Fig. 7

HSPB1 rescues the effect of MSI2 deficiency on CRC ferroptosis in vitro and in vivo. A Western blotting for MSI2, HSPB1, ACSL4, FTH1, PCNA and GPX4 expression in SW620 and LOVO stable cells rescued with vector or HSPB1 plasmids. B HSPB1, ACSL4, FTH1 and GPX4 mRNA expression was determined by qRT‒PCR in SW620 and LOVO stable cells rescued with vector or HSPB1 plasmids. C Representative images and statistical analysis of colony formation assays of stable SW620 and LOVO cells rescued with vector or HSPB1 plasmids. D-E FACS and statistical analysis of total ROS levels (H2DCFDA) (D) and lipid ROS levels (C11-BODIPY) (E) in SW620 and LOVO stable cells rescued with vector or HSPB1 plasmids, gray indicates the positive control. F, FACS and statistical analysis of cell death rate in SW620 and LOVO stable cells rescued with vector or HSPB1 plasmids and treated with erastin (5 µg/mL) for 8 h. G-I The levels of intracellular ferrous ions (G), total iron (H) and reduced GSH (I) were determined by treatment with erastin (10 µg/mL) for 8 h in SW620 and LOVO stable cells rescued with vector or HSPB1 plasmids. J-K Representative electron microscopy images and statistical analysis of mitochondrial abnormalities in stable SW620 and LOVO cells transfected with vector or HSPB1 rescue plasmids. Scale bars, 1 μm. L In vivo model of xenograft tumors from SW620 stable cells transfected with vector or HSPB1 rescue plasmids and statistical analysis of tumor volume in M-NSG mice, n = 4. M Representative IHC images of xenograft tumors from SW620 stable cells transfected with vector or HSPB1 rescue plasmids; these images show HSPB1, ACSL4, Ki67 and PCNA expression. Scale bars, 100 μm. These results are presented as the mean ± SD values; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; B-J, L One-way ANOVA