Effects of colchicine on cardiac pyroptosis following CoCl2 exposureColchicine attenuates chemical hypoxia-induced pyroptosis through downregulation of nuclear factor kappa B and caspase-1 in cardiomyocytes

This experiment was conducted using the H9c2 cardiac myoblast cell line. This study compared the H9c2 cell line under hypoxic conditions to those treated with both hypoxia and colchicine.Sheet 1: HIF-1 alpha-positive cells (flowcytometry analysis) on various concentrations of cobalt chloride and time pointsSheet 2: Cell viability (WST-1 assay) on various concentrations of cobalt chloride and time pointsSheet 3: Expression of HIF-1 alpha, IL-18, caspase-1, and NFkB following early (3 h) and late (24 h) hypoxia) using flowcytometry analysis. Cobalt chloride exposure was 600 micro Molar and the colchicine treatment was 1 micro Molar.Sheet 4: The percentage of pyroptotic H9c2 cells analyzed by flowcytometry after 3 h and 24 h hypoxic treatment. Early pyroptosis indicated cells expressing only caspase-1, while late pyroptosis indicated cells expressing both caspase-1 and propidium iodide.Sheet 5: Similar analysis as sheet 4, but the experiment was performed using confocal laser scanning microscope. Data are presented as arbitrary unit.本实验采用H9c2心肌成肌细胞系（H9c2 cardiac myoblast cell line）开展。本研究对比了缺氧条件下的H9c2细胞，以及同时接受缺氧与秋水仙碱（colchicine）处理的H9c2细胞。 工作表1：不同氯化钴（cobalt chloride）浓度与时间节点下的缺氧诱导因子-1α（HIF-1 alpha）阳性细胞比例（流式细胞术分析） 工作表2：不同氯化钴浓度与时间节点下的细胞活力（WST-1实验检测） 工作表3：分别于早期缺氧（3小时）与晚期缺氧（24小时）后，通过流式细胞术分析缺氧诱导因子-1α、白细胞介素-18（IL-18）、半胱天冬酶-1（caspase-1）与核因子κB（NFkB）的表达水平。本次实验中氯化钴暴露浓度为600微摩尔，秋水仙碱处理浓度为1微摩尔。 工作表4：经3小时与24小时缺氧处理后，通过流式细胞术分析的焦亡（pyroptosis）H9c2细胞占比。其中早期焦亡定义为仅表达半胱天冬酶-1的细胞，晚期焦亡定义为同时表达半胱天冬酶-1与碘化丙啶（propidium iodide）的细胞。 工作表5：与工作表4分析方法一致的实验，但采用激光共聚焦扫描显微镜（confocal laser scanning microscope）完成。数据以任意单位（arbitrary unit）呈现。