Journal: JCI Insight

Article Title: Mechanosensitive Piezo1 channels mediate renal fibrosis

doi: 10.1172/jci.insight.152330

Figure Lengend Snippet: ( A – C ) Representative immunoblots and corresponding densitometry analysis of collagen I, fibronectin, p-Smad2/3, and Smad2/3 protein abundance in the kidney of sham, 3UUO, 3UUO+GsMTx4, 7UUO, and 7UUO+GsMTx4 mice. Data are shown as mean ± SEM ( n = 4 in each group). ( D ) Photomicrographs of Masson’s staining and immunohistochemistry of collagen I in the kidney of sham, 7UUO, and 7UUO+GsMTx4 mice (original magnification, 400×). ( E ) mRNA levels of TGF-β 1 , α-SMA, and fibronectin in the kidney of sham, 3UUO, 3UUO+GsMTx4, 7UUO, and 7UUO+GsMTx4 mice. Data are shown as mean ± SEM ( n = 4 in each group); * P < 0.05 when compared with sham mice and # P < 0.05 when compared with 3UUO or 7UUO mice by 1-way ANOVA with Student-Newman-Keuls test. p-, phosphorylated; TGF-β1, transforming growth factor-β 1 ; SMA, smooth muscle actin.

Article Snippet: Western blotting was performed by electrophoresis and incubation with primary antibodies against fibronectin, α-SMA (ab5694, Abcam; BM0002, Boster; 14395-1-AP, Proteintech), collagen I (BA0325, Boster), calpain2 (11472-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), integrin β 1 (12594-1-AP, Proteintech), Piezo1 (15939-1-AP, Proteintech; 28511-1-AP, Proteintech), Smad2/3 (5678s, Cell Signaling Technology), p-Smad2/3 (8828, Cell Signaling Technology), p-FAK Tyr397 (sc-81593, Santa Cruz Biotechnology), talin1 (14168-1-AP, Proteintech), GAPDH (60004-1-Ig, Proteintech), and β-actin (66009-1-Ig, Proteintech), followed by the addition of horseradish peroxidase–labeled secondary antibodies goat anti–rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti–mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific).

Techniques: Western Blot, Staining, Immunohistochemistry

Journal: JCI Insight

Article Title: Mechanosensitive Piezo1 channels mediate renal fibrosis

doi: 10.1172/jci.insight.152330

Figure Lengend Snippet: ( A and B ) Representative immunoblots and corresponding densitometry analysis of Piezo1, fibronectin, and collagen I protein abundance in the kidney of CTL, FAN, and FAN+GsMTx4 mice. Data are shown as mean ± SEM ( n = 5 in CTL group, n = 6 in FAN and FAN+GsMTx4 group). ( C ) Photomicrographs of Masson’s staining in the kidney of CTL, FAN, and FAN+GsMTx4 mice (original magnification, 400×). ( D ) mRNA levels of TGF-β 1 , α-SMA, and fibronectin in the kidney of CTL, FAN, and FAN+GsMTx4 mice. Data are shown as mean ± SEM ( n = 5 in CTL group, n = 6 in FAN and FAN+GsMTx4 group). * P < 0.05 when compared with CTL mice and # P < 0.05 when compared with FAN mice by 1-way ANOVA with Student-Newman-Keuls test. CTL, control; FAN, folic acid nephropathy.

Article Snippet: Western blotting was performed by electrophoresis and incubation with primary antibodies against fibronectin, α-SMA (ab5694, Abcam; BM0002, Boster; 14395-1-AP, Proteintech), collagen I (BA0325, Boster), calpain2 (11472-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), integrin β 1 (12594-1-AP, Proteintech), Piezo1 (15939-1-AP, Proteintech; 28511-1-AP, Proteintech), Smad2/3 (5678s, Cell Signaling Technology), p-Smad2/3 (8828, Cell Signaling Technology), p-FAK Tyr397 (sc-81593, Santa Cruz Biotechnology), talin1 (14168-1-AP, Proteintech), GAPDH (60004-1-Ig, Proteintech), and β-actin (66009-1-Ig, Proteintech), followed by the addition of horseradish peroxidase–labeled secondary antibodies goat anti–rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti–mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific).

Techniques: Western Blot, Staining

Journal: JCI Insight

Article Title: Mechanosensitive Piezo1 channels mediate renal fibrosis

doi: 10.1172/jci.insight.152330

Figure Lengend Snippet: ( A ) mRNA level of Piezo1 in HK2 cells pretreated with 5 μM GsMTx4 followed by cyclic stretch. Data are shown as mean ± SEM ( n = 6 in each group). ( B and C ) Representative immunoblots and corresponding densitometry analysis of Piezo1 protein abundance in HK2 cells pretreated with 1 μM or 5 μM GsMTx4 followed by cyclic stretch. Data are shown as mean ± SEM ( n = 4 in each group). ( D ) Fluo-4-AM was added to HK2 cells transfected with Piezo1 siRNA followed by cyclic stretch, and the fluorescence signals of calcium were detected by flow cytometry analysis. ( E and F ) Representative immunoblots and corresponding densitometry analysis of fibronectin, α-SMA, and E-cadherin protein abundance in HK2 cells pretreated with 5 μM GsMTx4 followed by cyclic stretch. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with static and # P < 0.05 when compared with stretch by 1-way ANOVA with Student-Newman-Keuls test. ( G and H ) Representative immunoblots and corresponding densitometry analysis of fibronectin, α-SMA, and E-cadherin protein abundance in HK2 cells transfected with Piezo1 siRNA followed by cyclic stretch. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with scramble siRNA static and # P < 0.05 when compared with scramble siRNA stretch by 1-way ANOVA with Student-Newman-Keuls test. ( I and J ) Representative immunoblots and corresponding densitometry analysis of Piezo1, fibronectin, and α-SMA protein abundance in HK2 cells pretreated with 5 μM GsMTx4 followed by 15 mmHg compression for 24 hours. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with CTL and # P < 0.05 when compared with compression by 1-way ANOVA with Student-Newman-Keuls test.

Article Snippet: Western blotting was performed by electrophoresis and incubation with primary antibodies against fibronectin, α-SMA (ab5694, Abcam; BM0002, Boster; 14395-1-AP, Proteintech), collagen I (BA0325, Boster), calpain2 (11472-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), integrin β 1 (12594-1-AP, Proteintech), Piezo1 (15939-1-AP, Proteintech; 28511-1-AP, Proteintech), Smad2/3 (5678s, Cell Signaling Technology), p-Smad2/3 (8828, Cell Signaling Technology), p-FAK Tyr397 (sc-81593, Santa Cruz Biotechnology), talin1 (14168-1-AP, Proteintech), GAPDH (60004-1-Ig, Proteintech), and β-actin (66009-1-Ig, Proteintech), followed by the addition of horseradish peroxidase–labeled secondary antibodies goat anti–rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti–mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific).

Techniques: Western Blot, Transfection, Fluorescence, Flow Cytometry

Journal: JCI Insight

Article Title: Mechanosensitive Piezo1 channels mediate renal fibrosis

doi: 10.1172/jci.insight.152330

Figure Lengend Snippet: ( A and B ) Cell proliferation of HK2 cells cultured on polyacrylamide hydrogels with different stiffness, assessed by EdU. Scale bar: 500 μm. Data are shown as mean ± SEM ( n = 4 in 4 kPa group, n = 5 in 8 kPa group, n = 8 in 20 kPa group, n = 9 in 35 kPa group). ( C ) Piezo1 mRNA level of HK2 cells cultured on polyacrylamide hydrogels with different stiffness. Data are shown as mean ± SEM ( n = 4 in each group). ( D and E ) Representative immunoblots and corresponding densitometry analysis of Piezo1, fibronectin, and α-SMA protein abundance in HK2 cells cultured on polyacrylamide hydrogels with different stiffness. Data are shown as mean ± SEM ( n = 4 in each group). ( F and G ) Representative immunoblots and corresponding densitometry analysis of fibronectin and α-SMA protein abundance in HK2 cells cultured on 4 kPa and 20 kPa polyacrylamide hydrogels, followed by treatment with GsMTx4. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with 4 kPa and # P < 0.05 when compared with 20 kPa by 1-way ANOVA with Student-Newman-Keuls test. ( H and I ) Representative immunoblots and corresponding densitometry analysis of Piezo1, fibronectin, and α-SMA protein abundance in primary mPTCs cultured on polyacrylamide hydrogels with different stiffness. Data are shown as mean ± SEM ( n = 4 in each group). ( J and K ) Representative immunoblots and corresponding densitometry analysis of fibronectin and α-SMA protein abundance in primary mPTCs cultured on polyacrylamide hydrogels with 4 kPa and 35 kPa stiffness, followed by treatment with GsMTx4. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with 4 kPa and # P < 0.05 when compared with 35 kPa by 1-way ANOVA with Student-Newman-Keuls test. mPTCs, mouse proximal tubular cells; EdU, 5-ethynyl-2′-deoxyuridine.

Article Snippet: Western blotting was performed by electrophoresis and incubation with primary antibodies against fibronectin, α-SMA (ab5694, Abcam; BM0002, Boster; 14395-1-AP, Proteintech), collagen I (BA0325, Boster), calpain2 (11472-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), integrin β 1 (12594-1-AP, Proteintech), Piezo1 (15939-1-AP, Proteintech; 28511-1-AP, Proteintech), Smad2/3 (5678s, Cell Signaling Technology), p-Smad2/3 (8828, Cell Signaling Technology), p-FAK Tyr397 (sc-81593, Santa Cruz Biotechnology), talin1 (14168-1-AP, Proteintech), GAPDH (60004-1-Ig, Proteintech), and β-actin (66009-1-Ig, Proteintech), followed by the addition of horseradish peroxidase–labeled secondary antibodies goat anti–rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti–mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific).

Techniques: Cell Culture, Western Blot

Journal: JCI Insight

Article Title: Mechanosensitive Piezo1 channels mediate renal fibrosis

doi: 10.1172/jci.insight.152330

Figure Lengend Snippet: ( A ) Morphologic changes of HK2 cells pretreated with GsMTx4 followed by TGF-β 1 treatment for 48 hours. Scale bar: 100 μm. ( B and C ) Representative immunoblots and corresponding densitometry analysis of Piezo1, fibronectin, α-SMA, and E-cadherin protein abundance in HK2 cells pretreated with GsMTx4 followed by TGF-β 1 treatment for 48 hours. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with CTL and # P < 0.05 when compared with TGF-β 1 by 1-way ANOVA with Student-Newman-Keuls test. ( D and E ) Representative immunoblots and corresponding densitometry analysis of fibronectin, α-SMA, and E-cadherin protein abundance in HK2 cells transfected with Piezo1 siRNA followed by TGF-β 1 treatment for 48 hours. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with scramble siRNA CTL and # P < 0.05 when compared with scramble siRNA TGF-β 1 by 1-way ANOVA with Student-Newman-Keuls test. ( F and G ) Representative immunoblots and corresponding densitometry analysis of Piezo1, fibronectin, and α-SMA protein abundance in primary cultured mPTCs, pretreated with GsMTx4 followed by TGF-β 1 treatment for 48 hours. Data are shown as mean ± SEM ( n = 6 in each group). * P < 0.05 when compared with CTL and # P < 0.05 when compared with TGF-β 1 by 1-way ANOVA with Student-Newman-Keuls test.

Article Snippet: Western blotting was performed by electrophoresis and incubation with primary antibodies against fibronectin, α-SMA (ab5694, Abcam; BM0002, Boster; 14395-1-AP, Proteintech), collagen I (BA0325, Boster), calpain2 (11472-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), integrin β 1 (12594-1-AP, Proteintech), Piezo1 (15939-1-AP, Proteintech; 28511-1-AP, Proteintech), Smad2/3 (5678s, Cell Signaling Technology), p-Smad2/3 (8828, Cell Signaling Technology), p-FAK Tyr397 (sc-81593, Santa Cruz Biotechnology), talin1 (14168-1-AP, Proteintech), GAPDH (60004-1-Ig, Proteintech), and β-actin (66009-1-Ig, Proteintech), followed by the addition of horseradish peroxidase–labeled secondary antibodies goat anti–rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti–mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific).

Techniques: Western Blot, Transfection, Cell Culture

Journal: JCI Insight

Article Title: Mechanosensitive Piezo1 channels mediate renal fibrosis

doi: 10.1172/jci.insight.152330

Figure Lengend Snippet: ( A and B ) Yoda1 induced the cationic currents in HK2 cells. ( A ) I-V curve exhibited the currents activated by 10 μM Yoda1 recorded at different membrane potentials in voltage-clamp mode. ( B ) Representative traces: recorded at –90 and +90 mV by puffing 10 μM Yoda1 (short black arrow) for 350 ms to HK2 cells. ( C ) Morphologic changes of HK2 cells treated with Yoda1 for 3 hours, 6 hours, 12 hours, and 24 hours. Scale bar: 100 μm. ( D ) Immunofluorescence of fibronectin in HK2 cells treated with Yoda1 for 24 hours. Scale bar: 50 μm. ( E and F ) Representative immunoblots and corresponding densitometry analysis of fibronectin, α-SMA, and E-cadherin protein abundance in HK2 cells treated with Yoda1 for 3 hours, 6 hours, 12 hours, and 24 hours. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with CTL by 1-way ANOVA with Student-Newman-Keuls test. ( G and H ) Representative immunoblots and corresponding densitometry analysis of fibronectin, α-SMA, and E-cadherin protein abundance in HK2 cells transfected with Piezo1 siRNA followed by Yoda1 treatment for 24 hours. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with scramble siRNA CTL and # P < 0.05 when compared with scramble siRNA Yoda1 by 1-way ANOVA with Student-Newman-Keuls test. ( I ) mRNA expression of TGF-β 1 in HK2 cells treated with Yoda1 for 24 hours. Data are shown as mean ± SEM ( n = 6 in each group). * P < 0.05 when compared with CTL by unpaired Student’s t test. ( J and K ) Representative immunoblots and corresponding densitometry analysis of fibronectin and α-SMA protein abundance in primary cultured mPTCs treated with Yoda1 for 24 hours. Data are shown as mean ± SEM ( n = 6 in each group). * P < 0.05 when compared with CTL by unpaired Student’s t test. CTL, control; SMA, smooth muscle actin; TGF-β1, transforming growth factor-β 1 ; mPTCs: mouse proximal tubular cells.

Article Snippet: Western blotting was performed by electrophoresis and incubation with primary antibodies against fibronectin, α-SMA (ab5694, Abcam; BM0002, Boster; 14395-1-AP, Proteintech), collagen I (BA0325, Boster), calpain2 (11472-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), integrin β 1 (12594-1-AP, Proteintech), Piezo1 (15939-1-AP, Proteintech; 28511-1-AP, Proteintech), Smad2/3 (5678s, Cell Signaling Technology), p-Smad2/3 (8828, Cell Signaling Technology), p-FAK Tyr397 (sc-81593, Santa Cruz Biotechnology), talin1 (14168-1-AP, Proteintech), GAPDH (60004-1-Ig, Proteintech), and β-actin (66009-1-Ig, Proteintech), followed by the addition of horseradish peroxidase–labeled secondary antibodies goat anti–rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti–mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific).

Techniques: Immunofluorescence, Western Blot, Transfection, Expressing, Cell Culture

Journal: JCI Insight

Article Title: Mechanosensitive Piezo1 channels mediate renal fibrosis

doi: 10.1172/jci.insight.152330

Figure Lengend Snippet: ( A and B ) Fluo-4-AM was added to HK2 cells treated with Yoda1 for 1 hour, 3 hours, 6 hours, and 24 hours, and the fluorescence signals of Ca 2+ were detected by flow cytometry analysis and fluorescence microscope. Scale bar: 200 μm. “50% Show” means that flow cytometry data are presented based on 10,000 cells of 20,000 cells analyzed. ( C and D ) Representative immunoblots and corresponding densitometry analysis of calpain2, fibronectin, α-SMA, and E-cadherin protein abundance in HK2 cells pretreated with 2 mM EGTA followed by Yoda1 treatment for 24 hours. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with CTL and # P < 0.05 when compared with Yoda1 by 1-way ANOVA with Student-Newman-Keuls test. ( E and F ) Representative immunoblots and corresponding densitometry analysis of calpain2 protein abundance in HK2 cells transfected with Piezo1 siRNA followed by Yoda1 treatment for 24 hours. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with scramble siRNA CTL and # P < 0.05 when compared with scramble siRNA Yoda1 by 1-way ANOVA with Student-Newman-Keuls test. ( G and H ) Representative immunoblots and corresponding densitometry analysis of calpain2, fibronectin, α-SMA, and E-cadherin protein abundance in WT HK2 cells and CAPN2-KD HK2 cells treated with Yoda1 for 24 hours. Data are shown as mean ± SEM ( n = 4 in each group). * P < 0.05 when compared with CTL by 1-way ANOVA with Student-Newman-Keuls test. CAPN2-KD, calpain2 knockdown.

Article Snippet: Western blotting was performed by electrophoresis and incubation with primary antibodies against fibronectin, α-SMA (ab5694, Abcam; BM0002, Boster; 14395-1-AP, Proteintech), collagen I (BA0325, Boster), calpain2 (11472-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), integrin β 1 (12594-1-AP, Proteintech), Piezo1 (15939-1-AP, Proteintech; 28511-1-AP, Proteintech), Smad2/3 (5678s, Cell Signaling Technology), p-Smad2/3 (8828, Cell Signaling Technology), p-FAK Tyr397 (sc-81593, Santa Cruz Biotechnology), talin1 (14168-1-AP, Proteintech), GAPDH (60004-1-Ig, Proteintech), and β-actin (66009-1-Ig, Proteintech), followed by the addition of horseradish peroxidase–labeled secondary antibodies goat anti–rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti–mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific).

Techniques: Fluorescence, Flow Cytometry, Microscopy, Western Blot, Transfection

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Effect of Huai Qi Huang on Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells through miR-200a

doi: 10.1155/2016/8612190

Figure Lengend Snippet: Effects of Huai Qi Huang (HQH) on E-cadherin and α -SMA protein expression in NRK-52E cells stimulated with transforming growth factor- β 1 (TGF- β 1). (a) TGF- β 1 induces downregulated expression of E-cadherin but upregulated expression of α -SMA in NRK-52E cells; HQH displays inhibitory effect on phenotypic changes and protein expression of E-cadherin and α -SMA in NRK-52E cells at 24 h (immunofluorescence, 200x). (b and c) Western blot and quantitative analysis of E-cadherin and α -SMA protein expression in NRK-52E cells in each group at 6 h, 12 h, 24 h, and 48 h. ∗ p < 0.05 versus the TGF- β 1 group at the same time point. All experiments were repeated for three times. The bars show mean ± SEM.

Article Snippet: The anti- α -smooth muscle actin antibody was from Boster company (Wuhan, China).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Effect of Huai Qi Huang on Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells through miR-200a

doi: 10.1155/2016/8612190

Figure Lengend Snippet: Huai Qi Huang (HQH) inhibited epithelial-mesenchymal transition (EMT) in UUO rats. (a) Immunohistochemical staining shows E-cadherin + epithelial cells and α -SMA + myofibroblasts distributed in sham operation and obstructed kidney in each group (200x). (b) Representative bands for E-cadherin and α -SMA protein detected by Western blots in obstructed kidneys of UUO rats. (c) Relative protein levels of E-cadherin or α -SMA to GAPDH in each group. ∗ p < 0.05 versus vehicle-treated UUO rats on the same day after obstruction. Five rats in each dosage group. The bars show mean ± SEM.

Article Snippet: The anti- α -smooth muscle actin antibody was from Boster company (Wuhan, China).

Techniques: Immunohistochemical staining, Staining, Western Blot

Journal: PLoS ONE

Article Title: TRPV4 Channel Inhibits TGF-β1-Induced Proliferation of Hepatic Stellate Cells

doi: 10.1371/journal.pone.0101179

Figure Lengend Snippet: A. The level of the TRPV4 was analyzed by immunohistochemistry in human normal liver and liver fibrosis patients. Representative views from each group are presented (original magnification, ×50). B. Liver tissues from liver fibrosis patients tissue were double stained with TRPV4 and α-SMA antibodies. Representative photomicrographs are shown (original magnification, ×40) in B. C. Pathology observation of the experimental rat liver sections stained with hematoxylin and eosin (H&E) staining and massion staining (×200). D. The level of the α-SMA was analyzed by immunohistochemistry in vehicle control livers and liver fibrotic tissue. Representative views from each group are presented (original magnification, ×50). E-F. Total RNAs were isolated from the livers of CCl 4 -treated rats or vehicle-treated groups. The expression of TRPV4 mRNA was assessed by RT-PCR(E) and realtime PCR(F). Representative images of three independent experiments are shown. *p<0.05, **p<0.01 vs. vehicle control. G. Whole-cell extracts were isolated from the liver tissues of CCl 4 -treated rats or vehicle-treated groups, and subjected to immunoblot for TRPV4 and β-actin control. Representative images of three independent experiments are shown. *p<0.05, **p<0.01 vs. vehicle control.

Article Snippet: Mouse monoclonal antibodies against α-SMA and β-actin were obtained from Boster (Wuhan, China).

Techniques: Immunohistochemistry, Staining, Control, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: TRPV4 Channel Inhibits TGF-β1-Induced Proliferation of Hepatic Stellate Cells

doi: 10.1371/journal.pone.0101179

Figure Lengend Snippet: A. Total RNAs were isolated from TGF-β1-treated HSC-T6 cells, and subjected to qRT-PCR analyses. Representative images of three independent experiments are shown. *p<0.05 vs. non-treated cells. B. Whole-cell extracts were isolated from TGF-β1-treated HSC-T6 cells, and subjected to Western blot analyses with TRPV4 and β-actin antibodies. Representative blots of three independent experiments are shown. **p<0.01 vs. non-treated cells. C. Total RNAs were isolated from TGF-β1 treated HSC-T6 cells at different time points. The expression of α-SMA and Col1a1 mRNA was assessed by RT-PCR. Representative images of three independent experiments are shown. **p<0.01 vs. non-treated cells.

Article Snippet: Mouse monoclonal antibodies against α-SMA and β-actin were obtained from Boster (Wuhan, China).

Techniques: Isolation, Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: TRPV4 Channel Inhibits TGF-β1-Induced Proliferation of Hepatic Stellate Cells

doi: 10.1371/journal.pone.0101179

Figure Lengend Snippet: A. Total RNA extracts were made from HSC-T6 cells treated with or without TGF-β1 and Ru, and subjected to qRT-PCR analyses of TRPV4. Representative images of three independent experiments are shown. # p<0.05 vs. TGF-β1-treated cells. B. HSC-T6 cells were seeded in triplicate on day 0 and incubated in DMEM containing 10% fetal bovine serum or same media supplemented with Ru for further 24 h. Proliferation was measured by adding 5 mg/ml MTT reagent per well and incubating it for 4 h. # p<0.05 vs. TGF-β1-treated cells. C. Total RNA extracts were made from HSC-T6 cells treated with or without TGF-β1 and Ru, and subjected to qRT-PCR analyses of α-SMA. Representative images of three independent experiments are shown. # p<0.05 vs. TGF-β1-treated cells. D. Whole-cell protein extracts were made from HSC-T6 cells treated with or without TGF-β1 and Ru, and subjected to Western blot analyses of TRPV4. Representative images of three independent experiments are shown. ## p<0.01 vs. TGF-β1-treated cells. E. HSC-T6 cells were treated with TGF-β1 for 48 h, followed by transfection with TRPV4-siRNA for an additional 48 h, and cell viability was determined by MTT assay. Mean±SE of two HSC preparations in quadruplets is shown; *p<0.05 vs. non-treated cells, # p<0.05 vs. TGF-β1-treated cells. F. Whole cell extracts were isolated from TGF-β1-treated HSC-T6 cells with RNAi transfection, and subjected to Western blot analyses. Representative images of three independent experiments are shown. **p<0.01 vs. non-treated cells, ## p<0.01 vs. TGF-β1-treated cells.

Article Snippet: Mouse monoclonal antibodies against α-SMA and β-actin were obtained from Boster (Wuhan, China).

Techniques: Quantitative RT-PCR, Incubation, Western Blot, Transfection, MTT Assay, Isolation

Journal: Biomedicines

Article Title: Derazantinib Inhibits the Bioactivity of Keloid Fibroblasts via FGFR Signaling

doi: 10.3390/biomedicines11123220

Figure Lengend Snippet: KFs were treated with 2.5 μmol derazantinib or without derazantinib for 48 h. ( A ) The PCR showed that the expression of fibrotic genes was suppressed in the experiment group. ( B – D ) The immunofluorescence staining showed that collagen I, α-SMA, and PAI-1 expression were decreased in the derazantinib group. The length of the scale is 50 μm. ( E , F ) The WB showed that the protein production of a-SMA, collagen I, PAI-1, and FGFR1 in the derazantinib group was suppressed. ( G ) The PCR showed that the expression of PI3K and JNK genes was suppressed in the experiment group. ( H ) The WB showed that the expression of ERK, p-ERK, AKT, TGF-β, p-AKT, and PI3K in the derazantinib group was suppressed. No significant effect was found on the SMAD expression levels. * p < 0.05 compared to control group.

Article Snippet: The PVDF membrane was separately treated with the specific primary antibodies: α-SMA (AF1032, Affinity, Changzhou, China, rabbit), collagen I (66948, CST, USA, mouse), PAI-1 (13801-1-AP, Proteintech, IL, USA, rabbit), FGFR1 (60325-1-Ig, Proteintech, USA, mouse), GAPDH (GB11002, Servicebio, Wuhan, China, rabbit), PI3K (AF5112, Affinity, Changzhou, China, rabbit), p-AKT (AF0016, Affinity, Changzhou, China, rabbit), AKT (BS0115R, Bioss, Wuhan, China, rabbit), p-ERK (AF1015, Affinity, Changzhou, China, rabbit), ERK (AF0155, Affinity, Changzhou, China, rabbit), TGF-β (BA0290, Bosterbio, Beijing, China, rabbit), Smad (6367, Affinity, Changzhou, China, rabbit), and p-Smad (3367, Affinity, Changzhou, China, rabbit).

Techniques: Expressing, Immunofluorescence, Staining, Control