Journal: The Journal of Biological Chemistry

Article Title: Binding of Gtf2i-β/δ transcription factors to the ARMS2 gene leads to increased circulating HTRA1 in AMD patients and in vitro

doi: 10.1016/j.jbc.2021.100456

Figure Lengend Snippet: Action of HTRA1 on TGF-β/ALK5/SMAD2/3 signaling. A , we assayed the effects of HTRA1 on pSMAD2/3, SMAD2/2, VEGF, and TGFβRII by transfecting pCMV-Myc-HTRA1 vector in HEK-293, 661W and HeLa cells, followed by WB. Addition of the HTRA1 expression vector significantly inhibited SAMD2/3 phosphorylation but not SMAD 2/3 expression in HEK-293 cells. HTRA1 enhanced VEGF expression in HeLa cells and cleavage TGFβRII in all three cell lines. B , the band intensity was analyzed by Image J software. C , influence of overexpressed HTRA1 in mice. Phospho-Smad2/3 and TGFβRII, but not Smad2/3, decreased in 1-year-old Htra1 Tg mouse compared with WT mouse. VEGF enhanced in Htra1 Tg mouse compared with WT mouse. D , analysis of band intensities. E , mRNA levels of TGF II, TGFβRII, and ALK5 in HTRA1 transfected HEK-293, 661W, and HeLa cells, respectively. qRT-PCR analysis of TGF II, TGFβRII, and Alk5 mRNA levels ( F ) and VEGF isoforms (VEGF 120 , VEGF 164 , and VEGF 188 ) ( G ) in the retina of Htra1 Tg mouse. The expression of VEGF 120 isoform mRNA was significantly enhanced in 1-year-old Htra1 Tg mouse compared with WT. The other two VEGF isoforms were undetectable. Throughout, the results are expressed as the mean ± SEM. The p value was obtained by Student's t test.

Article Snippet: The primary antibodies used in WB included: TFII-I antibody (1:1000; CST; #4562), anti-hnRNP K antibody (1:10,000; Abcam; ab52600), EF-1r polyclonal antibody (1:1000; SAB; #40863), Lamin A/C (4c11) antibody (1:2000; CST; #4777); anti-actin (1/1000; Millipore; #MAB1501), ANTI-FLAG M2 (1:1000; SIGMA; F1804), anti-firefly luciferase antibody (1:1000; Abcam, ab21176), Samd2/3(D7F7) (1:1000; CST; #8685), Phospho-Samd2 (ser465/467)/smad3 (ser423/425) (D27F4) (1:1000; CST; #8828), anti-VEGF (1:1000; Abcam; ab46154), anti-HTRA1(1:500; R&D, MAB2916), anti-TGFβ RII(D-2) (1:300S; CB; sc-17799).

Techniques: Plasmid Preparation, Expressing, Phospho-proteomics, Software, Transfection, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo

doi: 10.1186/1465-9921-15-51

Figure Lengend Snippet: ATO inhibits Smad2/Smad3 and Akt phosphorylation but does not affect p38 phosphorylation. (A-B) NHLFs were pre-treated with ATO for 24 hrs. Then cells were treated with TGF-β1 (1 ng/ml) for 30 mins. TGF-β1 did not affect total Smad2 and Smad3 expression, but did increase their phosphorylation. TGF-β1 induced Smad2 and Smad3 phosphorylation were blocked by ATO. (C) NHLFs were pre-treated with ATO for 24 hrs, and then washed 5 times with PBS to remove ATO from the media. Thereafter cells were exposed to TGF-β1 (1 ng/ml) for 30 mins. The results indicate that the effects of ATO on TGF-β1 induced Smad2 and Smad3 phosphorylation are not due to disruption of the of the TGF-β1 ligand. (D) NHLFs were pre-treated with ATO for 24 hrs., and then treated with TGF-β1 (1 ng/ml) for 12 hrs. Akt phosphorylation was induced by TGF-β1 and this induction was diminished by ATO. (E) NHLFs were pre-treated with ATO for 24 hrs, then treated with TGF-β1 (1 ng/ml) for 30 mins. ATO increased TGF-β1 induced p38 phosphorylation. (F) TGF-β1 induces H 2 O 2 in NHLFs. ATO at the indicated concentrations did not induce H 2 O 2 , but did diminish TGF-β1-induced H 2 O 2 production. (G) NHLFs were pre-treated with ATO for 24 hrs, then treated with TGF-β1 (1 ng/ml) with or without H 2 O 2 for 30 mins. Smad3 phosphorylation was up-regulated by H 2 O 2. (H) TGF-β1 induced NOX-4 mRNA expression in NHLFs, whereas ATO at the indicated concentrations inhibited TGF-β1-induced NOX-4 mRNA expression. Western blot data represents consistent trend in three independent repeats with the best image quality. RT-PCR data represent results from three independent experiments with duplicate repeats. *P value < 0.05, **P value <0.01, ***P value < 0.001.

Article Snippet: Antibodies for Smad2, p-Samd2, Smad3, p-Smad3, Akt, p-Akt, Erk, p-Erk, p38, p-p38 were purchased from Cell signaling and used at a concentration of 1:1000.

Techniques: Phospho-proteomics, Expressing, Disruption, Western Blot, Reverse Transcription Polymerase Chain Reaction