Fig 2: 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.

Fig 3: Schematic of potential 17β-estradiol-mediated mechanisms of neuropathic pain regulation in ovariectomized female rats. 17β-estradiol may regulate the expression of ClC-3 mRNA through the 17β-estradiol receptor, including nuclear receptors α and β or membrane receptor GPER, via certain signaling pathways in the cell, thereby affecting the expression of ClC-3 mRNA and protein and relieving neuropathic pain. However, this effect may be blocked by Cltx, a specific blocker that affects the function of ClC-3 channel protein but does not affect the expression of mRNA. GPER, G protein-coupled 17β-estradiol receptor. ER-α and ER-β: nuclear 17β-estradiol receptors α and β.

Fig 4: 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 5: SOX2 promotes ClC-3 expression. (A) (Left) ClC-3 antibody or (right) SOX2 antibody was used to immunoprecipitate SOX2 or ClC-3 in A549 cells. IgG was used as the negative control. (B) Protein expression levels of ClC-3 and SOX2 in A549 cells transfected with (left) siClC-3 or (right) ClC-3 overexpression vector were examined by western blotting. (C) Protein expression levels of ClC-3 and SOX2 in A549 cells transfected with (left) siSOX2 or (right) SOX2 overexpression vector were examined by western blotting. (D) (Left) SOX2 binding peaks within 3 kb of the gene TSS determined by CUT&Tag analysis of A549 cells. (Right) Binding density of SOX2 was visualized using deepTools. The heatmap presents the CUT&Tag tag counts on the different SOX2 binding peaks in A549 cells. (E) Genome-wide distribution of SOX2-binding peaks in A549 cells. (F) Gene Ontology analysis of the SOX2-binding peaks at candidate target genes. (G) Genome browser tracks of CUT&Tag signal at the ClC-3 loci. The red area is the predicted SOX2 binding site in the promoter of CLCN3. (H) Changes of ClC-3-binding levels in A549 cells were determined by reverse transcription-quantitative PCR and presented as relative fold-change to the control after normalization as described in the materials and methods section. (I) (Above) Luciferase assay in A549 cells after co-transfection with the indicated plasmids. (Below) Protein expression levels of ClC-3 and SOX2 in A549 cells co-transfected with the indicated plasmids. GAPDH or tubulin were used as a loading control in western blotting. All data are presented as the mean ± standard deviation. ***P<0.001. Relative, vs. respective control. SOX2, SRY-box transcription factor 2; ClC-3, chloride voltage-gated channel 3; A549-PTX cells, paclitaxel-resistant A549 non-small cell lung cancer cells; siRNA/si, small interfering RNA; siNC, control siRNA; TSS, transcription start sites; CUT&Tag, cleavage under targets and tagmentation; IP, immunoprecipitation.