The effect of radiotherapy in combination with nifuroxazide on the proliferation, migration and apoptosis of Hep G2 cells. (A) The effect of the radiotherapy in combination with nifuroxazide on the viability of Hep G2 cells by CCK-8 assay. (B) The effect of the radiotherapy in combination with nifuroxazide on the proliferation of Hep G2 cells by cell clone formation assay. (C) The effect of the radiotherapy in combination with nifuroxazide on the migration of Hep G2 cells by Wound-Healing assay. (D) The effect of the radiotherapy in combination with nifuroxazide on the migration of Hep G2 cells by transwell assay. (E) The effect of the radiotherapy in combination with nifuroxazide on the apoptosis of Hep G2 cells by flow cytometry assay. One-way ANOVA was carried out and the data were presented as mean ± SD (n=3). Compared with the control group, *P<0.05; compared with “4Gy” group, #P<0.05.

The effect of radiotherapy in combination with nifuroxazide on the expressions of tumor-associated proteins in cells. After radiotherapy, Hep G2 cells were treated with nifuroxazide at the different dose. At 24 h or 48 h after being incubated, the expression of tumor-associated proteins was detected by Western blot. (A-E) The expression of Stat3, p-Stat3, PCNA, Ki67 and cyclin D1 related with cell proliferation was analyzed by Western blot. (F-N) The expression of cytochrome C, pro-caspase 3, c-caspase 3, pro-caspase 9, c-caspase 9, Bax, Bcl-2, PARP and c-PARP related with cell apoptosis was analyzed by Western blot. One-way ANOVA was carried out and the data are presented as mean ± SD (n=3). Compared with the control group, *P<0.05; compared with “4Gy” group, #P<0.05.

The effects of radiotherapy in combination with nifuroxazide on tumor growth and survival of tumor-bearing mice. At 7 days after establishing the tumor model, the mice are received distinct treatments. (A) Treatment scheme of different methods. (B) Tumor pictures and tumor weight of tumor-bearing mice under different treatments are measured for statistical analysis. (C) The tumor volume changes of tumor-bearing mice under different treatments. (D) The survival of tumor bearing mice under different treatments. (E) Pathological pictures of tumor-bearing mice under different treatments. The ruler in the top row of the images represents 200 nanometers; the ruler in the bottom row of the images represents 50 nanometers. (F) Statistical analysis of pathological score. One-way ANOVA was carried out and the data are presented as mean ± SD (n=3) except Figure 3D, which was analyzed by Kaplan-Meier method. Compared with the PBS group, *P<0.05; compared with the radiotherapy group, $P<0.05; compared with nifuroxazide group, #P<0.05.

The effects of radiotherapy in combination with nifuroxazide on the cell proliferation or apoptosis in tumor tissues. (A-C) The expression of Ki6, PCNA or c-caspase-3 in tumor tissues is detected by immunohistochemistry. The ruler in the top row of the images represents 200 nanometers; the ruler in the bottom row of the images represents 50 nanometers. (D) The cell apoptosis on tumor tissues is detected by TUNEL assay. (E-N) The protein expression of Stat3, p-Stat3, Ki67, PCNA, cyclin D1, cytochrome C, Bcl-2, Bax, pro-caspase 3 and c-caspase 3 in tumor tissues is detected by Western blot. One-way ANOVA was carried out and the data are presented as mean ± SD (n=3). Compared with the PBS group, *P<0.05; compared with the radiotherapy group, #P<0.05; compared with the nifuroxazide group, $P<0.05.

The effects of radiotherapy in combination with nifuroxazide on the infiltration of immune cells in tumor tissues. (A-C) Different isoforms of T lymphocytes infiltration in tumor tissues detected by immunofluorescence assay. (D) M1 macrophage infiltration in tumor tissues detected by immunofluorescence assay. (E) Statistical analysis about Semi quantitative of Figure A-D. (F-I) The protein expression of CD4, CD8, CD86 and Granzyme in tumor tissues is detected by Western blot. One-way ANOVA was carried out and the data are presented as mean ± SD (n=3). Compared with the PBS group, *P<0.05; compared with the radiotherapy group, #P<0.05; compared with the nifuroxazide group, $P<0.05.

The effect of radiotherapy in combination with nifuroxazide on the ratios of immune cells in spleens. The ratios of immune cells in the spleens of tumor-bearing mice were detected by flow cytometry. One-way ANOVA was carried out and the data are presented as mean ± SD (n=3). Compared with the PBS group, *P<0.05; compared with the radiotherapy group, #P<0.05; compared with the nifuroxazide group, $P<0.05.

Nifuroxazide in combination with the radiotherapy significantly increase the degradation of pd-l1 through ubiquitination proteasome pathway. (A) The expression of PD-L1 in cells combined treatment with radiotherapy and nifuroxazide is detected by Western blot. (B) The mRNA level of PD-L1 in cells is detected by PCR. (C-E) The effect of the radiotherapy combined with nifuroxazide on PD-L1 degradation through ubiquitination-proteasome pathway. (F) The schematic diagram of the mechanism that nifuroxazide degraded PD-L1 through ubiquitination-proteasome pathway. One-way ANOVA was carried out and the data are presented as mean ± SD (n=3). Compared with “0” group, *P<0.05; compared with “4Gy” group, #P<0.05.

The effect of radiotherapy in combination with nifuroxazide on the expression of pd-l1 in tumor tissues. (A) PD-L1 expression in tumor tissues of HCC patients treated with radiotherapy are detected by immunofluorescence. (B) PD-L1 expression in tumor tissues of mice combined treatment with radiotherapy and nifuroxazide is detected by immunofluorescence. (C-D) The expression of PD-L1 or GSK3β in tumor tissues of mice combined treatment with radiotherapy and nifuroxazide is detected by Western blot. One-way ANOVA was carried out and the data were expressed as mean ± SD (n=3). Compared with the before-radiotherapy samples, **** P<0.0001; compared with the PBS group, *P<0.05; compared with the radiotherapy group, #P<0.05; compared with the nifuroxazide group, $P<0.05.

The synergistic antitumor mechanism of radiotherapy in combination with nifuroxazide in tumor-bearing mice. NK cell: natural killer cell. Treg: regulatory T cells. TAM: tumor-associated macrophage. CD8+ CTL: CD8+ cytotoxic lymphocyte.