upstream analysis of Hdac3Isl1KO microarray data. E, top 30 upstream regulators, based on number of associated genes differentially expressed in E9.5 Hdac3Isl1KO hearts from IPA upstream analysis. F, transcripts for Sumo1, Nrp2, Smad4, Snai1, Tgf-1, and Kpnb1 were detected by real-time qPCR in Hdac3F/F and Hdac3Isl1KO outflow tract with right ventricle derived from E9.5 embryos (mean S.E. (error bars), n 3). G–J, ChIP-qPCR analysis of HDAC3 recruitment to promoter-proximal regions of TGF- pathway genes performed in wild-type E9.5 outflow tract (mean S.E., n 3). K and L, ELISA for TGF-1 (K) and phospho-SMAD2/3 (L) was performed in Hdac3F/F and Hdac3Isl1KO outflow tracts (mean S.E., n 3 (K) and n 4 (L)). M, phospho-SMAD2/3 immunostaining (arrows) in E9.5 Hdac3Isl1KO; R26R-LacZ/ hearts.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Microarray, Derivative Assay, ChIP-qPCR, Enzyme-linked Immunosorbent Assay, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 8. HDAC3 epigenetically silences TGF-1 within valvular mesenchymal cells by recruiting PRC2 complex to the NCOR complex. A, relative mRNA levels of TGF-1 in isolated cardiac endothelial cells (EC) or cardiac mesenchymal cells (MC) from E10.5 Hdac3F/F outflow tract cushion explants, infected either with CRE or GFP control lentivirus. B, relative mRNA levels of TGF-1 in isolated cardiac endothelial cells or dissected semilunar valves (SL) derived from Hdac3Isl1KO and Hdac3F/F E14.5 hearts. C, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in isolated cardiac endothelial cells or cardiac mesen- chymal cells from E10.5 outflow tract cushion explants. D, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in isolated cardiac endothelial cells or dissected semilunar valves from E14.5 hearts. E–K, ChIP-qPCR analysis of H3K27 trimethylation (E), H3K27 acetylation (F), RNA polymerase II (G), CREBBP (H), EZH2 (I), EED (J), and SUZ12 (K) upstream of TGF-1 in Hdac3Isl1KO and control E14.5 semilunar valves. L, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in E14.5 valvular mesenchymal cells infected with either control shRNA (sc-shRNA), EZH2 shRNA, or NCOR1 shRNA. M, ChIP-qPCR analysis of NCOR1 upstream of TGF-1 in Hdac3Isl1KO and control E14.5 semilunar valves. N, Co-ChIP for HDAC3 and either EZH2, NCOR1, H3K27me3, H3K27ac, or polymerase II upstream of TGF-1 in E14.5 wild-type dissected semilunar valves. O, total lysates from E14.5 wild-type pooled semilunar valves were immunoprecipitated (IP) by EZH2 antibody, and Western blot was performed using HDAC3 antibody. -Tubulin is shown as an input control. HDAC3 was quantified and normalized to total input -tubulin using ImageJ software (mean S.E. (error bars), n 3). P, ChIP-qPCR analysis of H3K27me3 upstream of TGF-1 in E14.5 valvular mesenchymal cells infected with either control shRNA, EZH2 shRNA, or NCOR1 shRNA.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Isolation, Infection, Control, Derivative Assay, ChIP-qPCR, shRNA, Immunoprecipitation, Western Blot, Software

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 9. HDAC3 functions in a deacetylase-independent manner to regulate EndMT and epigenetic silencing of TGF-1. A, HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression constructs were transfected in HEK-293T cells. Expression was detected by Western blot from whole cell lysates using FLAG antibody. GAPDH is shown as a loading control. B, HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression was quantified and normalized to total input GAPDH using ImageJ software (mean S.D. (error bars), n 3). C, HDAC activity of HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression was quantified against a pseudosubstrate. D, EndMT assay of control- or Cre-infected E10.5 Hdac3F/F outflow tract cushion explants co-infected with GFP, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses, imaged 24 h after isolation. E, quantification of average radial migration, measured in eight directions, of control- or Cre-infected E10.5 Hdac3F/F outflow tract cushion explants co-infected with GFP, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses, measured 24 h after isolation(mean S.E.(errorbars),n3).F,relativemRNAlevelsofTgf-1incontrol-orCre-infectedE14.5Hdac3F/Fvalvularmesenchymalcellsco-infectedwith control, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses (mean S.E., n 3). G, a 1309-bp TGF-1 promoter luciferase reporter (WT) or a truncated, 1267-bp TGF-1 promoter luciferase reporter, lacking an HDAC3-enriched region (mutant) were transfected in murine endothelial cells with and without an HDAC3-FLAG or HDAC3H134A,H135A-FLAG expression plasmid. Induction is represented as a ratio of firefly and Renilla luciferase activity. H–L, ChIP-qPCR analysis of H3K27 acetylation (H), H3K27 trimethylation (I), EZH2 (J), EED (K), and SUZ12 (L) upstream of TGF-1 in control- or Cre-infected E14.5 Hdac3F/F valvular mesenchymal cells co-infected with control, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses (mean S.E., n 3).

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Histone Deacetylase Assay, Expressing, Construct, Transfection, Western Blot, Control, Software, Activity Assay, Infection, Isolation, Migration, Luciferase, Mutagenesis, Plasmid Preparation, ChIP-qPCR

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 10. Summary of phenotypes and proposed model of HDAC3 function within second heart field progenitor cells and second heart field-derived mesenchymal cells. A, loss of HDAC3 in second heart field progenitor cells leads to outflow tract and semilunar valve pathologies. Strikingly, genetic deletion of HDAC3 in differentiated mesenchymal and smooth muscle cells (Hdac3TaglnKO) recapitulates the majority of these phenotypes. However, deletion of HDAC3 in differentiated cardiomyocytes (Hdac3Myh6KO) or endothelial cells (Hdac3Cdh5KO) did not recapitulate the cardiovascular defects observed in Hdac3Isl1KO embryos. B, Hdac3Isl1KO hearts exhibit disorganized collagen and elastin within dilated aortic walls and hyperplastic semilunar valves containing activated myofibroblasts and disorganized extracellular matrix. In both control and Hdac3Isl1KO cardiac endothelial cells, the upstream regulatory region of TGF-1 is occupied by RNA polymerase II and CREBBP and exhibits H3K27 acetylation concomitant with TGF-1 expression. In control semilunar valves, endothelial cells undergo EndMT to become mesen- chymalcells.Inthesemesenchymalcells,NCOR1,HDAC3,andPRC2complex(EZH2,EED,andSUZ12)arerecruitedtotheupstreamregulatoryregionofTGF-1,which becomes trimethylated on histone H3 Lys-27, and TGF-1 expression is epigenetically silenced. In Hdac3Isl1KO hearts, EZH2, EED, and SUZ12 are not recruited to the TGF-1 regulatory region, RNA polymerase II and CREBBP are present, and histone H3 Lys-27 remains acetylated, favoring aberrant expression of TGF-1 in mesen- chymal cells. TGF-1 activates mesenchymal cells to become myofibroblasts, which perpetuate EndMT and activation of mesenchymal cells through continued induction of TGF-1 and aberrant expression of extracellular matrix, including proteoglycans and collagen.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Derivative Assay, Control, Expressing, Activation Assay