Figure 6. E2-induced PKCδ activation is responsible for the ASL height decrease in CuFi-1 cells.

NuLi-1 (grey bars) and CuFi-1 (white bars) cells were treated basolaterally with E2 (1 nM) for 30 mins. After hormone treatment, cells were washed and total protein extracts were prepared in order to measure the level of phosphorylation of PKCα/βII and PKCζ/λ by immunoblot. Representative Western blots are shown in panel A and densitometric quantitation of phospho-PKCα/βII / β-actin and phospho-PKCζ/λ / β-actin are shown in panel B (n = 4, Error bars reflect standard error of the mean, Student’s t-test). B. NuLi-1 and CuFi-1 cells were treated basolaterally with E2 (1 nM) for the indicated times. After treatment, cells were washed and total protein extracts were prepared in order to measure the level of phosphorylation of PKCδ/θ. Representative images are shown in panel C and mean phosphorylation state of PKCδ/θ changes in control conditions, or following treatment with estrogen at different time points, are shown in panel D (n≥4, Error bars reflect standard error of the mean, ANOVA, * p<0.05). E. ASL height was measured in CuFi-1 (white bars) and NuLi-1 (grey bars) monolayers in response to estrogen and PKCδ inhibition. NuLi-1 and CuFi-1 monolayers were treated with E2 (1 nM) or pretreated with rottlerin (5 µM) and then treated with E2 (n = 5, Error bars reflect standard error of the mean, ANOVA, * p<0.05).