Journal: Frontiers in Oncology

Article Title: The MYC Paralog-PARP1 Axis as a Potential Therapeutic Target in MYC Paralog-Activated Small Cell Lung Cancer

doi: 10.3389/fonc.2020.565820

Figure Lengend Snippet: Effects of JQ1 and BMN673 on the HR repair pathway. (A) Representative images of Rad51 immunofluorescence staining in SCLC cells treated with drugs as indicated for 24 h. Scale bar, 20 μm. Quantification of Rad51 fluorescence intensities from three independent experiments was shown as mean ± S.D. **P < 0.01; ***P < 0.001. (B) Western blot analysis of Rad51 expression in SCLC cells treated with drugs as indicated for 24 h. (C) Western blot analysis of indicated proteins in DMS273 cell upon knockdown of BRD2 or BRD3 or BRD4 . (D) ChIP-PCR analysis displays the decreased association of BRD4 in the promoter of RAD51 in DMS273 and H526 cells treatment with JQ1 for 24 h. *P < 0.05; **P < 0.01; ns, no significance. (E) Western blot analysis of phosphorylated-DNA-PKcs and phosphorylated-RPA32 in MYC paralog-dependent and independent-SCLC cells treated with indicated drugs for 24 h. β-actin was used as a loading control.

Article Snippet: Antibodies against the following proteins were used for our studies: c-MYC (1:1,000, abcam ab32072), PARP1 (1:1,000, Affinity DF7198), GAPDH (1:10,000, Affinity AF7021), Rad51 (1:10,000, Abcam ab133534), PARP (1:1,000, CST 9542), γH2AX (1:1,000, CST 2577), p-CHK1 (1:1,000, Ser317, CST 12302), BRD2 (1:1,000, Proteintech 22236-1-AP), BRD3 (1:1,000, Proteintech 11859-1-AP), BRD4 (1:500, Bethyl A301-985A50), p-DNA-PKcs (1:1,000, Abcam ab124918), p-RPA32 (1:5,000, Ser4/Ser8, Novus), MYCN (1:1,000, CST 9405), MYCL (1:1,000 R&D, AF4050) and β-actin (1:10,000, Transgen HC201-02).

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing, Knockdown, Control

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: Melanocyte differentiation gene expression is suppressed by BET inhibition. a Melb-a cells were differentiated in the presence of vehicle (VC) or 500 nM (+)JQ1. Cells were harvested at the indicated time-points and subjected to qRT-PCR. Tyr , Tyrp1 , or Trpm1 levels were normalized to that of Rpl7 . The data are the average of three independent experiments. Standard error bars are shown. Statistically significant difference between VC and (+)JQ1 treated samples at each time point are shown (* p < 0.05, ** p < 0.01, *** p < 0.001). b Protein extracts from cells cultured in the absence (vehicle treated) or presence of (+)JQ1 were subjected to immunoblot analysis using the indicated antibodies. The figure is representative of three or more experiments. c Neonatal human epidermal melanocytes (NHEMs) were cultured in the absence (vehicle treated) or presence of 500 nM (+)JQ1 for the indicated number of days. Harvested cells were subjected to immunoblot analysis using the indicated antibodies. The figure is representative of three or more experiments. d NHEMs were cultured as in c and then subjected to qRT-PCR. The TYRP1 level was normalized to that of RPL9 . The data are the average of three independent experiments. Standard error bars are shown. Statistically significant difference between VC and (+)JQ1 treated samples at each time point are shown (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Gene Expression, Inhibition, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: Depletion of BRD4 or BRD2 in Melb-a cells suppresses expression of melanocyte-specific genes. a Melb-a cells were transfected with control siRNA or siRNA that targets BRD2, BRD3, or BRD4, then differentiated for 48 h. Immunoblot analysis was performed with the indicated antibodies. The immunoblot is representative of two or more experiments. b Melb-a cells were transfected with a control (siC) or a pool of siRNAs that targets BRD4 or BRD2 (distinct from those used in a ) then differentiated for 48 h. Immunoblot analysis was performed with the indicated antibodies. The immunoblot is representative of two or more experiments. c , d BRD4 or BRD2 depleted cells treated as described in b were subjected to qRT-PCR. Brd4 , Brd2 , Tyr , and Tyrp1 levels were normalized to that of Rpl7 . The data in c and d are the average of three independent experiments. Standard error bars are shown. Statistically significant differences between siC, siBRD4, and siBRD2 transfected cells are shown (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: BRD4 and BRD2 binding overlap H3K27ac and canonical MITF-binding sites at melanocyte-specific loci. Publicly available ChIP-seq data in normal human melanocytes, SK-MEL-5 melanoma cells (GSM1968282), 501 melanoma cells (GSM1517751), and neonatal human epidermal melanocytes (NHEM) (GSM2842798) with the indicated antibodies at the a TYR and b TYRP1 loci

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Binding Assay, ChIP-sequencing

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: BRD4 and BRD2 occupancy at MITF-binding sites is associated with chromatin accessibility in Meb-a cells. a , b Melb-a cells were differentiated for 24 h in the presence of vehicle (VC) or 500 nM (+)JQ1. Cells were cross-linked and control IgG or an antibody to BRD4 or BRD2 was used for ChIP. BRD4 and BRD2 binding to the MITF-binding sites to the promoter regions of Tyr and Tyrp1 was assayed by qPCR. BRD4 and BRD2 enrichment is shown relative to IgG enrichment and normalized to a non-specific region ( IgH enhancer). The data are the average of three independent experiments. Standard error bars are shown statistically significant differences between VC and (+)JQ1 treated samples are shown (*< 0.05, ** p < 0.01, *** p < 0.001). c Melb-a cells were differentiated as described in a , b . FAIRE enrichment at the MITF-binding sites in the Tyr and Tyrp1 promoters was determined by qPCR. Enrichment at the IgH enhancer is shown as a negative control. The data are the average of two independent experiments performed in triplicate Statistically significant differences between VC and (+)JQ1-treated samples are shown (*< 0.05, ** p < 0.01, *** p < 0.001). D. NHEMs were treated with 500 nM inactive (−)JQ1 or 500 nM active (+)JQ1 for 48 h, then harvested for FAIRE. FAIRE enrichment at the MITF-binding site in the TYRP1 promoter was determined by qPCR. FAIRE enrichment at the CD25 promoter is shown as a negative control. The data is the average of two independent experiments performed in triplicate Statistically significant differences between (−)JQ1 and (+)JQ1-treated samples are shown (*< 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Binding Assay, Control, Negative Control

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: BRD4 and BRD2 promote MITF occupancy at the Tyr and Tyrp1 promoters. a Melb-a cells were differentiated and cross-linked as described in Fig. . ChIPs were performed with a control IgG or an antibody to MITF. MITF enrichment is shown relative to IgG enrichment and normalized to a non-specific region ( IgH enhancer). The data are the average of three independent experiments. Standard error bars are shown. Statistically significant differences between (−)JQ1 and (+)JQ1 treated samples are shown (*< 0.05, ** p < 0.01, *** p < 0.001). b Melb-a cells were transfected with control siRNA (siC) or a pool of siRNAs that target either BRD4 or BRD2. Cells were differentiated for 24 h then harvested for ChIPs. MITF enrichment is shown relative to IgG enrichment and normalized to a non-specific region ( IgH enhancer). The data are the average of two independent experiments performed in triplicate. Standard error bars are shown. Statistically significant differences between siC-transfected cells and siBRD4 and siBRD2-transfected cells are shown (*< 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Control, Transfection