Fig 1: CB2R protects against iron-induced toxicity in hepatocytes by suppressing the STAT3/hepcidin pathway. a Immunofluorescence detection of CB2R localisation in liver (green). Nuclear counterstaining was performed with DAPI (blue, Scar bar = 100 and 20 μm). b Cell viability measured by CCK-8 assay across different concentrations of FAC, and comparison between WT and CB2R-KO hepatocytes (n = 6, 6 respectively). c Representatives of the flurometric assay on MMP and ROS in different treatment groups. d Summarized data demonstrated changes in MMP and ROS in hepatocytes (n = 3, 5 respectively). e Total cellular iron content (n = 4). f The level of LIP was detected by using flow cytometry (n = 5). g HAMP mRNA levels determined by qRT-PCR (n = 3, 3 respectively). h Representative Western blots of FPN1 protein, with β-actin as the loading control. i Representative Western blots of the upstream regulatory signaling molecules p-SMAD1/5/9, SMAD1/5/9, p-STAT3, and STAT3, with β-actin as the loading control. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± SD for each group. Individual data points are displayed as dots
Fig 2: CB2R deficiency aggravates iron retention while its overexpression alleviates accumulation. a Representative Prussian blue staining of liver sections from WT and CB2R-KO mice (scale bars = 100 and 20 µm). b Liver iron concentrations in WT and CB2R-KO mice (n = 4). c Serum hepcidin levels (n = 6). d Hepatic HAMP mRNA expression (n = 5). e Representative Western blots of FTL and FPN1 in the liver of WT and CB2R-KO mice, with β-actin as the loading control. f Representative Western blot verifying AAV-mediated CB2R overexpression in the liver, with GAPDH as the loading control. g Prussian blue staining of liver sections from AAV-empty and AAV-CB2R injected mice. h Liver iron concentrations following AAV injection (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± SD for each group. Individual data points are displayed as dots
Fig 3: Pharmacological CB2R activation limits hepatic iron accumulation by modulating the hepcidin-FPN1 axis. a Representative Prussian blue staining of liver sections from pharmacological treatment groups (black arrows indicate positive blue iron deposits; scale bars = 100 and 20 µm). b Liver iron concentrations quantified using a ferrozine-based assay (n = 4). c Serum ferritin levels determined using ELISA (n = 5). d Serum hepcidin levels using ELISA (n = 6). e Hepatic HAMP mRNA expression determined by qRT-PCR (n = 5). f Representative Western blots of iron-related proteins FPN1, FTH, and FTL in the liver across different treatment groups, with β-actin as the loading control. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± SD for each group. Individual data points are displayed as dots
Supplier Page from Abcam for Mouse Hepcidin ELISA Kit