FIGURE 3.

The majority of gefitinib-resistant persisters (GRPs) of PC9 fluorescent ubiquitination-based cell cycle indicator (FUCCI) exist in a quiescent state, maintained by F-box/WD repeat-containing protein 7 (FBXW7). Stable FUCCI-expressing cells were made by introducing FUCCI vector plasmid into PC9. (A, left) PC9-FUCCI cells were seeded in 35 mm glass bottom dishes, followed by treatment with or without gefitinib and subsequently subjected to cell cycle analysis. The cells in G0/G1 or S/G2/M phases appear red and green, respectively. (A, right) Histograms demonstrate the cell cycle phase distribution before and after gefitinib treatment. (B, left) The cell cycle profile of PC9-FUCCI was analyzed after knockdown of FBXW7 using siRNA transfection and gefitinib treatment for 72 h. (B, right) Histograms show the distribution of cells in G0/G1 phase and S/G2/M phase after knocking down FBXW7 under gefitinib treatment. Five pictures were taken from each group, and the cell number in G0/G1 phase as well as in S/G2/M phase was counted and divided by the total number of cells. (C) The number of GRPs was decreased after knockdown of FBXW7 in PC9-FUCCI. Five pictures were taken from each group and the number of cells in G0/G1 phase as well as in S/G2/M phase was counted. Scale bar indicates 50 µm.