Journal: Oncogenesis

Article Title: CRB3 downregulation confers breast cancer stem cell traits through TAZ/β-catenin

doi: 10.1038/oncsis.2017.24

Figure Lengend Snippet: CRB3 downregulation induces activation of TAZ and β-catenin. ( a ) Western blot of CRB3 in human mammary epithelial cells. ( b ) Western blot of the Hippo pathway components. ( c ) Western blot of β-catenin, β-TrCP and βTrcp substrates Smad4 after CRB3 knockdown. ( d ) Cytoplasmic and nuclear expression of TAZ and β-catenin. ( e ) MCF 10A cells were transfected with the TEAD-luciferase reporter and TEAD promoter activity was detected by luciferase assay. ( f ) Real-time PCR showed that CRB3 knockdown increased expressions of TAZ target gene CYR61 and CTGF. ( g ) Luciferase assay of TOPFLASH or control FOPFLASH as a measure of β-catenin/TCF activity. ( h ) Real-time PCR of TERT. ( i ) Localization of TAZ, β-catenin and YAP as shown by IF. All data are presented as mean±s.e.m. and statistical significance was calculated using a two-tailed t -test.

Article Snippet: CRB3 antibody (292449), SOX2 antibody (17320), cMyc antibody (764) and p-TAZ antibody (17610) were purchased from Santa Cruz (Dallas, TX, USA); NANOG antibody (55241) was purchased from Sangon Biotech (Shanghai, China); OCT4 antibody (WL1005a) was purchased from Wanleibio (Shenyang, China); Scribble antibody (4475), LKB1 antibody (3050), β-catenin antibody (9562 s), Snail antibody (3879); β-TrCP antibody (4394); Smad4 antibody (12747) and the Hippo signaling antibody sampler kit (8579) were purchased from Cell Signaling (Beverly, MA, USA); Vimentin antibody (10366), LaminA antibody (10298) and GAPDH antibody (HRP-60004) were obtained from Proteintech (Wuhan, China); E-cadherin antibody (610405) and N-cadherin antibody (610920) were purchased from BD Biosciences (San Jose, CA, USA); α-SMA antibody (A2547) and DLG5 antibody (000555) were purchased from Sigma-Aldrich; LGL1 antibody (H00003996-M01) and LGL2 antibody (H00003993-M06) were purchased from Abnova (Taiwan, China); TAZ antibody (MAB7210) was obtained from R&D Systems (Minneapolis, MN, USA); ZO-1 antibody (339100) was obtained from ThermoFisher Scientific (Carlsbad, CA, USA); The AMOT antibody was produced by Genemed Synthesis, Inc. (South San Francisco, CA, USA).

Techniques: Activation Assay, Western Blot, Knockdown, Expressing, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Control, Two Tailed Test

Journal: Thrombosis journal

Article Title: GDF15 affects venous thrombosis by promoting EndMT through smad2/p-smad2 pathway.

doi: 10.1186/s12959-023-00547-7

Figure Lengend Snippet: Fig. 4 Cell mRNA expression, WB and double luciflucidase assay. (A) After stimulating endothelial cells with TGF-β1 + TNF-α + IL-1β for 24 h, RT-qPCR was used to detect mRNA levels of related proteins. TF and PAI-1 expressions were up-regulated, while TFPI and PLAU expression levels were down. (B) After 24 h stimulation of endothelial cells by cytokine mixture, TF and PAI-1 expression decreased significantly after GDF15 knockout, *compared with CON group, # compared with CON + cytokine group. (C) Western-blot results, where CON is the control group and CON + C is the control + cytokine. After cyto kine stimulation, Smad pathway was activated in endothelial cells. Knocking down GDF15 inhibited the phosphorylation of Smad2 and the expression of the transcription factor snail, while TF expression decreased significantly. (D) Dual luciferase reporter gene, SBE4 transcription level is up-regulated after EndMT, and knocking down GDF15 can significantly inhibit this effect. (E, F) Results of p-smad2 immunohistochemical staining of mouse inferior vena cava tissue, the scale was 200 μm (100×). Average optical density value of the positive signal of immunohistochemistry analyzed by Image J. Significant post hoc effects were revealed by the Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: The primary antibodies used in this study were: β-actin ((CST, #8457, 1:1000), SM22 (Beyotime, AF5318, 1:1000), GDF15 (Abcam, ab206414,1:1000), CD31 (CST, #77,699, 1:1000), TF (Beyotime, AF2455, 1:1000), Snail (CST, #3879, 1:1000), SMAD2 (CST, #5339, 1:1000), p-SMAD2 (CST, #7348, 1:1000), Smad2/3 (CST, #8685, 1:1000).

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Western Blot, Control, Phospho-proteomics, Luciferase, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Prevention of Streptozotocin-Induced Diabetic Nephropathy by MG132: Possible Roles of Nrf2 and I κ B

doi: 10.1155/2017/3671751

Figure Lengend Snippet: Effects of MG132 on diabetes-induced renal fibrosis in wild-type mice (a) and Nrf2-KO mice (b) were determined by detecting the expression of FN and TGF- β with western blotting assay. Diabetes-induced fibrotic changes (fold) between wild-type and Nrf2-KO mice and the decreased percentages of these changes with MG132 between WT and Nrf2-KO diabetic mice were compared (c). Data are presented as mean ± SD. ∗ p < 0.05 versus WT/control or Nrf2-KO/control correspondingly; # p < 0.05 versus WT/DM or Nrf2-KO/DM correspondingly; & p < 0.05 versus WT mice.

Article Snippet: The primary antibodies were FN (1 : 200 dilution), TGF- β (1 : 1000 dilution), 3-NT (1 : 1000 dilution), 4-HNE (1 : 1000 dilution), IL-6 (1 : 500 dilution), NF- κ B (1 : 1000 dilution), I κ B- α (1 : 1000 dilution), Nrf2 (1 : 500 dilution), actin (1 : 3000 dilution), and α -tubulin (1 : 2000 dilution), all of which were purchased from Santa Cruz Biotechnology except for 3-NT (Millipore), 4-HNE (Alpha Diagnostic), and TGF- β , NF- κ B, I κ B- α , and α -tubulin (Cell Signaling).

Techniques: Expressing, Western Blot