Fig. 5: BHLHE40 alleviates ferroptosis by upregulating SLC7A11.

A HUVECs treated with Erastin (ferroptosis inducer). Western blotting detection of BHLHE40 and SLC7A11 protein expression. B BHLHE40-overexpressing (OE) and control HUVECs were treated with Erastin alone or in combination with ferrostatin-1 (Fer-1). Western blotting detection of BHLHE40 and SLC7A11 protein expression (C). Mitochondrial morphology in BHLHE40-OE and control HUVECs after Erastin treatment, examined by TEM. Enlarged view of the area indicated by the dashed box in the left panel. Scale bars, 2 μm and 500 nm. D JC-1 immunofluorescence in control, BHLHE40-OE, and BHLHE40-OE/SLC7A11-knockdown (KD) HUVECs treated with Erastin. JC-1 monomer/aggregate ratios were quantified using ImageJ and shown in the right panel. JC-1 monomer: green; JC-1 aggregate: red; Scale bars, 50 μm. E ROS detection in control, BHLHE40-OE, and BHLHE40-OE/SLC7A11-KD HUVECs treated with Erastin. DCFH-DA: green; Hoechst: blue; Scale bars, 50 μm. DCFH-DA/Hoechst ratios were quantified using ImageJ and shown in the right panel. F Control, BHLHE40-OE, and BHLHE40-OE-SLC7A11-KD HUVECs were treated with Erastin alone or in combination with ferrostatin-1 (Fer-1). 4-NHE were quantified by immunofluorescence. 4-NHE: red; DAPI: blue; Scale bars, 50 μm. Relative Fluorescence Intensity was calculated using ImageJ and shown in the right panel. G, H Control, BHLHE40-OE, and BHLHE40-OE-SLC7A11-KD HUVECs were treated with Erastin alone or in combination with ferrostatin-1 (Fer-1). Measurement of GPx activity and GSH/GSSG ratio (G) and qRT-PCR measurement of ACSL4, PTGS2, and TRFC mRNA levels (H). Results were representative of three independent experiments (mean ± SD). Statistical significance was determined by unpaired Student’s t-test (D–H).