Journal: Cell reports

Article Title: Bromodomain-containing protein 4 activates androgen receptor transcription and promotes ovarian fibrosis in PCOS.

doi: 10.1016/j.celrep.2023.113090

Figure Lengend Snippet: Figure 2. TGF-b/p-Smad2/3 signaling upregulates BRD4 transcription Primary rat ovarian granular cells (GCs) and human granulosa-like tumor cell line (KGN) were pretreated with LY2109761 (LY210), a p-Smad2/3-selective inhibitor, and subsequently with 500 nM DHT or 10 ng/mL TGF-b. (A) Western blot analysis of GCs for p-Smad2, p-Smad3, TGF-b, and BRD4 proteins after treatment with LY210 (0, 2, 5 mM)/DHT. (B) Quantitative analysis of (A). (C) Western blot analysis of KGN for p-Smad2, p-Smad3, BRD4, Ac-H4, Ac-H3, and Col1a proteins after treatment with LY210 (10 mM)/DHT. (D) Quantitative analysis of (C). (E) Western blot analysis of KGN for p-Smad2, p-Smad3, and BRD4 proteins after treatment with LY210 (10 mM)/TGF-b1. (F) Quantitative analysis of (E). (G) ChIP assay. KGN cells were treated with LY210 (10 mM)/TGF-b1, followed by immunoprecipitation with antibody to isotype-matched immunoglobulin (V) or p- Smad2, respectively, and then the genomic DNA (input) and the antibody-bound DNA fragments of BRD4 promoter were PCR amplified. The PCR products were analyzed on agarose gel. (H) Quantitative analysis of (G), which was normalized with input DNA. (I) CoIP assay. KGN cells were treated with LY210 (10 mM)/TGF-b1, then tested for p-Smad2 and p-Smad3 (input). The same cell lysates were immunoprecipitated with isoform-matched immunoglobulin (Ig) or antibody to p-Smad2, and then immunoprecipitants were analyzed for p-Smad3 and p-Smad2 by western blot assay. (J) Quantitative analysis of (I). Three independent experiments were performed with similar results. Data are shown as mean ± SEM. *p < 0.05.

Article Snippet: The lysates of KGN and rat ovaries were first immunoprecipitated with antibody to p-Smad2 (p-Smad2, ET1702-34, HUABIO), HIF-1a (NB100-479SS, NOVUS), BRD4 (83375S, CST, USA), or isoform-matched immunoglobulin (Ig) followed by Protein A + G Agarose (P2019, Beyotime), and then the immunoprecipitants were assayed by Western blot with antibody to p-Smad3, BRD4, HIF-1a, or Ac-H4, respectively.

Techniques: Western Blot, Immunoprecipitation, Agarose Gel Electrophoresis, Co-Immunoprecipitation Assay

Journal: Cell reports

Article Title: Bromodomain-containing protein 4 activates androgen receptor transcription and promotes ovarian fibrosis in PCOS.

doi: 10.1016/j.celrep.2023.113090

Figure Lengend Snippet: Figure 7. A schematic diagram of BRD4 ab- erration, sequential AR activation, and ovarian fibrosis Persistent AR activation in the ovary promotes oxidative stress and the release of TGF-b, which causes ovarian fibrosis in PCOS. Epigenetic fibrotic factors promote TGF-b/Smad2/3 signaling activation, and increased BRD4 transcription. Upregulated BRD4 is associated with HIF-1a and acetylated histones and binds to AR promoter, activating AR transcription. Selective BRD4 in- hibitor JQ1 suppresses BRD4 activity and down- regulates AR, resulting in the amelioration of ovarian fibrosis and ovarian function.

Article Snippet: The lysates of KGN and rat ovaries were first immunoprecipitated with antibody to p-Smad2 (p-Smad2, ET1702-34, HUABIO), HIF-1a (NB100-479SS, NOVUS), BRD4 (83375S, CST, USA), or isoform-matched immunoglobulin (Ig) followed by Protein A + G Agarose (P2019, Beyotime), and then the immunoprecipitants were assayed by Western blot with antibody to p-Smad3, BRD4, HIF-1a, or Ac-H4, respectively.

Techniques: Activation Assay, Activity Assay

Journal: BMC Cancer

Article Title: NSDHL contributes to breast cancer stem-like cell maintenance and tumor-initiating capacity through TGF-β/Smad signaling pathway in MCF-7 tumor spheroid

doi: 10.1186/s12885-024-13143-3

Figure Lengend Snippet: Fig. 3 NSDHL knockdown led to a significant reduction in the secretion of TGF-β 1 and 3, phosphorylation of Smad2 and 3, and expression of SOX2 and NANOG in MCF-7 spheroids. A-C. Scatter plot, volcano plot, and Venn diagram of differentially expressed genes (DEGs) comparing NSDHL siRNA (siNSDHL)-and control siRNA (siCtrl)-transfected MCF-7 spheroids. D. Top 10 enriched KEGG pathway analyses of DEGs. E-F. Representative images of MCF-7 spheroids and western blotting images of phopho-Smad2 and 3, Smad2, and Smad3 in the presence or absence of A-83-01 (50 µM) for 7 days. Scale bar: 100 μm. G. Immunoassays of secreted TGF-β1, 2, and 3 levels measured from conditioned medium of siNSDHL- or siCtrl-spheroids (n = 6). H-K. Representative western blot images and quantification data of phopho-Smad2 and 3, Smad2, Smad3, SOX2, and NANOG in siNSDHL spheroids relative to siCtrl spheroids (n = 3). L. Relative mRNA levels of SOX2 and NANOG were assessed by qRT-PCR analysis in siNSDHL-spheroids and siCtrl-spheroids (n = 4). Bar graphs show the mean ± standard deviation of at least three independent experiments. *P < 0.05, ** P < 0.01, ***P < 0.001 as compared to monolayer using unpaired t-tests

Article Snippet: For western blot, immunofluorescence staining, immunohistochemistry, and flow cytometry we used the following antibodies: β-actin (sc-47778), Vimentin (sc6260), SOX2 (sc-365823), and NANOG (sc-293121) from Santa Cruz (CS, USA); NSDHL (ab190353), mCherry (ab167453), CD24 (ab202073), CD44 (ab6124), EpCAM (ab20160) and ALDH1A1 (ab227964) from Abcam (Cambridge, UK); Snail (#3879), PARP (46D11) (#9532), Cleaved PARP (Asp214) (#9544), Smad2 (#5339), Smad3 (#9523), Phospho-Smad2 (Ser465/467) (#3108) and Phospho-Smad3 (Ser423/425) (#9520) from Cell Signaling (Beverly, MA, USA); CD49f (NBP1-85747) from Novus Biologicals (Centennial, CO, USA); FITC-conjugated CD24 (555427), PE-conjugated CD44 Monoclonal Antibody (IM7) (550989), APC-conjugated CD44 Monoclonal Antibody (G44-26) (559942), and APC-conjugated EpCAM (347200) from BD Biosciences (Mansfield, MA, USA); FITC-conjugated CD49f (313606) from Biolegend (San Diego, CA, USA).

Techniques: Knockdown, Phospho-proteomics, Expressing, Control, Transfection, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: BMC Cancer

Article Title: NSDHL contributes to breast cancer stem-like cell maintenance and tumor-initiating capacity through TGF-β/Smad signaling pathway in MCF-7 tumor spheroid

doi: 10.1186/s12885-024-13143-3

Figure Lengend Snippet: Fig. 5 NSDHL knockdown reduced the BCSC population with CD44+/CD24 − phenotype and ALDH activity in tumor spheroid-injected xenograft tumor tissues, accompanied by decreased Smad2/3 phosphorylation and SOX2 expression. A-D. Representative flow cytometry histograms, dot plots, and quantification of CD44 + and CD24- cells in control (shCtrl) and NSDHL shRNA (shNSDHL)-transduced MCF-7 tumors (n = 4). E-F. Representative flow cytometry dot plots and quantification of ALDH + cells in shCtrl- and shNSDHL MCF-7 tumors using ALDEFLUOR assay (n = 4). G. Representative double immunohistochemistry images of CD44 + and ALDH1A1 + cells in shCtrl and shNSDHL MCF-7 tumors. CD44 (green), ALDH1A1(red). Double CD44+/ ALDH1A1 + cells (arrow). H-O. Representative western blot images and quantification data of phopho-Smad2 and 3, Smad2, Smad3, SOX2, and NANOG levels in shNSDHL MCF-7 tumors relative to shCtrl MCF-7 tumors (n = 5). All graphs show the mean ± standard deviation. *P < 0.05, compared with shCtrl using unpaired t-tests

Article Snippet: For western blot, immunofluorescence staining, immunohistochemistry, and flow cytometry we used the following antibodies: β-actin (sc-47778), Vimentin (sc6260), SOX2 (sc-365823), and NANOG (sc-293121) from Santa Cruz (CS, USA); NSDHL (ab190353), mCherry (ab167453), CD24 (ab202073), CD44 (ab6124), EpCAM (ab20160) and ALDH1A1 (ab227964) from Abcam (Cambridge, UK); Snail (#3879), PARP (46D11) (#9532), Cleaved PARP (Asp214) (#9544), Smad2 (#5339), Smad3 (#9523), Phospho-Smad2 (Ser465/467) (#3108) and Phospho-Smad3 (Ser423/425) (#9520) from Cell Signaling (Beverly, MA, USA); CD49f (NBP1-85747) from Novus Biologicals (Centennial, CO, USA); FITC-conjugated CD24 (555427), PE-conjugated CD44 Monoclonal Antibody (IM7) (550989), APC-conjugated CD44 Monoclonal Antibody (G44-26) (559942), and APC-conjugated EpCAM (347200) from BD Biosciences (Mansfield, MA, USA); FITC-conjugated CD49f (313606) from Biolegend (San Diego, CA, USA).

Techniques: Knockdown, Activity Assay, Injection, Phospho-proteomics, Expressing, Flow Cytometry, Control, shRNA, Immunohistochemistry, Western Blot, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: Treatment with a PPAR-γ Agonist Protects Against Hyperuricemic Nephropathy in a Rat Model

doi: 10.2147/DDDT.S247091

Figure Lengend Snippet: RGTZ abrogates TGF-β-Smad3 signaling in the kidneys of HN rats. ( A ) Kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against TGF-β1 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ( B ) Expression levels of TGF-β1 were quantified by densitometry and normalized to GAPDH. ( C ) The kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against p-Smad3, Smad3, or GAPDH. ( D ) Expression levels of p- Smad3 were quantified by densitometry and normalized to Smad3. RGTZ, rosiglitazone. Data are represented as the mean ± SEM. *p < 0.05 vs sham group; #p< 0.05 vs sham + RGTZ group. **p < 0.05 vs HN group.

Article Snippet: Antibodies to Smad3 and p-Smad3 were purchased from Hangzhou HuaAn Biotechnology Co., Ltd. (Hangzhou, China).

Techniques: Western Blot, Expressing