HAIs act via matriptase to regulate degradation of EpCAM, TROP2, and claudins in lysosomes. HaCaT cells were transfected using electroporation with control or SPINT1 plus SPINT2, matriptase, or SPINT1, SPINT2 plus matriptase siRNAs (A), or control or SPINT1 and SPINT2 siRNAs (B). Before being harvested at 72 h after transfection (A,B), cells were treated with or without 100 μM chloroquine for 20 h (B). RIPA cell lysates were resolved using SDS-PAGE and immunoblotted with anti-HAI-1, anti-HAI-2, anti-matriptase, anti-EpCAM anti-TROP2, anti-claudin-1, or anti-claudin-7. β-actin was used as a loading control. Representative data from 1 of 5 experiments is shown. (C) Band intensities corresponding to full-length (FL) EpCAM, FL TROP2, claudin-1 and claudin-7 (left panel) and cleaved EpCAM and cleaved TROP2 (right panel) were quantified and normalized to β-actin. Data are plotted as ratios (means ± SEM) relative to corresponding untreated siControls (n = 5). A two-way ANOVA was used to calculate p values for multiple group comparisons to assess mean differences between groups (* p < 0.05, ** p < 0.01, *** p < 0.001 or 0.0001, n.s. p > 0.05).