human primary lung microvascular endothelial cells (lmvecs)  (Lonza)

Journal: bioRxiv

Article Title: Endothelial PHD2 deficiency induces apoptosis resistance and inflammation via AKT activation and AIP1 loss independent of HIF2α

doi: 10.1101/2024.02.01.578286

Figure Lengend Snippet: Cells were transfected with scrambled control (siCTRL), PHD2 (siPHD2), AIP1 (siAIP1) siRNA or their combination. A and B , 48 h post-transfection, cells were further incubated for 24 h in complete medium with specific PI3Kd/AKT inhibitor leniolisib (5 mM) or vehicle control. Activation of STAT1 (pSTAT1-Y701), STAT3 (pSTAT3-Y705), and AKT (pAKT-S473) was assessed by Western blotting (n = 5). C , 48 h post-transfection, cells were further incubated for 24 h in serum- and growth factor-free medium with leniolisib or vehicle and assessed for apoptosis by Annexin V/Propidium Iodide staining (n = 4). D , 72 h post-transfection, cell culture supernatants were collected for measuring secreted CXCL10 levels by ELISA (n = 5). E , Lung microvascular endothelial cells isolated from congenital associated PAH (APAH) patients and failed donor (FD) controls were cultured in complete medium and assessed activation of pSTAT1-Y701 and pSTAT3-Y705 (n = 5). Western blot protein levels were normalized to β-actin. Data are presented as mean ± SEM. #p = 0.07; *p < 0.05; **p ≤ 0.01 Two-way ANOVA with post-hoc t-test (A-D), pooled t-test (E).

Article Snippet: Human primary lung microvascular endothelial cells (LMVECs) and pulmonary artery endothelial cells (PAECs) from different donors were purchased from Lonza (Walkersville, MD.

Techniques: Transfection, Incubation, Activation Assay, Western Blot, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation