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: The FASEB Journal

Article Title: Mitigation of Ferroptosis in Diabetic Kidney Disease Through Mesenchymal Stem Cell Intervention via the Smad2/3/METTL3/S1PR1 Axis

doi: 10.1096/fj.202403207r

Figure Lengend Snippet: FIGURE 1 | Effects of MSC intervention on renal pathology, inflammation, Smad2/3 activation, and ferroptosis in DKD Mice: (A, B) Schematic representation of the modeling and intervention process in C57 and db/db mice. (C) Blood glucose levels monitored across the experimental groups. (D) Comparison of Kidney weight/Body weight ratios between groups. (E) H&E staining to assess renal pathological changes, and Masson staining to quantify renal fibrosis. Representative histopathological images are provided, with Masson staining quantification depicted on the right. (F) ELISA detection of IL-6 and TNF-α levels as markers of inflammation. (G) Western blot analysis of Smad2, Smad3, and their phosphorylated forms (p- Smad2, p-Smad3), with β-Actin as the loading control. (H) Immunohistochemistry to visualize p-Smad2/3 nuclear translocation, with magnification at 200× and nuclear staining shown in blue. (I) Western blot analysis of ferroptosis-related proteins (ACSL4, GPX4, and SLC7A11), using β-Actin as the loading control. Control Group: C57 Mice on a standard diet, administered an equal volume of physiological saline via tail vein injection. DKD Group: db/db mice with an established T2DM model, receiving physiological saline via tail vein injection. DKD + MSCs Group: db/db mice with an established T2DM model, treated with MSCs (1 × 106 cells/0.2 mL per mouse) via weekly tail vein infusion. Interventions were conducted over an 8- week period. Each group consisted of 6 mice, and all mice participated in and underwent all experimental assessments. All in vitro experiments were performed with at least three independent replicates (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: Primary antibodies targeting Smad3 (Affinity, 1:100) and Smad2 (Huabio, 1:100) were applied and incubated overnight at 4°C.

Techniques: Activation Assay, Comparison, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Immunohistochemistry, Translocation Assay, Saline, Injection, In Vitro

Journal: The FASEB Journal

Article Title: Mitigation of Ferroptosis in Diabetic Kidney Disease Through Mesenchymal Stem Cell Intervention via the Smad2/3/METTL3/S1PR1 Axis

doi: 10.1096/fj.202403207r

Figure Lengend Snippet: FIGURE 3 | MSCs inhibit the activation of the Smad2/3 pathway and ferroptosis. (A, B) Western blot analysis of Smad2, Smad3, and their phos- phorylated forms (p-Smad2, p-Smad3), with β-Actin as the loading control. (C) Immunofluorescence analysis of Smad2/3 nuclear localization. Nuclei stained white, while Smad2/3 are labeled green using goat anti-rabbit IgG H&L (Alexa Fluor 488) secondary antibody. (D) Western blot analysis of ferroptosis-related proteins (ACSL4, GPX4, and SLC7A11), with β-Actin as the loading control. Grouping: Control Group: MPC5 Cells cultured in standard medium. HG Group: MPC5 Cells treated with 25 μmol/L glucose in standard medium for 72 h. HG + MSCs Group: MPC5 Cells co-cultured with MSCs in medium containing 25 μmol/L glucose for 72 h. All in vitro experiments were performed with at least three independent replicates (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: Primary antibodies targeting Smad3 (Affinity, 1:100) and Smad2 (Huabio, 1:100) were applied and incubated overnight at 4°C.

Techniques: Activation Assay, Western Blot, Control, Immunofluorescence, Staining, Labeling, Cell Culture, In Vitro