HSC-4

Induction of Autophagy Impairs Migration and Invasion in HSC-4 Cells

To investigate the involvement of autophagy in cell migration and invasion, serum deprivation (serum-free media) was performed to induce autophagy in HSC-4 cells. As shown in Fig. 1 A-C, the intensity of MDC-stained autophagic vacuoles, the number of MDC-positive cells, LC3 gene expression and LC3 immunofluorescence intensity were distinctly increased in serum-free medium whereas chloroquine (CQ), an inhibitor of autophagy, pre-incubation significantly reduced MDC staining, expression level of LC3 and immunofluorescence intensity of LC3 gene compared to serum-free medium. In addition, HSC-4 cells in serum-free medium displayed a significant decrease in migration percentage, invaded the area, and number of invaded cells. In contrast, the wound area distance significantly increased compared to control cells and untreated cells. In contrast, CQ pre-treatment led to significantly abolishing the effect of autophagy on the percentage of migration, the invaded area, and the invaded cells (Fig. 1 D-F, respectively). These results demonstrate that the induction of autophagy impaired the migration and invasion capacity of HSC-4 cells.

Fig. 1 Autophagy induction impairs migration and invasion in HSC-4 cells. (A) MDC staining, (B) mRNA expression of p62 and LC3 genes, and (C) LC3 immunofluorescence staining in HSC-4 cells pre-treated with 50 μM CQ for 1 h, followed by incubation in serum-free medium (serum deprivation) for 24 h. (D) Migratory ability was assessed by wound healing assay, (E and F) invasive ability was examined by the agarose spot and Transwell invasion assays in HSC-4 cells pre-treated with 50 μM CQ for 1 h, followed by incubation in serum-free medium for 24 h. (Binlateh T, et al., 2022)

Silencing of FANCD2 Increased the Bax/Bcl2 Ratio and Activated the p38 MAPK Signaling Pathway in HSC-4 Cells

Western blot analysis revealed that after the FANCD2 gene expression in HSC-4 cells was silenced by shRNA interference, the expression of the Bax and p-p38 proteins was significantly higher in the cells of the experimental group than in the control groups (P<0.05), whereas the expression of the p38 and Bcl2 proteins was significantly decreased (P<0.05; Fig. 2A and B). After radiotherapy, the same results were obtained; additionally, a significant difference was observed in the expression of these proteins before and after radiotherapy (P<0.05; Fig. 2A and B). After the nude mice with transplanted HSC-4 cells received radiotherapy, western blotting, and immunohistochemistry were performed, which showed an increase in the expression of Bax and p-p38 proteins in the tumor tissues of mice in the experimental group compared with the control group (P<0.05); in contrast, the expression of p38 and Bcl2 proteins was decreased (P<0.05; Figs. 2C and D, 3 and 4). This finding is consistent with the results of the in vitro cell culture experiments. These results suggest that the silencing of FANCD2 increases the Bax/Bcl2 ratio and activates the p38 MAPK signaling pathway in HSC-4 cells after radiotherapy.

Fig. 2 (A and B) Western blot analysis was used to determine the expression of the Bax, Bcl2, p38, and p-p38 proteins in the three groups of HSC-4 cells before and after radiotherapy. (C and D) Western blot analysis was used to examine the expression of the Bax, Bcl2, p38, and p-p38 proteins in the tumors derived from transplanted HSC-4 cells after radiotherapy. (Feng HJ, et al., 2017)

Fig. 3 H&E staining of the tumors derived from HSC-4 cells and immunohistochemical analysis of the Bax, Bcl2, p38, and p-p38 protein expression after radiotherapy. (Feng HJ, et al., 2017)

Fig. 4 Image-Pro Plus 6 software was used to quantitatively analyze the expression of the Bax, Bcl2, p38, and p-p38 proteins using immunohistochemistry (IHC) in the tumor sections from the three groups of mice after radiotherapy. (Feng HJ, et al., 2017)