Fig. 7
Silencing BTB and CNC homolog 1 (BACH1) alleviates glutaminase kidney isoform, mitochondrial (GLS) levels to rescue tert-butyl hydroperoxide (TBHP)-induced ferroptosis in bone mesenchymal stem cells (BMSCs). A total of 1 × 106 BMSCs were used in each group. Nontargeting negative control BMSCs (Sh-NC) and a short hairpin RNA (shRNA) targeting BACH1 BMSCs (Sh-BACH1) were incubated with PBS, TBHP (50 μM), BPTES (5 μM), or TBHP (50 μM) in the presence of BPTES (5 μM) for 24 h. A Cell proliferation was detected by 5-ethynyl-2’-deoxyuridine (EdU) assay (n = 5). Representative EdU-positive cells (red) are shown. White Bars = 100 μm. B EdU-positive cells (red) were counted to calculate the percentage. C The ROS levels were measured by 2’−7’-dichlorofluorescein diacetate (1:1000, 20 μmol/L) (n = 5). White Bars = 100 μm. D Relative reactive oxygen species (ROS) intensity (green staining) was statistically analyzed. E Malondialdehyde (MDA) assay was used to quantify lipid peroxidation in BMSCs (n = 5). F Quantitative analysis of glutamate content levels was measured by using the Glutamate Assay Kit (n = 5). G Quantitative analysis of glutathione (GSH) levels was measured by a GSH analysis kit ((n = 5). H BMSCs were harvested, BACH1 and GLS were evaluated by Western Blot (n = 5). I and J Quantitative analysis of the protein levels of BACH1 and GLS. All data are representative of at least five separate experiments. All these experiments were repeated by at least five times. Analysis: ANOVA followed by a post hoc analysis (B, D, E, F, G, I and J); *P < 0.05, **P < 0.01, ***P < 0.001