Fig. 7: The CSLTN1 isoform with exon 11 inclusion promotes EMT.

a RNA pull-down assay showing that AKAP8 binds to the cis-element located in the intron upstream of CLSTN1 variable exon 11. A schematic of the CLSTN1 pre-mRNA and the cis-element sequence (CI10) are shown in the top panel. RNA pull down was performed using lysates from 293FT and HMLE/Twist-ER cells. Inp: Input. b A schematic of the location of CLSTN1 isoform-specific shRNAs (black line, top panel), and semi-qPCR analysis (bottom panel) showing CLSTN1 isoform-specific knockdown efficiency in HMLE/Twist-ER cells. shL: CLSTN1-L knockdown; shS: CLSTN1-S knockdown. c Phase-contrast images (× 10) indicating accelerated cell morphology changes in control shRNA (Ctrl) and CLSTN1-S knockdown (shCLSTN1-S) cells 8 days after tamoxifen (TAM) induction. White line represents scale bar at 100 μm. d Western blot analysis of EMT markers showing that CLSTN1-S silencing resulted in decreased expression of epithelial markers E-cadherin and γ-catenin and increased expression of the mesenchymal marker N-cadherin after 8 days of TAM induction. e Immunofluorescence images showing the loss of E-cadherin expression at cell junctions in the CLSTN1-S knockdown cells after 8 days of TAM treatment. f Plot of the mesenchymal marker vimentin (VIM) expression and CLSTN1 alternative splicing in the breast cancer TCGA data set showing a significant positive correlation in luminal breast cancer. PSI: Percent Spliced In. P value was determined by person correlation test. g Kaplan–Meier plot showing correlation between CLSTN1 alternative splicing, represented as PSI levels, and breast cancer patient survival in TCGA data set. The average PSI for the high group is 0.49 and low group is 0.15, which were determined by k-means clustering. P value was determined by log-rank test. Source data are provided as a Source Data file.