To simultaneously detect the fluorescein ingress and the expression of transmembrane MUC1, HCECs that had been maintained in KSFM or MPM were shifted to culture condition with 2 mM fluorescein-containing KSFM or MPM, respectively, for subsequent cell incubation at 37°C for 15 minutes. Cells were then washed with warm PBS twice, harvested with TrypLE Express Enzyme (1×), and resuspended in PBS containing 1% bovine serum albumin (BSA) in a 1.5 mL Eppendorf tube. The cell suspension was stained with anti-MUC1 antibody [EP1024Y] (ab45166; Abcam, Cambridge, MA, USA) for one hour at room temperature and fixed in 2% paraformaldehyde for 15 minutes at room temperature. To detect the total mucin 1 (including intracellular and membrane bound mucin1), the cell suspension was stained with anti-MUC1 antibody [HMFG2] (ab245693; Abcam) for one hour at room temperature and fixed in 2% paraformaldehyde for 15 min at room temperature. After being washed with 1% BSA/PBS, the cell suspension was incubated with a goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 647 (A21244; Invitrogen) for 30 minutes at room temperature. To detect the expression of K3, K12 or vimentin, cell suspensions were fixed in 2% paraformaldehyde for 15 minutes at room temperature before being stained with an anti-cytokeratin 3/CK-3 antibody [AE5] (ab77869; Abcam), anti-keratin 12/K12 antibody [EPR17882] (ab185627, Abcam), or an anti-vimentin (D21H3) antibody (CS no. 5741, cell signaling) for one hour at room temperature. To detect the expression of GAL3, cell suspensions were fixed and incubated with anti-galectin-3 (GAL3) mouse antibody (no. A3A12; Novus Biologicals). Then, the cell suspensions were incubated with a goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, and an Alexa Fluor 647 (A21244, Invitrogen) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (A11001, Invitrogen) for 30 minutes at room temperature. Flow cytometry was performed with BD LSRFortessa Flow Cytometer (BD Bioscience, Franklin Lakes, NJ, USA). Data were analyzed using the FlowJo software program.