Fig. 6.

Anta-miR-744 transfection-induced HBE ferroptosis was mitigated by GPX4, ACSL4, and ALOX15 inhibitors. (A) qPCR results for miR-744 expression in HBE cells after transfection with miR-NC, miR-744, or anta-miR-744. (B) Representative images of anta-miR-744 transfection experiment. Representative images of miR-744 transfection experiment are shown in Fig. S4A. (C–I) HBE cells were analyzed after transfection with anta-miR-744, with or without the corresponding inhibitors. The experiments were performed with three independent technical replicates. (C)TEM results. Red arrows: mitochondrial shrinkage and denser membranes; yellow arrows: widened endoplasmic reticulum; dark-red arrows: increased number of vesicles. (D) Representative LIP images; (E) LIP images quantified using ImageJ. (F) Flow cytometry data for ROS, including representative images (left) and quantitative analysis (right). (G) Immunofluorescence assay results for ROS levels. (H) qPCR results for GPX4, ACSL4, and ALOX15 expression. (I) Left panel: Representative western blots for GPX4, ACSL4, and ALOX15; Right panel: Blots were quantified using ImageJ. ∗P < 0.05, ∗∗∗P < 0.001 (ANOVA, followed by multiple comparison tests). ACSL4, long-chain acyl-CoA synthetase 4; ALOX15, 15-lipoxygenase; GPX4, glutathione peroxidase 4; HBE, human bronchial epithelial; LIP, labile iron pool; PBS, phosphate-buffered saline; TEM, transmission electron microscopy; ROS, reactive oxygen species. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)