Journal: International journal of oncology

Article Title: Mast cell chymase promotes angiogenesis and lymphangiogenesis mediated by activation of melanoma inhibitory activity gene family members in oral squamous cell carcinoma.

doi: 10.3892/ijo.2020.4996

Figure Lengend Snippet: Figure 5. Effects of chymase on angiogenesis and lymphangiogenesis in OSCC cells. Effects of chymase and MIA gene family member siRNA transfection on (A) VEGF‑A, (B) VEGF‑C, (C) VEGF‑C and (D) PDGFB secretion in OSCC cells. Changes in the (E and F) adhesive and (G and H) transmigra tory abilities of HSC3 cells to endothelial cells following chymase treatment and/or MIA gene family member siRNA transfection. *P<0.05. OSCC, oral squamous cell carcinoma; MIA, melanoma inhibitory activity; TANGO, transport and Golgi organization protein 1; PDGFB, platelet‑derived growth factor β polypeptide; siRNA, small interfering RNA; VEGF, vascular endo thelial growth factor.

Article Snippet: ELISA kits were used to analyze MIA (cat. no. 11976826001; Roche Diagnostics), MIA2 (cat. no. LS‐F16959; LifeSpan BioSciences, Inc.), TANGO/MIA3 (cat. no. LS‐F52248; LifeSpan BioSciences, Inc.), VEGF‐A (cat. no. RAB0508; Calbiochem; Merck KGaA), VEGF‐C (cat. no. DVEC00; R&D Systems, Inc.), VEGF‐D (cat. no. DVED00; R&D Systems, Inc.) and PDGFB (cat. no. DBB00; R&D Systems, Inc.).

Techniques: Transfection, Adhesive, Activity Assay, Small Interfering RNA

Journal: Journal of Biological Chemistry

Article Title: Spatial Compartmentalization of Tumor Necrosis Factor (TNF) Receptor 1-dependent Signaling Pathways in Human Airway Smooth Muscle Cells

doi: 10.1074/jbc.m605738200

Figure Lengend Snippet: FIGURE 4. Disruption of lipid rafts inhibits TNF--induced RhoA activa- tion. A, human airway smooth muscle cells were treated with () or without () 10 mM MCD for 1 h at 37 °C prior to extraction with cold Triton X-100 and sucrose density gradient fractionation. The distribution of TNFR1, RhoA, and the raft-resident proteins flotillin-1 and caveolin-1 was determined by immu- noblotting. B, cells, treated with () or without () 10 mM MCD for 30 min at 37 °C, were stimulated with TNF- (200 ng/ml) for 5 min at 37 °C, extracted, and analyzed by immunoblotting for TNFR1 and RhoA expression and TNF- binding.ActivatedRhoA(RhoA-GTP)wasisolatedusingaglutathioneS-trans- ferase fusion protein containing the Rho binding domain of Rhotekin as describedunder“ExperimentalProcedures.”C,humanairwaysmoothmuscle cells were surface-biotinylated with sulfo-NHS-biotin prior to treatment with () or without () MCD as in B. Cell extracts were immunoprecipitated (IP) with control (con Ig) or anti-TNFR1 antibody, and cell surface-associated TNFR1 was detected by blotting with HRP-streptavidin. To control for protein input, total cell extracts (total) were analyzed by immunoblotting for expres- sion of TNFR1. D, cells treated with () or without () MCD as in B were stimulated for 5 min at 37 °C with TNF- (200 ng/ml), PDGFBB (50 ng/ml), or S1P (1 M). Cell extracts were separated by SDS-PAGE and analyzed by immu- noblotting with antibodies against the indicated proteins. RhoA-GTP was iso- lated as described in B.

Article Snippet: Reagents—Recombinant human TNF- , biotin-TNF- , biotin-soybean trypsin inhibitor, polyclonal goat anti-human TNF- blocking antibody, and PDGFBB were purchased from R & D Systems (Abingdon, UK).

Techniques: Disruption, Extraction, Fractionation, Western Blot, Expressing, Binding Assay, Immunoprecipitation, Control, SDS Page

Journal: Cell Death & Disease

Article Title: RASSF1A inhibits PDGFB-driven malignant phenotypes of nasopharyngeal carcinoma cells in a YAP1-dependent manner

doi: 10.1038/s41419-020-03054-z

Figure Lengend Snippet: a A heat map generated using the significantly changed genes categorized in the “cytokine-cytokine receptor interaction pathway” is shown. b , c mRNA expression ( b ) was evaluated by qRT-PCR and protein concentration by ELISA ( c ) in CM of RASSF1A-overexpressing CNE-2 cells, RASSF1A-depleted CNE-1 cells and their corresponding control cells, The data are presented as the mean ± S.D. values, ** p < 0.01, Student’s t test. d – g PDGFB was transiently knocked down with a pool of siRNA or treated with a neutralizing antibody for PDGF-BB (10 µg/mL) in RASSF1A-depleted CNE-1 cells. PDGF-BB secretion in the CM was measured by ELISA ( d ), ** p < 0.01, Student’s t test. e Number of spheroids formed was determined via microscopy, and representative images ( e left panel) are shown. The formed spheroids were compared ( e right panel), the data are presented as the mean ± S.D. values, * p < 0.05, ** p < 0.01, Student’s t test; ns: non-sinificant. Scale bar: 200 µm. Representative images of the migration assay ( f ) and invasion assay ( g ) are shown, the data are presented as the mean ± S.D. values, ** p < 0.01, Student’s t test. Scale bar: 100 µm. h – j Recombinant PDGF-BB or IgG was added to RASSF1A-overexpressing CNE-2 cells. Representative images of sphere formation ( h ) (Scale bar: 200 µm.), migration ( i ) and invasion ( j ) assays (Scale bar: 100 µm) of RASSF1A-overexpressing CNE-2 cells treated with PDGF-BB (the culture medium was supplemented with 20 ng/ml or an equal volume of control IgG) are shown, The data are presented as the mean ± S.D. values, * p < 0.05, ** p < 0.01, Student’s t test.

Article Snippet: Cells were plated in 6-well plates (Corning, USA) and treated with humane recombinant PDGF-BB (220-BB-010, R&D Systems, USA) or Immunoglobulin G (IgG) control (AB-108-C, R&D Systems, USA) or neutralizing antibody against PDGF-BB (AB-220-NA, R&D Systems, USA) or latrunculin b (LTB, ab144291, Abcam, UK) 12 h after plating.

Techniques: Generated, Expressing, Quantitative RT-PCR, Protein Concentration, Enzyme-linked Immunosorbent Assay, Control, Microscopy, Migration, Invasion Assay, Recombinant

Journal: Cell Death & Disease

Article Title: RASSF1A inhibits PDGFB-driven malignant phenotypes of nasopharyngeal carcinoma cells in a YAP1-dependent manner

doi: 10.1038/s41419-020-03054-z

Figure Lengend Snippet: a – c YAP1 was transiently knocked down in RASSF1A-depleted CNE-1 cells. a In the indicated cells, YAP1 protein expression was assessed by using western blotting; b PDGFB, CYP61 and CTGF mRNA expression was assessed by qRT-PCR; c Concentration of PDGF-BB secreted in CM was measured by ELISA. The data are presented as the mean ± S.D. values, * p < 0.05, ** p < 0.01, Student’s t test. d – f Recombinant PDGF-BB or IgG was added to YAP1-silenced NPC cells. d The formed spheroids were counted via microscopy, and ( e ) representative images are shown. f The impact of PDGF-BB treatment on the migration and invasion of RASSF1A-depleted cells was determined by Transwell assays. The data are presented as the mean ± S.D. values, * p < 0.05, ** p < 0.01, Student’s t test. Scale bar: 200 µm.

Article Snippet: Cells were plated in 6-well plates (Corning, USA) and treated with humane recombinant PDGF-BB (220-BB-010, R&D Systems, USA) or Immunoglobulin G (IgG) control (AB-108-C, R&D Systems, USA) or neutralizing antibody against PDGF-BB (AB-220-NA, R&D Systems, USA) or latrunculin b (LTB, ab144291, Abcam, UK) 12 h after plating.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Microscopy, Migration