Fig 2: T24/DOX and T24/CIS cells were resistant to DOX and CIS, respectively, but sensitive to NTX. (A) T24 and T24/DOX cells were treated with different concentrations of DOX (0, 0.1, 1, 10, 20, or 100 μM) for 24 h, followed by the evaluation of cell viability using XTT assay. (B) T24 and T24/CIS cells were treated with different concentrations of CIS (0, 10, 20, 40, or 80 μM) for 24 h, followed by the evaluation of cell viability using XTT assay. Data are expressed as the mean ± SD (n=6). *P < 0.05 vs T24 parental cells. (C) The expression levels of STAT3 and P-gp in T24, T24/DOX, and T24/CIS cells were analyzed by western blot analysis. β-actin was used as a loading control. (D) T24, (E) T24/DOX, and (F) T24/CIS cells were exposed to different concentrations of NTX (0, 2.5, 5, 10, 20, and 40 μM) for 24, 48, and 72 h. The cell viability was determined by XTT assay. Data are shown as the mean ± SD (n=6). *P < 0.05 vs the control group.

Fig 3: 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 4: WS-716 stimulated ATP hydrolysis without changing the expression or subcellular localization of P-glycoprotein (P-gp). (A) WS-716 did not change the protein level of P-gp in SW620/Ad300 cells. (B) Effect of WS-716 on the subcellular localization of P-gp (green) after treatment for 76 h. The cell nuclei (blue) were stained by Hoechst 33258. The scale bar is 10 μm. (C) WS-716 stimulated ATP hydrolysis and reached the maximal value at 20 μmol/L. Data are presented as the mean ± SD, n = 3.

Fig 5: ABCB1 up-regulation mediated by CCL20-induced NF-κB activation reduced the intracellular abundance of taxane and promoted chemoresistance in breast cancer cells.(A) Transcriptome sequencing (RNA-seq) utilizing the next-generation technology was conducted with RNA from vector (“VEC”) and CCL20v1-overexpressing (“CCL20 OE”) SUM159 cells and MDA-MB-231 cells. Heat map shows expression levels of the most up-regulated genes (>1.5-fold) and significantly down-regulated genes (decline more than 40%) both in CCL20v1-overexpressing cells of SUM159 and MDA-MB-231 versus vector. Values are the fold change to vector. (B-C) The mRNA and protein levels of ABCB1 were determined by qRT-PCR or western blot in vector and CCL20v1-overexpressed cells of SUM159 and MDA-MB-231. (D) Level of ABCB1 was determined by qRT-PCR in MDA-MB-231 cells. (E) Pathway of response to chemical stimulus was positively correlated with CCL20 overexpression in GSEA from RNA-seq in (A). Nominal P value was shown. (F) Vector and CCL20-overexpressing MDA-MB-231 cells were treated with 200 nM DOC for 12 hours. Then, supernatants and cells were collected for quantitative analysis of DOC abundance with HPLC-MS. (G) MDA-MB-231 cells were cultured in the presence or absence of DOC (10 nM) or CAPE (5 μM); then, the mRNA level of CCL20 was determined by qRT-PCR. (H) A cartoon to illustrate the pattern of CCL20-induced NF-κB activation through PKCζ or p38 MAPK pathway to up-regulate expression of ABCB1 for taxane pumping-out, leading to chemoresistance. Data are shown as mean ± SEM and are representative of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired t test of triplicates. ABCB1, ATP-binding cassette subfamily B member 1; CAPE, caffeic acid phenethyl ester; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor type 6; DOC, docetaxel; GSEEA, Gene Set Enrichment Analysis; HPLC-MS, high-performance liquid chromatography–mass spectrometry; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; PKCζ, protein kinase Cζ; qRT-PCR, quantitative real-time PCR; RNA-seq, RNA sequencing; TBP, TATA-box binding protein.