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. 2022 Apr 14;23(8):4367. doi: 10.3390/ijms23084367

Figure 1.

Figure 1

Establish acquired concurrent chemoradiotherapy resistance (CCRTR) ESCC cell lines and validate their viability to CCRT treatment. (A) A schematic diagram of two chemoradioresistant ESCC cell lines (CE48T and Kyse70) established by fractionated irradiation (5 Gy) and cisplatin treatment. Total effective dosage of irradiation exposure was 65–75 Gy. (B) Cell viability was examined in CCRT-selected CE48T cells. Different CCRT intensity-selected CE48T cells (5th, 10th and 15th) and control cells were treated with the indicated CCRT conditions, 5 Gy irradiation and various concentrations of cisplatin (0, 5, 10, 20, 30, 40, 50 and 100 μM) to determine their CCRT response by MTT assay. These three independent cell lines harbored a consistent resistance potential but differential resistance ability to CCRT treatment. (C) Validation of chemoradioresistant ability in acquired CE48T CCRT-resistance line (CCRTR) by colony formation assay. CE48T parental control and CCRTR cells were treated with the combination of 5 Gy irradiation exposure and indicated concentrations of cisplatin (0, 5, 10, 20, 30, 40 μM). At the end of 12 days incubation period, viable cell colonies were fixed, stained with crystal violet, and calculated. Upper panel: Representative images of viable colonies after CCRT treatment; lower panel: Quantification of CCRT-mediated colonies formation in control and CCRTR cells at 12 days post-CCRT treatment. (D) Examine the effect of CCRT treatment on cell viability using CE48T control and CCRTR cells by MTT assay. The 1 × 104 control and CCRTR cells were plated into a 96-well plate, cells were treated with 5 Gy irradiation combined with the indicated concentrations of cisplatin (0, 5, 10, 20, 30, 40, 50, and 100 μM) for 48 h. Relative quantification of cell viability in control and CCRTR cells post 48h-CCRT treatment compared to cells without CCRT treatment were shown. The IC50 to cisplatin is 2.3 μM in control cells and 28.6 μM in CCRTR cells. (E) Validation of Chemoradioresistant ability in acquired Kyse70 CCRT-resistance line (CCRTR) by colony formation assay. Experimental protocol was similar to (C), whereas the treatment concentrations of cisplatin were 0, 0.1, 0.3, 0.5, 1, and 5 μM. Representative images of viable colonies at 14 days post-CCRT treatment (top) and quantification of CCRT-mediated colonies formation in control and CCRTR cells (bottom) were shown. (F) Examine the effect of CCRT treatment on cell viability using Kyse70 control and CCRTR cells by MTT assay. The 1 × 104 control and CCRTR cells were plated into a 96-well plate, cells were treated with 5 Gy irradiation combined with the indicated concentrations of cisplatin (0, 0.1, 0.3, 0.5, 1, and 5 μM). The IC50 to cisplatin is 0.4 μM and 1.3 μM in control and CCRTR of Kyse70 cells, respectively. * p < 0.05, ** p < 0.01, n.s.: not significant.