Fig. 1: Establishment and characterization of sequential gemcitabine-resistant cancer (GRC) cell lines.

A Schematic overview showing the establishment of sequential GRC cell lines. B Four types of the GRC cell lines (GRC1, GRC2, GRC3, and GRC4) were constructed from P1 to P15. Early phase P3, intermediate phase P7, and late phase P15 of the GRC1 cell line were used as sequential GRC models (blue squares). C Changes in the GRC1 cell line when the phase increases by one step after gemcitabine treatment. The first two pictures show the elapsed time (d, day) and colony morphology during proliferation from P2 to P3, the middle pictures from P6 to P7, and the last pictures from P14 to P15. D Gemcitabine sensitivity curves for each concentration of the GRC1 cell line. Various gemcitabine concentrations [μM] were obtained by MTT analysis after 72 h of treatment. The average of the experimental values repeated at least three times are shown. E Cell survival assays were performed to determine the cell viability of early phase P3, intermediate phase P7, and late phase P15 compared to parental phase P0 at 0, 24, 48, and 72 h. F Cell survival assays were performed in the late phase P15 of GRC2-4 cell lines and GRC1 cell line compared to parental P0 with 300 nM of gemcitabine treatment condition. G Anchorage-dependent growth assay was performed in the GRC1 cell line with treated 300 nM of gemcitabine. H, I Anchorage-dependent (H) and anchorage-independent (I) growth assays were performed in the GRC1 cell line and parental phase P0 for 7 days. J Cell invasion and migration abilities of the GRC1 cell line were enhanced compared to parental phase P0. Cells were detected and calculated by counting cells per field. ×400. K A wound-healing assay was performed to examine the ability of wound closure of the GRC1 cell line compared to parental phase P0. Monolayers were scratched using a 200 μl pipette tip and then photographed at 0 and 20 h. The extent of wound healing was quantified using ImageJ software and the percentage of wound closure was calculated. **P < 0.01; ***P < 0.001.