Figure 4.

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.