Fig 1: Higher levels of SOX2 confer PTX resistance in A549 non-small cell lung cancer cells. (A) mRNA expression levels of stemness-related genes were measured by reverse transcription-quantitative PCR in A549 and A549-PTX cells. (B) (Left) Protein expression levels of NANOG, KLF4, OCT4 and SOX2 were examined by western blotting in A549 and A549-PTX cells. (Right) Protein expression was semi-quantified using ImageJ. (C) Protein expression levels of SOX2 were examined by western blotting in A549-PTX cells transfected with siNC or SOX2 siRNA (siSOX2-1, siSOX2-2 and siSOX2-3). (D) Viability was examined using CCK-8 assays in A549-PTX cells transfected with siSOX2 or its control and treated with PTX for 48 h. (E) (Left) Protein expression levels of MDR1, ABCC10, ABCC2 and SOX2 were examined by western blotting in A549-PTX cells transfected with siSOX2 or its control. (Right) Protein expression was semi-quantified using ImageJ. (F) Protein expression levels of SOX2 were examined by western blotting in A549 cells overexpressing SOX2 or its vector control. (G) Viability was examined using CCK-8 assays in A549, A549-PTX and SOX2-overexpressing A549 cells treated with PTX for 48 h. (H) (Left) Protein expression levels of MDR1, ABCC10 and ABCC2 were examined by western blotting in A549 cells overexpressing SOX2 or its vector control. (Right) Protein expression was semi-quantified using ImageJ. GAPDH or Tubulin were used as a loading control in western blotting. All data are presented as the mean ± standard deviation. *P<0.05, ***P<0.001. ns, not significant. Relative, vs. respective control. SOX2, SRY-box transcription factor 2; PTX, paclitaxel; A549-PTX cells, PTX-resistant A549 non-small cell lung cancer cells; KLF4, Krüppel like factor 4; OCT4, octamer-binding transcription factor 4; siRNA/si, small interfering RNA; siNC, control siRNA; CCK-8, Cell Counting Kit-8; MDR1, multidrug resistance mutation 1; ABCC10, ATP binding cassette subfamily C member 10; ABCC2, ATP binding cassette subfamily C member 2; Ctl, control.
Fig 2: Knockdown of ClC-3 expression in A549 cells prevents PTX resistance induced by upregulation of SOX2 expression. (A) (Left) Protein expression levels of MDR1, ABCC2, ABCC10 and tubulin in A549 cells transfected with ClC-3 plasmid or siSOX2 and its control vector or siNC. (Right) Protein expression was semi-quantified using ImageJ. (B) Viability was examined using a CCK-8 assay in A549 cells transfected with ClC-3 plasmid or siSOX2 and its control vector or siNC. Cells were treated with PTX for 48 h. (C) (Left) Protein expression levels of MDR1, ABCC2, ABCC10 and tubulin in A549 cells transfected with SOX2 plasmid or siClC-3 and its control vector or siNC. (Right) Protein expression was semi-quantified using ImageJ. (D) Viability was examined using a CCK-8 assay in A549 cells transfected with SOX2 plasmid or siClC-3 and its control vector or siNC. Cells were treated with PTX for 48 h. Tubulin were used as a loading control in western blotting. All data are presented as the mean ± standard deviation. **P<0.005, ***P<0.001. ns, not significant. Relative, vs. respective control. ClC-3, chloride voltage-gated channel 3; A549-PTX cells, paclitaxel-resistant A549 non-small cell lung cancer cells; PTX, paclitaxel; MDR1, multidrug resistance mutation 1; ABCC2, ATP binding cassette subfamily C member 2; ABCC10, ATP binding cassette subfamily C member 10; siRNA/si, small interfering RNA; CCK-8, Cell Counting Kit-8.
Fig 3: Establishment of PTX-resistant A549 non-small cell lung cancer cells. (A) Viability was assessed using CCK-8 assays in A549 and A549-PTX cells treated with PTX for 48 h. (B) (Left) Cell proliferation was examined by colony formation assays in A549 and A549-PTX cells treated with PTX. (Right) Colony numbers as quantified using ImageJ. (C) mRNA expression levels of drug resistance-related genes were measured by reverse transcription-quantitative PCR in A549 and A549-PTX cells. (D) (Left) Protein expression levels of ClC-3, MDR1, ABCC10, ABCC2 and GAPDH were examined by western blotting in A549 and A549-PTX cells. (Right) Protein expression was semi-quantified using ImageJ. (E) Viability was assessed using CCK-8 assays in A549, A549-PTX or DIDS-treated A549-PTX cells treated with PTX for 48 h. DIDS, 10 µM for 48 h. GAPDH was used as a loading control in western blotting. All data are presented as the mean ± standard deviation. **P<0.01, ***P<0.001. Relative, vs. respective control. PTX, paclitaxel; CCK-8, Cell Counting Kit-8; A549-PTX cells, PTX-resistant A549 NSCLC cells; CIC-3, chloride voltage-gated channel 3; MDR1, multidrug resistance mutation 1; ABCC10, ATP binding cassette subfamily C member 10; ABCC2, ATP binding cassette subfamily C member 2; DIDS, 4,4-diisothiocyanatostilbene-2,2-disulfonate.
Fig 4: ClC-3 is upregulated in PTX-resistant A549 cells. (A) ClC-3 protein expression was measured by western blotting in A549-PTX cells transfected with siNC or ClC-3 siRNA (siClC-3-1, siClC-3-2 and siClC-3-3). (B) Viability was examined by CCK-8 assays in A549-PTX cells transfected with siClC-3 or control treated with PTX for 48 h. (C) (Left) Protein expression levels of MDR1, ABCC10, ABCC2 and ClC-3 were examined by western blotting in A549-PTX cells transfected with siClC-3 or control. (Right) Protein expression was semi-quantified using ImageJ. (D) Protein expression levels of ClC-3 were examined by western blotting in A549 cells overexpressing ClC-3 or its vector control. (E) Viability was examined by CCK-8 assays in A549, A549-PTX and ClC-3-overexpressing A549 cells treated with PTX for 48 h. (F) (Left) Protein expression levels of MDR1, ABCC10 and ABCC2 were examined by western blotting in A549 cells overexpressing ClC-3 or its vector control. (Right) Protein expression was semi-quantified using ImageJ. Tubulin was used as a loading control in western blotting. All data are presented as the mean ± standard deviation. **P<0.01, ***P<0.001. ns, not significant. Relative, vs. respective control. ClC-3, chloride voltage-gated channel 3; PTX, paclitaxel; A549-PTX cells, PTX-resistant A549 non-small cell lung cancer cells; siRNA/si, small interfering RNA; siNC, control siRNA; ClC-3, chloride voltage-gated channel 3; MDR1, multidrug resistance mutation 1; ABCC10, ATP binding cassette subfamily C member 10; ABCC2, ATP binding cassette subfamily C member 2; CCK-8, Cell Counting Kit-8; Ctl, control.
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