Figure 1.
CFTR depletion reduces the availability of Rab5 and the recruitment of Rab5 effector EGFP-tagged-FYVESARA to early endosomes. (a) Confocal microscopic images of 16HBE14o- cells transfected with a plasmid encoding EGFP-tagged-FYVESARA and with CFTR-specific or scrambled siRNA (50 nM) in the presence or absence of cystamine (250 μM) with/without 3-MA (3 mM). Left, confocal microscopic images of EEA1 and EGFP-tagged-FYVESARA. Insets: boxed area show merged images of EGFP-tagged-FYVESARA (green) and EEA1 (red). Scale bar, 10 μm. Right, number of FYVESARA spots per cell were counted using AnalySIS software. Means±S.D. (n=30 cells per experiment; analysis of three independent experiments). *P<0.05 compared with scrambled siRNA-treated cells. In this analysis, the total fluorescence intensity per cell was used to monitor EGFP-tagged-FYVE transfection efficiency and was calculated for each sample using Zeiss LSM 510 software (version 3.2). (b) Effects of CFTR depletion on Rab5 and Rab7 protein levels. Effects of cystamine (250 μM), HA-Beclin-1 overexpression or SQSTM1 siRNA (50 nM) in CFTR depleted 16HBE14o- cells. Left, Rab5 and Rab7 protein expression versus whole cell lysates. β-actin expression was measured as a loading control. Right, densitometric analysis of protein levels expressed as percentage of control. Mean±S.D., *P<0.05 compared with untreated cells, #P<0.01 versus CFTR siRNA (analysis of variance (ANOVA)). (c) The cells were treated with or without CFTR siRNA (50 nM) in the presence or absence of cystamine (250 μM), after transfection with the indicated siRNAs (50 nM each). Left, expression of Rab5 and Rab7 in 16HBE14o- cells. Right, densitometric analysis of protein levels expressed as percentage of control. Mean±S.D., *P<0.05, compared with untreated cells, #P<0.01 compared with cystamine-treated cells (ANOVA)