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

Journal: Frontiers in Medicine

Article Title: Aging-enhanced autophagy activity promotes fibrotic progression via the TGF-β2/Smad signaling pathway in trabecular meshwork cells—a new insight from POAG

doi: 10.3389/fmed.2024.1534120

Figure Lengend Snippet: Antibodies used for Western blot.

Article Snippet: Smad3 rabbit mAb , Abmart , T55013 , 1:1,500.

Techniques: Western Blot

Journal: Frontiers in Medicine

Article Title: Aging-enhanced autophagy activity promotes fibrotic progression via the TGF-β2/Smad signaling pathway in trabecular meshwork cells—a new insight from POAG

doi: 10.3389/fmed.2024.1534120

Figure Lengend Snippet: Autophagy enhances TGF-β2/Smad-mediated TM cell fibrosis. (A–C) The protein levels of FN, αSMA, Smad3, and p-Smad3 were detected by a Western blot. GAPDH was used as an internal control. Quantified protein levels were displayed as bar graphs (below). (A) TM cells were stimulated with different concentrations of TGF-β2 for 48 h. (B) TM cells before and after pre-treating with 2 mM 3-MA for 24 h, then incubated in the presence or absence of 5 ng/mL TGF-β2 for 48 h. Data are presented as Mean ± SEM from three independent experiments (* P < 0.05, *** p < 0.001, **** p < 0.0001 vs. control group; ### p < 0.001 vs. 3-MA treatment group; one-way ANOVA) Rapa, rapamycin; 3-MA, 3-methyladenine. (C) TM cells before and after pre-treating with 10 μM SB-431542 for 2 h, then incubated in the presence or absence of (72 h) H 2 O 2 or (48 h) H 2 O 2 plus (24 h) Rapa. Data are presented as Mean ± SEM from three independent experiments (ns, not significant; **** p < 0.0001 vs. control group; #### p < 0.0001 vs. H 2 O 2 -treated group; &&&& p < 0.0001 vs. H 2 O 2 -Rapa cotreatment group; one-way ANOVA) Rapa, rapamycin; SB-4, SB-431542. (D) TM cells were treated with 5 ng/mL TGF-β2 for 48 h. The protein levels of LC3 and SQSTM1 were detected by a Western blot. GAPDH was used as an internal control. Quantified protein levels were displayed as bar graphs (below). (E) Confocal images of LC3 (green), SQSTM1 (red), and DAPI (blue). Scale bar, 50 μm.

Article Snippet: Smad3 rabbit mAb , Abmart , T55013 , 1:1,500.

Techniques: Western Blot, Control, Incubation