Journal: Experimental and Therapeutic Medicine

Article Title: Triptolide protects against TGF-β1-induced pulmonary fibrosis by regulating FAK/calpain signaling

doi: 10.3892/etm.2019.8127

Figure Lengend Snippet: TPL regulates TGF-β1-induced expression of FAK/calpain in lung fibroblasts. Lung fibroblasts were treated with 50 ng/ml TGF-β1 for 48 h and addition of 5 nmol/l TPL and incubation for 48 h. Cells that were untreated served as a control. The protein levels of p-FAK/FAK, calpain 1 and calpain 2 in lung fibroblasts was determined using western blot analysis. Protein levels of (A) p-FAK/FAK, (B) calpain 1 and (C) calpain 2. Values are expressed as the mean ± standard error of the mean (n=3/group). (D) Western blot analysis of FAK, p-FAK, calpain 1 and calpain 2. ***P<0.001 vs. the control group; ### P<0.001 vs. the model group. TGF-β1, transforming growth factor-β1; TPL, triptolide; p-FAK, phosphorylated-focal adhesion kinase.

Article Snippet: Anti-FAK (cat. no. CA36131), anti-phospho-(p)-FAK (cat. no. CN893300), anti-calpain 2 (cat. no. BS3696) and anti-β-actin (cat. no. 17AV0303) antibodies were obtained from Bioworld Technology, Inc. Anti-calpain 1 (cat. no. 00016377) was obtained from ProteinTech Group, Inc. Penicillin/streptomycin solution (X100), 0.05% trypsin-EDTA and DMSO were purchased from Sigma-Aldrich (Merck KGaA).

Techniques: Expressing, Incubation, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Triptolide protects against TGF-β1-induced pulmonary fibrosis by regulating FAK/calpain signaling

doi: 10.3892/etm.2019.8127

Figure Lengend Snippet: TPL inhibits cytokine release of TGF-β1-induced lung fibroblasts by downregulating FAK and upregulating calpain. Lung fibroblasts were treated with 50 ng/ml TGF-β1 for 48 h and addition of 5 nmol/l TPL and incubation for 48 h. In addition, cells were cultured with 50 ng/ml TGF-β1 and 5 nmol/l TPL for 48 h prior to the addition of 20 µM FAK inhibitor or 50 µM calpeptin for 24 h. Cells that were untreated served as a control. The protein levels of FAK, p-FAK, calpain 1 and calpain 2 in lung fibroblasts were determined using western blot analysis. The concentration of IL-6 in the cell culture supernatant was detected by ELISA. Quantified protein levels of (A) p-FAK/FAK, (B) calpain 1 and (C) calpain 2. Values are expressed as the mean ± standard error of the mean (n=3/group). (D) Western blotting of FAK, p-FAK, calpain 1 and calpain 2. (E) Concentration of IL-6 in the cell culture supernatant. ***P<0.001 vs. the control group; ### P<0.001 vs. model group; ∆∆∆ P<0.001 vs. TPL group. TGF-β1, transforming growth factor-β1; TPL, triptolide; p-FAK, phosphorylated focal adhesion kinase; IL, interleukin.

Article Snippet: Anti-FAK (cat. no. CA36131), anti-phospho-(p)-FAK (cat. no. CN893300), anti-calpain 2 (cat. no. BS3696) and anti-β-actin (cat. no. 17AV0303) antibodies were obtained from Bioworld Technology, Inc. Anti-calpain 1 (cat. no. 00016377) was obtained from ProteinTech Group, Inc. Penicillin/streptomycin solution (X100), 0.05% trypsin-EDTA and DMSO were purchased from Sigma-Aldrich (Merck KGaA).

Techniques: Incubation, Cell Culture, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Triptolide protects against TGF-β1-induced pulmonary fibrosis by regulating FAK/calpain signaling

doi: 10.3892/etm.2019.8127

Figure Lengend Snippet: TPL inhibits TGF-β1-induced pulmonary fibrosis by downregulation of the expression of ColIα and ColIII via regulating of FAK/calpain. Lung fibroblasts were treated with 50 ng/ml TGF-β1 for 48 h and addition of 5 nmol/l TPL and incubation for 48 h. In addition, cells were cultured in 50 ng/ml TGF-β1 and 5 nmol/l TPL for 48 h prior to the addition of 20 µM FAK inhibitor or 50 µM calpeptin for 24 h. Cells that were untreated served as a control. The expression of ColIα mRNA and ColIII mRNA in lung fibroblasts was detected using reverse transcription-quantitative PCR. (A) ColIα mRNA and (B) ColIII mRNA. Values are expressed as the mean ± standard error of the mean (n=5/group). ***P<0.001 vs. the control group; ### P<0.001 vs. the model group; ∆∆∆ P<0.001 vs. TPL group. TGF-β1, transforming growth factor-β1; TPL, triptolide; p-FAK, phosphorylated focal adhesion kinase; Col, collagen.

Article Snippet: Anti-FAK (cat. no. CA36131), anti-phospho-(p)-FAK (cat. no. CN893300), anti-calpain 2 (cat. no. BS3696) and anti-β-actin (cat. no. 17AV0303) antibodies were obtained from Bioworld Technology, Inc. Anti-calpain 1 (cat. no. 00016377) was obtained from ProteinTech Group, Inc. Penicillin/streptomycin solution (X100), 0.05% trypsin-EDTA and DMSO were purchased from Sigma-Aldrich (Merck KGaA).

Techniques: Expressing, Incubation, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Anti-PTK7 Monoclonal Antibodies Inhibit Angiogenesis by Suppressing PTK7 Function

doi: 10.3390/cancers14184463

Figure Lengend Snippet: Effect of PTK7 mAbs on VEGF-induced activation of KDR and its downstream signaling proteins in HUVECs. HUVECs were starved in M199 medium with 1% FBS for 6 h and pre-treated with mAb-32 or mAb-43 (10 μg/mL) or sPTK7 (4 μg/mL) 30 min before VEGF stimulation. The cells were stimulated with 10 ng/mL VEGF for 2 min to analyze the phosphorylation of KDR, 60 min to analyze the phosphorylation of FAK, or 10 min to analyze the phosphorylation of other signaling proteins. The phosphorylation of KDR and its downstream signaling proteins was examined using Western blotting. Solid and open triangles indicate full-length PTK7 and sPTK7, respectively. “–”: no treatment with anti-PTK7 mAb or sPTK7. The graph shows the phosphoprotein/total protein ratio in the mAb-treated and sPTK7-treated groups relative to that in the VEGF-alone group (n = 3). Data are represented as mean ± standard deviation. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. VEGF-alone control. The absence of the p value between antibody-treated groups indicates statistical insignificance.

Article Snippet: Antibodies were purchased from the following vendors: Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-phospho-ERK (sc-7383) and anti-FAK (sc-557) antibodies; Cell Signaling Technology (Beverly, MA, USA), anti-phospho-KDR (Tyr1175; 2478S), anti-KDR (2479S), anti-phospho-Src family (Tyr416; 2101S), anti-Src (2109S), anti-phospho-JNK (Thr183/Tyr185; 4668S), and anti-JNK (9252S) antibodies; Merck Millipore (Burlington, MA, USA), anti-phospho-FAK antibodies (Tyr396; abt135); Bioss (Boston, MA, USA), anti-ERK2 antibodies (bms-52068R); Sigma-Aldrich, anti-FLAG-M2 antibodies (F1804); BioLegend (San Diego, CA, USA), anti-HA antibodies (902302); Qiagen (Cambridge, MA, USA), anti-penta-His antibodies; AbClone (Seoul, Korea), anti-GAPDH antibodies (abc2003); KOMA Biotech (Seoul, Korea), horseradish peroxidase-conjugated goat anti-mouse-IgG and anti-rabbit-IgG antibodies.

Techniques: Activation Assay, Western Blot, Standard Deviation