Journal: Antioxidants

Article Title: Oxidized High-Density Lipoprotein Induces Endothelial Fibrosis Promoting Hyperpermeability, Hypotension, and Increased Mortality

doi: 10.3390/antiox11122469

Figure Lengend Snippet: OxHDL induces endothelial fibrosis through the TGF-β1/2 secretion/ALK-5/Smad protein pathway. ( A , B ) ECs were exposed to vehicle, HDL 50 μg/mL, and oxHDL 50 μg/mL for 48 h, and mRNA expression ( A ) and protein secretion ( B ) of TGF-β1 (left panels) and TGF-β2 (right panels) were measured. ( C , D ) ECs were exposed to vehicle and oxHDL 50 μg/mL for 48 h in the absence or presence of the NF-κB inhibitors SC-3060 (5 μM) and JSH-23 (30 μM), and mRNA expression ( C ) and protein secretion ( D ) of TGF-β1 (left panels) and TGF-β2 (right panels) were measured. (N = 11). ( E , F ) ECs were transfected with specific siRNA against TGF-β1 (siTGF-β1) and TGF-β2 (siTGF-β2) or non-transfected for 72 h, and then, cells were exposed to vehicle and oxHDL 50 μg/mL for 48 h, and mRNA ( E ) and protein ( F ) expression of VE-cadherin (left panels), α-SMA (middle panels), and FN (right panels) was measured. (N = 16). ( G , H ) ECs were exposed to vehicle and oxHDL 50 μg/mL for 48 h in the absence or presence of the ALK-5 inhibitor GW-788388 (5 μg/mL) and SB-431542 (30 μM), and mRNA ( G ) and protein ( H ) expression of VE-cadherin (left panels), α-SMA (middle panels), and FN (right panels) was measured. (N = 16). ( I , J ) ECs were exposed to vehicle and oxHDL 50 μg/mL for 48 h in the absence or presence of the Smad3 inhibitor SIS3 (10 μM) and (E)-SIS3 (5 μM), and mRNA ( I ) and protein ( J ) expression of VE-cadherin (left panels), α-SMA (middle panels), and FN (right panels) was measured. (N = 16). Statistical differences were assessed by one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Results are expressed as the mean ± SD.

Article Snippet: The following reagents and inhibitors were used: LOX-1 inhibitor κ-carrageenan (250 μg/mL, Sigma-Aldrich), neutralizing anti-LOX antibody (1:50, Abcam, Cambridge, UK), NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI, 10 μM, Sigma-Aldrich), NAD(P)H oxidase inhibitor, apocynin (Apo, 10 mM, Sigma-Aldrich), a reduced form of glutathione (GSH, 1 mM, Sigma-Aldrich), cell permeable antioxidant N-Acetylcysteine (NAC, 5 mM, Tocris, Bristol, UK), NF-κB inhibitor (SC-3060, 5 μM, Santa Cruz Biotechnology, Dallas, TX, USA), JSH-23 (30 μM, Sigma-Aldrich, USA), ALK5 inhibitor, GW-788388 (5 μg/mL) and SB-431542 (30 μM) (MedChemExpress, Monmouth Junction, NJ, USA), Smad3 inhibitor SIS3 (10 μM) and (E)-SIS3 (5 μM) (MedChemExpress, USA), and FITC-dextran 40 kDa (0.5 mg/mL, Sigma-Aldrich, USA).

Techniques: Expressing, Transfection

Journal: Nature cancer

Article Title: Targeting PAK4 to reprogram the vascular microenvironment and improve CAR-T immunotherapy for glioblastoma

doi: 10.1038/s43018-020-00147-8

Figure Lengend Snippet: a–d, GBM ECs were lentivirally transduced to express SMA-fLuc and CMV-rLuc, followed by shRNA library-based kinomic screening. fLuc and rLuc bioluminescence was then analyzed. a, fLuc/rLuc ratios. LTR, long terminal repeats; RRE, rev response element. b, Effects of kinase knockdown on global ratio changes. c, Positive and negative regulators denoted in the human kinome. In b and c, positive regulators indicate ratio decreases of >50% by the kinase knockdown, whereas negative regulators indicate ratio increases of >50% by the kinase knockdown. In a–c, the values of fLuc/rLuc ratios were averaged. d, Effects of PAK family kinase knockdown (means ± s.e.m.; n = 4 individual shRNAs for PAK1; n = 7 individual shRNAs for PAK2; n = 5 individual shRNAs for PAK3, PAK4, PAK5/7 and PAK6). e,f, ECs isolated from human GBM tumors or from normal brains were lentivirally transduced to express SMA-fLuc, CMV-rLuc and either CRISPR sgRNA targeting PAK4 or a control random sequence. e, Cell lysates were immunoblotted. This experiment was repeated independently twice with similar results. f, fLuc and rLuc bioluminescence was analyzed in GBM ECs (n = 8 independent cell assays; means ± s.e.m.). Statistical significance was determined by two-tailed Student’s t-test. g–k, ECs were lentivirally transduced to express CRISPR sgRNA targeting PAK4 or a random sequence. GBM ECs were subjected to proliferation (g; means ± s.e.m.), migration (h; means ± s.d.) and invasion analyses (i; means ± s.e.m.) (n = 3 EC samples each derived from a distinct human GBM tumor). Statistical significance in g–i was determined by two-tailed Student’s t-test. j, GBM ECs were seeded on transwells. FITC-dextran was loaded into the upper chamber and diffused FITC–dextran in the lower chamber was analyzed by fluorospectrometry (n = 3 EC samples, each derived from a distinct human GBM tumor; means ± s.d.). Statistical significance was determined by two-tailed Student’s t-test. k, GBM ECs were cultured on dishes to form monolayers, then imaged (left) or stained with phalloidin for visualizing F-actin (middle). Right, GBM ECs were seeded on Matrigel for 24 h to form capillary-like tubes. Scale bars, 20 μm. This experiment was repeated independently twice with similar results.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated dextran (molecular weight = 70,000 Da; 10 μg ml −1 ; Santa Cruz; sc-263323) was added to the top chamber.

Techniques: shRNA, Knockdown, Isolation, CRISPR, Control, Sequencing, Two Tailed Test, Migration, Derivative Assay, Cell Culture, Staining