Figure 5.

Stat3-mediated regulation of the lysosomal compartment. A, validation by Western blot analysis of the lysosomal localization of flotillin 1 and flotillin 2 using iron nanoparticles to isolate EpH4 lysosomes (Mag+) after 72 h of OSM stimulation. White asterisk, indicates the expected molecular weight of flotillin 2. Representative blots of n = 3 (flotillin 1) and n = 2 (flotillin 2) independent experiments are shown. B, microarray analysis of 12 different timepoints during the mammary gland pregnancy cycle showing the involution-related expression profiles of flotillin 1 and flotillin 2. V, virgin; d G, days gestation; d L, days lactation; h I, hours involution. C, fluorescence microscopy of LAMP1 and LAMP2 immunostaining in EpH4 cells treated with OSM for 72 h. Scale bars: 10 μm. D, Western blot analysis of OSM-induced changes to the lysosomal proteins LAMP1, LAMP2, CD63, and cathepsin B in whole-cell lysates from normal (WT) and two independent Stat3 KO EpH4 cell lines after 72 h of stimulation. Proteins were separated by SDS-PAGE under reducing or non-reducing conditions as indicated. pro-Ctsb, pro form of cathepsin B; sc Ctsb, single-chain cathepsin B; Scram, scrambled sgRNA control EpH4 cells. E, microarray analysis of 12 different timepoints during the mammary gland pregnancy cycle showing the involution-related expression profiles of CD63 and LAMP2.