TNF-α inhibited A549 cell viability, increased paracellular permeability, HIF-1α expression and decreased VASP expression.

(A) Cells were evenly seeded in 96-well plates at a density of 103–104 cells per well (200 µL per well) and then incubated with TNF-α (0 – 100 ng/mL) for 24 hours. The cell viability rates were examined using an MTT assay, as shown in the bar graph. Compared with 0 ng/mL TNF-α (vehicle control), the stimulation with 0.1–8 ng/mL TNF-α did not affect cell viability (p>0.05). In contrast, 10 ng/mL and 100 ng/mL TNF-α significantly inhibited the viability of A549 cells (*p5/mL A549 cell suspension was seeded in the upper chamber of a Transwell that was previously covered with 1% gelatin until 80% confluence. After the well was pre-incubated with 10 ng/mL TNF-α for 24 hours, the Pa was determined according to the rate of fluorescence intensity of FITC-Dextran in the upper and lower chambers by a multifunctional microplate reader, as shown in the bar graph, TNF-α treatment increased Pa by 23.4% compared with the control group. (C) A549 cells were exposed to 10 ng/mL TNF-α for 24 hours, and the total protein of HIF-1α was then extracted. Western Blotting analysis was then used to evaluate the total protein level with each antibody against total HIF-1α, as shown in the bar graph, which represents a graphical representation of the relative density of HIF-1α to GADPH. TNF-α dramatically increased HIF-1α expression by 30.7% compared with the vehicle control. (D) A549 cells were exposed to 10 ng/mL TNF-α for 24 hours, and the total VASP protein was then extracted. Western Blotting analysis was then used to evaluate the total protein level with each antibody against total VASP, as shown in the bar graph. TNF-α inhibited VASP expression by 56.6% compared with the vehicle control. GAPDH served as a loading control; representative blots from three independent experiments with similar results are shown. The relative protein expression levels obtained for HIF-1α or VASP/GAPDH were used in bar graphs C and D. The test was repeated three times with identical results. The data are presented as the mean ± SEM. *p**p

(A) 将细胞以10³–10⁴个/孔（每孔200 μL）的密度均匀接种于96孔板，随后用浓度范围为0–100 ng/mL的肿瘤坏死因子-α（TNF-α）处理24小时。采用MTT法检测细胞存活率，结果如柱状图所示。与0 ng/mL TNF-α（溶剂对照组）相比，0.1–8 ng/mL TNF-α刺激对细胞存活率无显著影响（p>0.05）；与之相反，10 ng/mL与100 ng/mL TNF-α可显著抑制A549细胞存活率（*p<0.05，原文此处存在笔误残留）。(B) 将浓度为5×10⁵个/mL的A549细胞悬液接种于预先用1%明胶包被的Transwell小室上层，待细胞汇合度达到80%。在上室用10 ng/mL TNF-α预孵育24小时后，通过多功能酶标仪检测异硫氰酸荧光素-葡聚糖（FITC-Dextran）在上下小室的荧光强度比值，以此计算通透系数（Pa），结果如柱状图所示。与对照组相比，TNF-α处理使Pa升高23.4%。(C) 将A549细胞用10 ng/mL TNF-α处理24小时后，提取总蛋白，采用蛋白质印迹（Western Blotting）分析，以抗总缺氧诱导因子-1α（HIF-1α）抗体检测总蛋白水平，结果如柱状图所示，该图代表HIF-1α相对于甘油醛-3-磷酸脱氢酶（GAPDH）的相对灰度密度。与溶剂对照组相比，TNF-α可使HIF-1α表达显著上调30.7%。(D) 将A549细胞用10 ng/mL TNF-α处理24小时后，提取总血管扩张刺激磷蛋白（VASP），采用蛋白质印迹（Western Blotting）分析，以抗总VASP抗体检测总蛋白水平，结果如柱状图所示。与溶剂对照组相比，TNF-α可使VASP表达显著下调56.6%。GAPDH作为内参加载对照；图中展示了3次独立重复实验的代表性印迹，结果一致。柱状图C与D分别采用HIF-1α或VASP/GAPDH的相对蛋白表达水平进行绘制。实验重复3次，结果均一致。数据以平均值±标准误（mean ± SEM）表示。原文末尾存在未完成的笔误残留。