Fig. 7: BBR promotes ferritin proteolysis to increase ferrous overload in HSCs.

A HSC-LX2 cells were treated with BBR (21 μM) for 24 h, and FTH1 and FTL mRNA levels were determined by RT–qPCR. B HSC-LX2 cells were exposed to BBR (21 μM) with or without MG-132 (10 μM), and the protein levels of FTH1, FTL, and ubiquitin were determined. C HSC-LX2 cells were exposed to BBR (21 μM) with or without cycloheximide (CHX, 100 μM) at the indicated time points, and FTH1 and FTL were assayed. D HSC-LX2 cells were treated with BBR (21 μM), CQ (10 μM), or BBR combined with CQ with or without MG-132 (10 μM); FTH1 and FTL levels were determined. E HSC-LX2 was treated with BBR (21 μM) in the presence of MG-132 (10 μM); ubiquitinated FTH1 and FTL were precipitated and detected with anti-ubiquitin. F, G HSC-LX2 cells were exposed to BBR (21 μM) for 24 h; lipid ROS, Fe²⁺, GSH, and MDA were assayed. H HSC-LX2 cells were transfected with vector, FTH1, or FTL plasmid; the protein expression of FTH1 and FTL was determined. I–K HSC-LX2 cells were transfected with vector or FTH1 plasmid and then treated with BBR (21 μM), and ROS, cell viability and Fe²⁺ were investigated. L HSC-LX2 cells were transfected with Flag-FTH1, HA- K48-Ub, and HA-K63-Ub plasmids and then treated with BBR (21 μM) and MG-132 (10 μM) for 9 h; an IP assay was used to detect the K48 and K63 sites of FTH1 ubiquitination. M HSC-LX2 cells were treated with or without BBR (21 μM); p97/VCP, p62, and S403-pp62 were precipitated. The results are expressed as mean ± SD. Data are representative of three independent experiments; n = 3–6 in every group; compared with the control group, **P < 0.01. NS, not significant.