Journal: Annals of Transplantation

Article Title: Antifibrotic Effects of Hepatocyte Growth Factor on Endothelial-to-Mesenchymal Transition via Transforming Growth Factor-Beta1 (TGF-β1)/Smad and Akt/mTOR/P70S6K Signaling Pathways

doi: 10.12659/AOT.906700

Figure Lengend Snippet: HGF decreases α-SMA expression in HUVECs through TGF-β/Smad and Akt/mTOR/p70S6K signaling pathways. ( A, B ) HUVECs treated with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours. We collected equal amounts of protein from whole cell lysates, which we analyzed by western blotting with antibodies against phosphorylated Smad 2, Smad 3, Akt, mTOR, p70 S6K, Erk1/2, c-Jun. ( C, D ) HUVECs were pretreated for one hour with MK2206 (10 μmol/L) ( C ), and SB431542 (10 μmol/L) ( D ), which are specific chemical inhibitors for Akt and Smad, respectively. Subsequently, we treated cells with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours. We collected cells one hour after HGF and/or TGF-β1 stimulation. We analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against phosphorylated and total Akt ( C ), phosphorylated and total mTOR ( C ), phosphorylated and total p70S6K ( C ), phosphorylated and total Smad 2 ( D ), phosphorylated and total Smad 3 ( D ). ( C, D ) We collected HUVECs 48 hours after HGF and/or TGF-β1 stimulation and analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against α-SMA and GAPDH.

Article Snippet: Then, we performed western blotting by incubation with primary antibodies of anti-GAPDH (1: 200, Abcam, Cambridge, MA, USA), anti-α-SMA (1: 2,500, Abcam), anti-CD31 (1: 1,000, Abcam), anti-TGF-β1 (1: 250, Abcam), anti-HGF (1: 500, Abcam), anti-Akt (1: 1,000, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt (Ser473, 1: 1,000, Cell Signaling Technology), anti-mTOR (1: 1,000, Cell Signaling Technology,), anti-phospho-mTOR (Ser2448, 1: 1,000, Cell Signaling Technology), anti-p70S6K (1: 1,000, Cell Signaling Technology), anti-phospho-p70S6K (Thr389, 1: 1,000, Cell Signaling Technology), anti-Erk 1/2 (1: 1,000, Cell Signaling Technology) anti-phospho-Erk 1/2 (Thr202/Tyr204, 1: 1,000, Cell Signaling Technology), anti-c-Jun (1: 1,000, Cell Signaling Technology) and anti-phospho-c-Jun (Ser73, 1: 1,000, Cell Signaling Technology), anti-Smad 2 (1: 1,000, Cell Signaling Technology), anti-phospho-Smad 2 (Ser465/467, 1: 1,000, Cell Signaling Technology), anti-Smad 3 (1: 1,000, Cell Signaling Technology) and anti-phospho-Smad 3 (Ser423/425, 1: 1,000, Cell Signaling Technology), followed by incubation with anti-rabbit or anti-mouse secondary antibody (1: 1,000, Abcam).

Techniques: Expressing, Western Blot

Journal: FEBS Open Bio

Article Title: Suppression of intrahepatic cholangiocarcinoma cell growth by SKI via upregulation of the CDK inhibitor p21

doi: 10.1002/2211-5463.13489

Figure Lengend Snippet: Sloan Kettering Institute suppresses cell proliferation and regulates key proliferation–related signaling proteins in human cholangiocarcinoma. SKI overexpression in (A) OZ and (B) KKU100 human cholangiocarcinoma cell lines were assessed by western blot analysis (top, 0–500 ng·mL −1 plasmid) and cell proliferation assays (bottom, MTT and CCK‐8, 500 ng·mL −1 plasmid; n = 4). (C) SKI knockdown in OZ cells was assessed by western blot analysis (top, 0–10 n m siRNA) and cell proliferation assays (bottom, MTT and CCK‐8, 10 n m siRNA, n = 6). (D) Transwell migration assay comparing SKI‐overexpressing (500 ng·mL −1 plasmid) KKU100 cells with control cells ( n = 6); scale bar, 100 μm. (E) Qualitative western blot analysis of cell proliferation–related proteins, including phosphorylated SMAD3, total SMAD3, phosphorylated MEK1/2, total MEK1/2, phosphorylated ERK1/2, total ERK1/2, p21, and p27 in SKI‐overexpressing (170/340 ng·mL −1 plasmid) OZ cells and control cells. GAPDH (A–C, E) served as a loading control. Symbols and bars represent the mean ± standard deviation (A–D). All cells were transfected with equal amounts of total plasmid or RNA through the addition of empty plasmid or siCNT, respectively (A–E). ** P < 0.01, *** P < 0.001; unpaired t ‐test (A–D). CCK‐8, cell counting kit 8; MEK, MAPK/ERK kinase.

Article Snippet: Primary antibodies were obtained as follows: anti‐SKI (#33693), anti‐dual‐specificity phosphatase (DUSP)2 (#32776), anti‐DUSP6 (#377070), anti‐lamin B1 (#374015), and antichromatin licensing and DNA replication factor 1 (CDT1; #365305) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); antiphosphorylated‐smooth muscle actin plus mothers against decapentaplegic (SMAD)3 (#9520), anti‐SMAD3 (#9513), antiphosphorylated mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) kinase (MEK) 1/2 (#9154), anti‐MEK1/2 (#8727), antiphosphorylated‐ERK1/2 (#4370), anti‐ERK1/2 (#4695) anti‐p21 (#2947) and anti‐p27 (#3686) (Cell Signaling Technology); anti‐β‐actin (#A2228), and anti‐GAPDH (#374) (Merck).

Techniques: Over Expression, Western Blot, Plasmid Preparation, CCK-8 Assay, Transwell Migration Assay, Standard Deviation, Transfection, Cell Counting

Journal: International Journal of Molecular Sciences

Article Title: Lithium Treatment Improves Cardiac Dysfunction in Rats Deprived of Rapid Eye Movement Sleep

doi: 10.3390/ijms231911226

Figure Lengend Snippet: Transforming growth factor beta (TGF-β) signaling pathway in REM sleep deprivation with and without lithium treatment. The REM sleep-deprived hearts ( n = 5) had greater expression levels of TGF-β, phosphorylated Smad 2/3, and α-smooth muscle actin (α-SMA) compared with the control ( n = 5) and lithium-treated REM sleep-deprived ( n = 5) hearts. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Blots were probed with the following antibodies against regulator proteins involved in cardiac fibrogenesis: TGF-β (1:2000, polyclonal, #3711; Cell Signaling Technology, Beverly, MA, USA), TGF-β RI (1:1000, polyclonal, #sc-398; Santa Cruz Biotechnology, Santa Cruz, CA, USA), total Smad 2/3 (1:2000, monoclonal, #8685; Cell Signaling Technology), phosphorylated Smad 2/3 (1:2000, monoclonal, #8828; Cell Signaling Technology), α-SMA (1:5000, monoclonal, #ab7817; Abcam, Cambridge, UK), AGTR1 (1:2000, polyclonal, #AAR-011; Alomone Labs, Jerusalem, Israel), total NF-κB p65 (1:2000, monoclonal, #8242; Cell Signaling Technology), phosphorylated NF-κB p65 (1:2000, monoclonal, #3033; Cell Signaling Technology), Orai1 (1:2000, polyclonal, #4281; ProSci Incorporated, Poway, CA, USA), STIM1 (1:2000, monoclonal, #610954; BD Transduction Laboratories, San Diego, CA, USA), TRPC 1 channel (1:2000, polyclonal, #ACC-010; Alomone Labs), TRPC3 (1:5000, polyclonal, #ACC-016; Alomone Labs), and TRPC6 (1:2000, polyclonal, #ACC-017; Alomone Labs).

Techniques: Expressing

Journal: Biomedicines

Article Title: The Upregulation of Caffeic Acid Phenethyl Ester on Growth Differentiation Factor 15 Inhibits Transforming Growth Factor β/Smad Signaling in Bladder Carcinoma Cells

doi: 10.3390/biomedicines10071625

Figure Lengend Snippet: TGFβ downregulates expressions of GDF15, maspin and NDRG1 is dependent on TGFβ/Smad signaling in bladder carcinoma cells. The expressions of Smad 2/3, p-Smad 2/3, Smad 1, Smad 5, and p-Smad 1/5 were determined using immunoblot assays (( A ), left) and quantitative analysis (( A ), right) after treated with (+) or without (−) 10 ng/mL rhTGFβ for 1 h in HT1376 cells. The expressions of GDF15, maspin, NDRG1, and β-actin were determined using immunoblot assays (( B ), left) and quantitative analysis (( B ), right) after being treated with various concentrations of rhTGFβ as indicated for 18 h in HT1376 cells. ( C ) HT1376 cells were treated with various concentrations of rhTGFβ as indicated for 18 h. The relative mRNA levels of GDF15, NDRG1, and maspin were determined by RT-qPCR assays. ( D ) The expressions of maspin, NDRG1, and β-actin were determined using immunoblot assays (( D ), left) and quantitative analysis (( D ), right) after being treated with 400 ng/mL rhGDF15, 20 μM LY364947, and 20 μM SB431542 as indicated for 18 h. The quantitative data were expressed as the intensity of protein bands of the target genes/β-actin relative to the control solvent-treated group ( n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: The blotting membranes were probed using antiserum of GDF15 (Ab206414, Abcam, Cambridge, MA, USA), NDRG1 (42-6200; Invitrogen, Carlsbad, CA, USA), Maspin (#554292, BD Biosciences, San Jose, CA, USA), GFRAL (ab107719, Abcam, Cambridge, MA, USA), β-actin (MAB1501, Merck Millipore, Burlington, MA, USA), Smad 2/3 (SC-398844, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Smad 1 (#6944, Cell Signaling, Danvers, MS, USA), Smad 5 (#9517, Cell Signaling, Danvers, MS, USA), phospho-Smad 2/3 (#8828, Cell Signaling, Danvers, MS, USA), or phospho-Smad 1/5 (#9516, Cell Signaling, Danvers, MS, USA) antiserum.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Biomedicines

Article Title: The Upregulation of Caffeic Acid Phenethyl Ester on Growth Differentiation Factor 15 Inhibits Transforming Growth Factor β/Smad Signaling in Bladder Carcinoma Cells

doi: 10.3390/biomedicines10071625

Figure Lengend Snippet: GDF15 inhibits the effect of TGFβ on Smas signaling activation and the expressions of GDF15, maspin, and NDRG1 in bladder carcinoma cells. The expressions of Smad 2/3, p-Smad 2/3, Smad 1, Smad 5, and p-Smad 1/5 were determined using immunoblot assays (( A ), left) and quantitative analysis (( A ), right) after treated with (+) or without (−) 10 ng/mL rhTGFβ and 400 ng/mL rhGDF15 as indicated for 1 h in T24 cells. The expressions of maspin, NDRG1, and β-actin were determined using immunoblot assays (( B ), left) and quantitative analysis (( B ), right) after being treated with (+) or without (−) 10 ng/mL rhTGFβ and 400 ng/mL rhGDF15 as indicated for 18 h in T24 cells. The expressions of Smad 2/3, p-Smad 2/3, Smad 1, Smad 5, and p-Smad 1/5 were determined using immunoblot assays (( C ), top) and quantitative analysis (( C ), bottom) after being treated with (+) or without (−) 10 ng/mL rhTGFβ as indicated for 1 h in T24-DNA and T24-GDF15 cells. The expressions of maspin, NDRG1, and β-actin were determined using immunoblot assays (( D ), top) and quantitative analysis (( D ), bottom) after treated with (+) or without (−) 10 ng/mL rhTGFβ and 400 ng/mL rhGDF15 as indicated for 18 h in T24-DNA and T24-GDF15 cells. The quantitative data were expressed as the intensity of protein bands of the p-target gene/target gene or target genes/β-actin relative to the control solvent-treated group ( n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: The blotting membranes were probed using antiserum of GDF15 (Ab206414, Abcam, Cambridge, MA, USA), NDRG1 (42-6200; Invitrogen, Carlsbad, CA, USA), Maspin (#554292, BD Biosciences, San Jose, CA, USA), GFRAL (ab107719, Abcam, Cambridge, MA, USA), β-actin (MAB1501, Merck Millipore, Burlington, MA, USA), Smad 2/3 (SC-398844, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Smad 1 (#6944, Cell Signaling, Danvers, MS, USA), Smad 5 (#9517, Cell Signaling, Danvers, MS, USA), phospho-Smad 2/3 (#8828, Cell Signaling, Danvers, MS, USA), or phospho-Smad 1/5 (#9516, Cell Signaling, Danvers, MS, USA) antiserum.

Techniques: Activation Assay, Western Blot

Journal: Biomedicines

Article Title: The Upregulation of Caffeic Acid Phenethyl Ester on Growth Differentiation Factor 15 Inhibits Transforming Growth Factor β/Smad Signaling in Bladder Carcinoma Cells

doi: 10.3390/biomedicines10071625

Figure Lengend Snippet: CAPE acts as the inhibitor of TGFβ receptor kinase in human bladder carcinoma cells. ( A ) The Smad 2/3, p-Smad 2/3, Smad 1, Smad 5, and p-Smad 1/5 expressions were determined using immunoblot assays after one hour of 10 ng/mL TGFβ treatments with (+) or without (−) 20 μM CAPE or 20 μM SB431542 pretreatment as indicated in T24 cells. ( B ) The quantitative data were expressed as the intensity bands of the phosphorylation proteins relative to the total protein levels for Smad 2/3 and Smad 1 (mean ± S.E.; n = 3). HT1376 cells were treated with 10 ng/mL rhTGFβ treatments with (+) or without (−) 20 μM CAPE or 20 μM SB431542 pretreatment as indicated for 18 h. ( C ) The relative mRNA levels ( n = 3) of GDF15 were determined by RT-qPCR assays and ( D ) the supernatants of culture media ( n = 4) were collected and GDF15 secretions were determined by ELISA. ( E ) The reporter activity of the GDF15 reporter vector treated with (+) or without (−) 10 ng/mL rhTGFβ, 20 μM CAPE or 20 μM SB431542, as indicated, in the HT1376 cells. ( F ) The reporter activity of the SEB4 reporter vector treated with 10 ng/mL rhTGFβ treatments with (+) or without (−) 20 μM CAPE or, 20 μM SB431542, or rhGDF15 pretreatment as indicated, in the HT1376 cells. Data are expressed as the mean percentage of luciferase activity relative to the mock-treated group ( n = 6). * p < 0.05, ** p < 0.01.

Article Snippet: The blotting membranes were probed using antiserum of GDF15 (Ab206414, Abcam, Cambridge, MA, USA), NDRG1 (42-6200; Invitrogen, Carlsbad, CA, USA), Maspin (#554292, BD Biosciences, San Jose, CA, USA), GFRAL (ab107719, Abcam, Cambridge, MA, USA), β-actin (MAB1501, Merck Millipore, Burlington, MA, USA), Smad 2/3 (SC-398844, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Smad 1 (#6944, Cell Signaling, Danvers, MS, USA), Smad 5 (#9517, Cell Signaling, Danvers, MS, USA), phospho-Smad 2/3 (#8828, Cell Signaling, Danvers, MS, USA), or phospho-Smad 1/5 (#9516, Cell Signaling, Danvers, MS, USA) antiserum.

Techniques: Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Plasmid Preparation, Luciferase

Journal: Experimental and Therapeutic Medicine

Article Title: Spironolactone alleviates myocardial fibrosis via inhibition of Ets-1 in mice with experimental autoimmune myocarditis

doi: 10.3892/etm.2022.11296

Figure Lengend Snippet: Effect of spironolactone on TGF-β1 and Smad-2/3 in EAM myocardium. (A) Immunostaining of TGF-β1 (magnification, x400). (B) Semi-quantification analysis of TGF-β1 staining. (C and D) Western blot analysis of p-Smad-2/3 and Smad-2/3 expression. * P<0.05 vs. control and # P<0.05 vs. EAM. Data are representative of three independent experiments. EAM, experimental autoimmune myocarditis.

Article Snippet: Separated protein was then transferred onto a PVDF membrane, which was incubated overnight at 4˚C with the following primary antibodies: total Ets-1 (1:1,000; cat. no. 6258; Cell Signaling Technology, Inc.), p-Ets-1 (1:500; cat. no. CBP1153; Assay Biotechnology Co., Inc.), Smad-2/3 (1:1,000; cat. no. 8685; Cell Signaling Technology, Inc.) and p-Smad-2/3 (1:1,000 dilution; cat. no. 8828; Cell Signaling Technology, Inc.).

Techniques: Immunostaining, Staining, Western Blot, Expressing