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