Fig 1.
The drug-resistant PC9 and HCC827 cells underwent EMT.
(A) Parental PC9 and gefitinib-resistant PC9 (PC9-GR) cells were treated with gefitinib at the indicated concentrations for 72 hours, and the relative cell viability was determined by MTT assay. (B) Viability of HCC827 and gefitinib-resistant HCC827 cells. (C) Bright-field microscopy images of parental PC9/HCC827 and gefitinib-resistant PC9/HCC827 cells. (D) Protein levels of the indicated EMT biomarkers and the internal control actin was determined by Western blot. (E) mRNA levels of E-cadherin, N-cadherin and Vimentin in parental PC9/HCC827 and gefitinib-resistant PC9/HCC827 cells were determined by qPCR. (F) qPCR analysis for the indicated epithelial (left panels) and mesenchymal (right panels) biomarkers in parental PC9/HCC827 and gefitinib-resistant PC9/HCC827 cells.
Fig 2.
Snail- and Slug-induced EMT promoted drug resistance of parental PC9 and HCC827 cells.
(A) The protein levels of EMT biomarkers and ectopically expressed Slug, Snail and Twist1 in the parental PC9 cells. (B) The pLex-, pLex-Slug- and pLex-Snail-expressing parental PC9 cells were treated with gefitinib at the indicated concentrations for 72 hours, and the relative cell viability was determined by MTT assay. (C) The protein levels of EMT biomarkers and the ectopically expressed Slug, Snail and Twist1 in the parental HCC827 cells. (D) The pLex-, pLex-Slug- and pLex-Snail-expressing parental HCC827 cells were treated with gefitinib at the indicated concentrations for 72 hours, and the relative cell viability was determined by MTT assay. (E) Endogenous E-cadherin (CDH1) was knocked down by two shRNA clones (#1 and #2). The protein levels of EMT biomarkers were examined by Western blot. (F) Stable clones of scramble shRNA-, shRNA #1- and shRNA #2-transduced PC9 cells were treated with gefitinib at the indicated concentrations for 72 hours, and the relative cell viability was determined by MTT assay.
Table 1.
The IC50 values of the PC9-WGR and HCC827-WGR cells decreased after prolonged withdrawal of gefitinib.
Fig 3.
Prolonged gefitinib withdrawal sensitized the gefitinib-resistant cells and induced reverse EMT.
(A) Bright-field microscopy images of the parental PC9/HCC827, gefitinib-resistant PC9 (PC9-GR)/HCC827 (HCC827-GR), and gefitinib-withdrawn PC9-GR (PC9-WRG)/HCC827-GR (HCC827-WGR) cells at different passages. (B) Parental, GR and WGR PC9 (left panel) and HCC827 cells (right panel) at different passages were treated with gefitinib at the indicated concentrations for 72 hours, and the relative cell viability was determined by MTT assay. (C) The protein levels of EMT biomarkers in the parental, GR and WGR PC9 (left panel) and HCC827 cells (right panel) at different passages were analyzed by Western blot.
Fig 4.
Blockage of gefitinib withdrawal induced reverse EMT, and the forced expression of Snail and Slug inhibited the regain of gefitinib sensitivity.
(A) The vectors pLex, pLex-Slug and pLex-Snail were introduced into the PC9-GR and PC9-WGR cells at different passages. The protein levels of EMT biomarkers were examined by Western blot. (B) The vectors pLex, pLex-Slug and pLex-Snail were introduced into the HCC827-GR and HCC827-WGR cells at different passages. The protein levels of EMT biomarkers were examined by Western blot. (C) Parental, GR and WGR PC9 cells at different passages were treated with gefitinib at the indicated concentrations for 72 hours, and the relative cell viability was determined by MTT assay. The IC50 values at each passage after gefitinib withdrawal were determined and plotted. (D) Parental, GR and WGR HCC827 cells at different passages were treated with gefitinib at the indicated concentrations for 72 hours, and the relative cell viability was determined by MTT assay. The IC50 values at each passage after gefitinib withdrawal were determined and plotted.