Figure 4: Effects of Chronic (24 h) Cyclic Stretch on [Ca

<b>(A)</b> Control <b>(top panel)</b> and stretched <b>(bottom panel)</b> AE cells were stained with Fluo-4 following administration of hyperforin to activate TRPC-6. In control cells, strong and continuous Ca²⁺ influx through the evenly distributed TRPC-6 channels at the cell surface is indicated <b>(white arrows, top panel inset)</b>. In chronically stretched cells, Ca²⁺ influx at the cell membrane is restricted to small, localized areas that form a <b>green punctate pattern (white arrows, bottom panel inset)</b>. <b>(B)</b> Bar graph showing the effects of chronic stretch, hyperforin, and 1-oleoyl-2-acetyl-sn-glycerol (OAG), a mechanosensitive channel activator, on the [Ca²⁺]i (respective cell numbers = 26, 30, 32, 25, 33, 40, in the order shown in graph). Ca²⁺ uptake by TRPC-6 and mechanosensitive Ca²⁺ channels, in general, is lower in the 24-h stretched AE cells compared with the non-stretched (control) cells. <b>(C)</b> Representative TRPC-6 immunoblot of biotin-labeled/streptavidin-immunoprecipitated cell membrane fraction (M) and total cell lysate (T) from 0-, 1-, and 24-h stretched AE cells, showing depleted levels of TRPC-6 in the cell membrane fraction of the 24-h stretched cells. Bar graph shows quantification of TRPC-6 levels from 3 separate experiments. β-tubulin immunolabeling was used as loading control. See Supplemental Figure 7C for full Western blots. All values are mean ± SEM. *p &lt; 0.001 versus control cells. See Supplemental Videos 2, 3, and 4. Abbreviations as in Figures 1, 2, and 3.

<b>(A)</b> 采用贯叶金丝桃素（hyperforin）激活瞬时受体电位阳离子通道亚家族C成员6（TRPC-6）后，使用Fluo-4对对照组（上图）与慢性牵拉组（下图）的AE细胞进行染色。对照组细胞中，细胞表面均匀分布的TRPC-6通道介导的强而持续的Ca²⁺内流，已通过白色箭头标注（上图内嵌图）。经慢性牵拉处理的细胞中，细胞膜处的Ca²⁺内流仅局限于局部微小区域，形成绿色点状染色模式（下图内嵌图白色箭头标注）。
<b>(B)</b> 柱状图展示了慢性牵拉、贯叶金丝桃素，以及机械敏感性通道激活剂1-油酰-2-乙酰基-sn-甘油（OAG）对细胞内游离钙离子浓度[Ca²⁺]i的影响（按图中所示顺序，对应检测细胞数依次为26、30、32、25、33、40）。总体而言，相较于未牵拉的对照组AE细胞，经24小时牵拉处理的AE细胞中，TRPC-6与机械敏感性钙通道所介导的Ca²⁺摄取水平更低。
<b>(C)</b> 对分别经0小时、1小时及24小时牵拉处理的AE细胞的生物素标记/链霉亲和素免疫沉淀细胞膜组分（M）与全细胞裂解液（T）进行TRPC-6代表性免疫印迹检测，结果显示24小时牵拉组细胞的细胞膜组分中TRPC-6的表达水平显著下调。柱状图展示了3次独立实验中TRPC-6表达水平的定量分析结果，以β-微管蛋白免疫标记作为上样内参。完整免疫印迹结果参见补充图7C。所有数值均以平均值±标准误（mean ± SEM）表示。与对照组相比，*p < 0.001。相关实验视频参见补充视频2、3及4。缩写含义同图1、图2及图3。