Figure 2.

U18666A-induced endosomal/ysosomal accumulation of non-esterified sterols. (A) 1c1c7 cultures were incubated with dextran-10000 tetramethylrhodamine (D10K-TMR) overnight prior to being washed and refed with fresh medium. At the time of refeeding some cultures were treated with 1 µM U18666A. After an additional 8 h of incubation the cultures were fixed and processed for detection of D10K-TMR (red) and LAMP1 (green). Colocalization of D10K-TMR and LAMP1 is indicated by orange – yellow punctate spots. (B) 1c1c7 cultures were incubated with D10K-TMR overnight prior to being washed, and refed with fresh medium. Some cultures were subsequently treated with 1 µM U18666A and incubated an additional 8 h before being stained with filipin. Colocalization of filipin (green) and D10K-TMR (red) structures is indicated by orange – yellow punctate spots. (C) 1c1c7 cultures were treated with nothing or 1 µM U18666A for 24 h prior to being fixed and processed for filipin (green) and LAMP1 (red) staining. (D) 1c1c7 cultures were treated with nothing or 1 µM U18666A for ~22 h prior to the addition of MitoTracker Green (MTG). After an additional 5 min of incubation cultures were washed, fixed and stained with filipin. The pictures presented in panels A–D are representative of what was observed in multiple fields of cells. White bar in panels A, B and D represents 20 microns. White bar in panel C represents 10 microns.