Journal: Scientific Reports
Article Title: Angiotensin II induces connective tissue growth factor expression in human hepatic stellate cells by a transforming growth factor β-independent mechanism
Figure Lengend Snippet: Ang II induces a rapid upregulation of CTGF expression independently of TGF- β in LX-2 cells. ( A ) Serum-starved LX-2 cells were stimulated with Ang II (10 −8 –10 −6 M) for 4 h. Whole cell lysates were immunoblotted with CTGF antibody and a representative immunoblot band of CTGF from 3 independent experiments is shown. β -Actin was used as an internal control for equal protein loading. ( B ) Results of total CTGF expression were obtained from densitometric analysis and expressed as ratio CTGF/ β -actin, as n -fold increase over that in unstimulated control cells. ( C,F ) Serum-starved LX-2 cells were exposed to Ang II (10 −7 M) for 0, 0.25, 0.5, 1, 2 and 4 h. The mRNA expression of the TGF-β1 and CTGF gene was measured by the qRT-PCR method as described in the Materials and Methods section. ( D,G ) Serum-starved LX-2 cells were exposed to Ang II (10 −7 M) for 0, 0.5, 1, 2, 4, 8, 16, 24 and 48 h. Whole cell lysates were prepared and immunoblotted with anti-CTGF or anti-TGF- β 1 antibodies, respectively. Anti- β -actin antibody was used to demonstrate equal protein loading. Similar results were observed in 3 independent experiments, and representative immunoblot bands of CTGF and TGF- β 1 are shown. ( E,H ) Quantitative determination of CTGF and TGF- β 1 protein levels at various time points as indicated, which were converted to arbitrary densitometric units, normalized by the value of β -actin and finally expressed as n -fold change over that of unstimulated control cells (defined as 1.0). ( I,J ) TGF- β signaling was blocked by pretreatment for 1 h with SB-431542 (a TGF- β receptor kinase inhibitor; 10 −5 M), ( K,L ) or by transfection with TGF- β 1 siRNA, before incubation with or without Ang II (10 −7 M) for 1, 4 and 24 h. The CTGF and TGF- β 1 protein levels were analyzed by Western blotting. As a positive control of CTGF production, TGF- β 1 (10 ng/mL) was used. Data are presented as mean ± SD of 3 independent experiments. # P
Article Snippet: Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was isolated from unstimulated control or Ang II-stimulated cell pellets using a RNApure high-purity total RNA rapid extraction kit (spin-column, BioTeke, Beijing, China) according to the manufacturer’s protocols.
Techniques: Expressing, Quantitative RT-PCR, Transfection, Incubation, Western Blot, Positive Control