Fig. 3: NEDD4L promotes ferroptosis in ESCC cells.

A Volcano plot of significantly up and down-regulated genes after NEDD4L depletion. B Heat map of mRNA changes in siControl (n = 3) and siNEDD4L (n = 3) of EC9706 by bulk RNA-seq. C–G Publicly available data showed that NEDD4L expression is positively correlated with that of the ferroptosis related genes HMOX1, IREB2, KEAP1, LPCAT3 and BECN1 in esophageal cancer (http://gepia.cancer-pku.cn/). H, I ROS level in Eca109 and EC9706 cells transfected with siControl/siNEDD4L for 48 h were detected. n = 3 per group. J Silencing NEDD4L promoted GSH level in ESCC cells. Eca109 and EC9706 cells were transfected with siControl/siNEDD4L for 48 h. GSH level was determined at 412 nm. n = 3 per group. K Silencing NEDD4L reduced MDA content in ESCC cells. Eca109 and EC9706 cells were transfected with siControl/siNEDD4L 48 h. MDA content was measured at 532 nm and 600 nm. n = 3 per group. L Representative cell and mitochondrial ultrastructural images of EC9706 cells transfected with siContrlo/siNEDD4L under the treatment of erastin for 24 h. Scale bar = 5 μm. M, N Overexpressing NEDD4L promoted ROS level in ESCC cells. Eca109 and EC9706 cells were transfected with Flag-vector or Flag-NEDD4L plasmids for 48 h. n = 3 per group. O Overexpressing NEDD4L decreased GSH level in ESCC cells. Eca109 and EC9706 cells transfected with Flag-vector or Flag-NEDD4L plasmids for 48 h. n = 3 per group. P Overexpressing NEDD4L increased MDA content in ESCC cells. Eca109 and EC9706 cells transfected with Flag-vector or Flag-NEDD4L plasmids for 48 h. n = 3 per group. The data are presented as the mean ± SD. Statistical significance in I–K were determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. Statistical significance in N–P Statistical analysis were performed using Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001.