Journal: International Journal of Molecular Sciences

Article Title: Activin A and Cell-Surface GRP78 Are Novel Targetable RhoA Activators for Diabetic Kidney Disease

doi: 10.3390/ijms22062839

Figure Lengend Snippet: RhoA mediates the profibrotic effects of activins in diabetic kidney disease (DKD). ( A ) Activin A (20 ng/mL, 3 h) and activin B (20 ng/mL, 3 h) both activated RhoA in mesangial cells (MC) ( n = 2). ( B ) Activation of downstream Rho-kinase was also seen, assessed by phosphorylation of myosin phosphatase target (MYPT) (pMYPT Thr696) in MC ( n = 6–8 * p < 0.05). ( C ) Rho-kinase inhibition with Y27632 (10 μM) or HA1077 (10μM) prevented activin A (20 ng/mL)-induced α-smooth muscle actin (αSMA) promoter luciferase activation ( n = 6–11 *** p < 0.001, **** p < 0.0001) and ( D ) protein expression of both αSMA and the fibrotic protein fibronectin (FN) ( n = 4, * p < 0.05, ** p < 0.01). ( E ) The activin inhibitor follistatin (FST, 500 ng/mL) prevented high glucose (HG) (3 h)-induced RhoA activation ( n = 2) and ( F ) Rho-kinase activation ( n = 3, * p < 0.05, ** p < 0.01). ( G ) RhoA activation in kidneys was also significantly inhibited by follistatin in type 1 diabetic Akita mice, as assessed by IHC with an antibody which specifically detects active RhoA ( n = 4, * p < 0.05, ** p < 0.01).

Article Snippet: The following antibodies were used for immunoblotting: α-smooth muscle actin (αSMA) (Pierce, 1:5000), phosphorylated myosin phosphatase target (pMYPT) Thr696 (Cell Signaling; 1:1000), fibronectin (FN) (BD Biosciences, 1:1000), total RhoA (Santa Cruz, 1:500), MTJ-1 (Santa Cruz, 1:1000), GRP78 (BD Biosciences, 1:1000), PDGFRβ (Cedarlane, 1:1000), vinculin (Sigma, 1:1000) and tubulin (Santa Cruz, 1:5000).

Techniques: Activation Assay, Inhibition, Luciferase, Expressing

Journal: bioRxiv

Article Title: MEST induces epithelial-mesenchymal transition through IL-6/JAK/STAT3 signaling in breast cancers

doi: 10.1101/295832

Figure Lengend Snippet: (A) Western blot analysis of the expression of E-cadherin, N-cadherin, fibronectin, and vimentin proteins in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. β-actin was used as a loading control. (B) Western blot (Left) and RT-PCR (Right) analysis of the expression of CAR, caludin3, occludin, E-cadherin, α-catenin, N-cadherin, fibronectin, and β-catenin proteins in MCF10A control or MCF10A-MEST cells. GAPDH and β-actin were used as a loading control. (C) Immunofluorescence images of E-cadherin and N-cadherin in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. The red or green signal represents staining of the corresponding protein, while the blue signal represents DAPI staining. (D) Immunofluorescence images of V5, E-cadherin, β-catenin, fibronectin, α-catenin, and N-cadherin in MCF10A control or MCF10A-MEST cells. The red signal represents the staining of the corresponding protein, while the blue signal represents DAPI staining.

Article Snippet: Antibodies were obtained from the following sources: Anti-MEST (SAB2501254) and anti-Flag (F3165) were from Sigma-Aldrich; anti-V5 (R960-CUS) was from Invitrogen; anti-JAK2 (ab108596), anti-Twist-1 (ab50887), anti-phosphoserine/threonine (ab17464), and anti-phosphotyrosine (ab10321) were from Abcam; anti-STAT3 (9139), anti-phospho-STAT3 (4113), and anti-phospho-JAK2 (3771) were from Cell Signaling Technology; and anti-E-cadherin (610405), anti-fibronectin (610078), anti-N-cadherin (610920), anti-alpha-catenin (610194), anti-vimentin (550513) and anti-beta-catenin (610154) were from BD Phamingen™.

Techniques: Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: MEST induces epithelial-mesenchymal transition through IL-6/JAK/STAT3 signaling in breast cancers

doi: 10.1101/295832

Figure Lengend Snippet: (A) The relative expression levels of mRNA encoding E-cadherin, N-cadherin, Fibronectin, and Vimentin in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells, as determined by quantitative RT-PCR. 18S was used as a loading control. P < 0.05, t -test. (B) The relative expression levels of mRNA encoding E-cadherin, N-cadherin, Fibronectin, and Vimentin in MCF10A control or MCF10A-MEST cells, as determined by quantitative RT-PCR. 18S was used as a loading control P < 0.005, t -test.

Article Snippet: Antibodies were obtained from the following sources: Anti-MEST (SAB2501254) and anti-Flag (F3165) were from Sigma-Aldrich; anti-V5 (R960-CUS) was from Invitrogen; anti-JAK2 (ab108596), anti-Twist-1 (ab50887), anti-phosphoserine/threonine (ab17464), and anti-phosphotyrosine (ab10321) were from Abcam; anti-STAT3 (9139), anti-phospho-STAT3 (4113), and anti-phospho-JAK2 (3771) were from Cell Signaling Technology; and anti-E-cadherin (610405), anti-fibronectin (610078), anti-N-cadherin (610920), anti-alpha-catenin (610194), anti-vimentin (550513) and anti-beta-catenin (610154) were from BD Phamingen™.

Techniques: Expressing, shRNA, Quantitative RT-PCR