Fig 2: NUPR1 is upregulated in HCC tissues and predicts radiotherapeutic resistance of HCC. a NUPR1 mRNA expression in unpaired HCC tissues and non-tumor liver tissues from TCGA and GSE14520 datasets were shown (left and middle). NUPR1 mRNA expression in 50 paired HCC tissues and the adjacent matched noncancerous tissues was displayed on the right. b Representative IHC images show the NUPR1 expression in HCC (n = 50) and corresponding adjacent liver tissues (n = 50, left). Scale bar: 200 µm in 4 × magnification and 50 µm in 20 × magnification. Quantification of IHC score was shown in boxplot (right). c Gene set enrichment analysis (GSEA) with relatively low-expressed versus high-expressed NUPR1 from TCGA LIHC and GSE14520 datasets showed that the oxygen species pathway was positively correlated with NUPR1 expression. d The mRNA level of NUPR1 was negatively correlated with the CYP1B1 and CYP3A4 expression in the GSE15654 dataset by using Pearson correlation analysis. e Kaplan-Meier survival analysis was used to assess the correlation between NUPR1 expression or combined with AhR, CYP1B1 level, and the overall survival of HCC patients in TCGA LIHC dataset (n = 366) and GES15654 dataset (n = 216). f NUPR1 and AhR expression in 13 HCC tissues from the patients who accepted RT treatment were analyzed by IHC. Representative IHC images (left) and quantified IHC score (right) are shown

Fig 3: A schematic model of the NUPR1/AhR/CYP signal axis showing it promotes radiation resistance of HCC

Fig 4: AhR/CYP signal axis is required for NUPR1-mediated radioresistance in HCC. a Western blot analysis was utilized to examine the expression levels of AhR, CYP1A1, and CYP1B1 in different NUPR1 expressing cell lines transfected with AhR-overexpressing vector or control vector. b Relative ROS levels in different NUPR1 expressing cells with AhR overexpression or negative control were measured after exposure to 8 Gy of IR. c Quantification of cell death in indicated cells with AhR overexpression or negative control was measured after IR (8 Gy). d Cells with different NUPR1 expressions were pretreated with 5 µM FICZ or CH223191 for 24 h, followed by being exposed to 8 Gy of IR. The cells were collected to measure relative ROS levels. e, f Colony formation assays were performed in stably transfected NUPR1 overexpression or knockdown cell lines pretreated with FICZ or CH223191, followed by exposure to 6 Gy of IR. Data are the mean of biological triplicates and are shown as the mean ± SD. P values: *P < 0.05; **P < 0.01; ***P < 0.001 and ns, not significant by two-tailed Student’s t-test