Fig. 1
Derivative HT-29/EGFP/FUR cell line prepared from human HT-29 cells is chemoresistant and incapable to communicate with the MSC via gap-junctional intercellular communication . We prepared chemoresistant cell line HT-29/EGFP/FUR by stable exposure of initially chemonaive HT-29/EGFP cells to increasing concentrations of the 5-FU till stable propagation in 2 µg/ml. Chemonaive HT-29/EGFP cells and their resistant derivative HT-29/EGFP/FUR were used for this study (a). b Direct enumeration of the red nuclei of the tumor cells by the kinetic imaging system showed significantly decreased proliferation rate of chemoresistant cells (here referred to as FUR/NLR) with 1.7-fold less cells presented in the culture after 140-h-long cultivation under the standard culture conditions. Values were expressed as means of decaplicates ± SD. Mann–Whitney U-test was used for statistical analysis. ****p < 0.0001. c Prolonged treatment of tumor cells with the 5-FU did increase the resistance of FUR/NLR cells as evaluated by direct counting of the tumor cell nuclei after 6-day-long exposure to 5-FU concentration gradient. Values were expressed as means of sextaplicates ± SD. d For the detection of gap-junctional intercellular communication, tumor cells were stained with DiI, adipose tissue-derived MSC (AT-MSC) were stained with calcein AM. HT-29 cells and MSCs were mixed immediately before measurement (fresh mix), as well as co-cultured for 24 h. We did not observe double-positive tumor cells indicating calcein AM transfer via gap junctions during co-culture. e Human breast cancer cell line T47D was used as a positive control. f The expression of Cx43 was determined by qPCR in the HT-29/EGFP and HT-29/EGFP/FUR cells. T47D cells were used as a positive control, HPRT1 served as an internal control
