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Fig. 1. LAMP2 knockdown sensitizes to vacuolization and cell death by nutrient depletion. (A,B) Immunoblot confirmation of the efficacy of LAMP1-(A) or LAMP2-specific (B) small interfering RNA (siRNA). HeLa cells were transfected with the indicated constructs specific for emerin (E), LAMP1 (A,B), and LAMP2 (64, 69), and the expression level of LAMP1 or LAMP2 was determined by immunoblot, after 24 or 48 hours. GAPDH expression was determined as a loading control. (C) Vacuolization induced by LAMP2 depletion. Twenty-four hours after transfection, the cells were cultured for 16 hours in nutrient-free (NF) medium – that is, in the absence of serum and amino acids, and then stained with CMFDA. Cytoplasmic vacuoles are visualized as holes. (D,E) Vesicular LC3 accumulation induced by LAMP2 depletion. Cells were transfected with siRNAs, then 24 hours later with an LC3-GFP fusion construct, and were nutrient deprived for 16 hours. Representative fluorescence microphotographs are shown in D, and the frequency of cells exhibiting the accumulation of LC3-GFP in vacuoles and apoptotic chromatin condensation (as determined by counterstaining with Hoechst 33342) was quantified (X±s.d., n=3) in E. Data in C and D are shown for LAMP1-A and LAMP2-64 siRNAs, and similar results were obtained for the LAMP1-B and LAMP2-69 siRNAs. (F) Electron microscopy of representative cells 6 hours after nutrient and serum depletion after siRNA specific for emerin (right) or siRNA specific for LAMP2 (64)+LAMP1 (A). Bars in C and D, 10 µm.
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