Figure 3.

The inducible knockdown of c-Cbl promotes EGF-EGFR recycling and enhances corneal epithelial cell migration. Parental hTCEpi (top) and hTCEpi (-c-Cbl) (bottom) cells were incubated without and with 1 μg/mL doxycycline for 72 hours. (A) Recycling of the ¹²⁵I-EGF-EGFR complex was monitored by measuring the amount of secreted intact radioligand. Data are plotted as the average ± SEM percentage of intact, extracellular radioligand at each time point (n = 3). (B) Brightfield images of hTCEpi and hTCEpi (-c-Cbl) in an in vitro healing assay taken at 16 hours after treatment with or without EGF (1.6 nM) and doxycycline (1 μg/mL). (C) Cell migrations were analyzed as the difference from the plug's removal (0 hour) compared with the growth after 16 hours of treatment. Data are plotted as the average ± SEM percentage of area covered (wound healed) (n = 3). Analysis by a two-way ANOVA with Bonferroni post hoc test. ***P < 0.001, n ≥ 3 with three to five replicate values. (D) Representative images 8 hours post wounding from a Scratch assay using confluent dishes of parental hTCEpi cells or hTCEpi(-Cbl) cells (bottom). Doxycycline and EGF treatments are indicated. Quantification of Scratch assays at (E) 8 hours and (F) 16 hours after wounding. Data were analyzed with a two-way ANOVA with Bonferroni post hoc test. *P < 0.05; ****P < 0.001, n ≥ 3 with three to five replicate values.