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. 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. 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. F Statistical analysis of pathological score. 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 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. 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. 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 Image A-D. F-I The protein expression of CD4, CD8, CD86 and Granzyme in tumor tissues is detected by Western blot. 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. 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. 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. 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.