Figure 4. Inhibition of RA signaling in cultured ER upregulates ductal cytokeratin KRT5.

Immunostain analysis reveals dramatically elevated level of KRT5 in RARE-LacZ reporter ER specimens cultured on medium containing BMS 493 relative to specimens grown on control medium. ER cultured for 48 hours on medium containing BMS 493 (B) were smaller with fewer branches and endbuds relative to their counterparts grown on control medium (A) as visualized by staining for KRT8. RA signaling, visualized by β-galactosidase fluorescence signal was reduced in ER cultured on BMS 493 (D) relative to control specimens (C). (I) The amount of RA signaling, as measured by the sum of relative fluorescence intensity signal for β-galactosidase, is reduced ≥2-fold, p = 0.02, N = 6 ER. KRT5 is dramatically upregulated in ER cultured on BMS 493 (F) relative to controls (E). For ER grown on control medium KRT5 signal is limited to a few cells at the tip of the main duct (E, G). In contrast, ER grown on medium containing BMS 493 had highly elevated KRT5 signal in all endbuds and ducts (F, H). (J) The amount of KRT5 protein, as measured by the sum of relative fluorescence intensity signal, was elevated ~ 5-fold in BMS 493-treated ER relative to control specimens, N = 6 ER, p=0.002. (K) Elevated KRT5 expression in BMS 493-treated ER was restricted to cells of the basal epithelium. White scale bars 50 μm, yellow scale bar = 20 μm.