Massey et al. 10.1073/pnas.0507436103. |
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Methods
Fig. 6. Characterization of cells stably RNA interfered for LAMP-2A. (A) (Upper) Filipin staining of WT and LAMP-2A(-) mouse fibroblasts maintained in medium supplemented (serum +) or not (serum -) with newborn calf serum. (Lower) Quantification of total cellular content of cholesterol by the Amplex Red Cholesterol Assay in the same cells. Values are the mean + SE of triplicate samples in two different experiments. (B) Activity of two lysosomal glycosidases (b-hexosaminidase and b-N-acetylglucosaminidase) in WT and three LAMP-2A(-) mouse fibroblasts clones. Values are expressed as total activity in fluorescence units in 106 cells and are the mean + SE of three different experiments. (C) Immunoblot of total cellular extracts (150 mg of protein) of the same cells for cathepsin B and cathepsin L. p, precursor; i, intermediate; m, mature (forms of the enzymes). (D) Immunofluorescence of WT and LAMP-2A(-) mouse fibroblasts for different organelle markers (BIP, endoplasmic reticulum; GM130, Golgi; phalloidin, a-actin; Mitotracker, mitochondria; caveolin-1, caveolae).
Fig. 7. Up-regulation of macroautophagy in cells with defective CMA. (A) WT and LAMP-2A(-) mouse fibroblasts were incubated in the presence of serum with monodansylcadaverine, and, at the indicated times, cells were washed, mounted in DAPI-containing mounting medium, and observed under a fluorescence microscope. (B) Immunoblot of the same cells for two different autophagy-related proteins (beclin and Atg12). The high-molecular-weight band corresponding to the Atg12/Atg5-conjugated complex is shown. (Right) Changes in the levels of autophagy-related proteins were calculated after densitometric quantification of three immunoblots such as the ones shown here. Values are expressed as fold increase of the values in the WT mouse fibroblasts.
Fig. 8. Endocytosis in cells stably RNA interfered for LAMP-2A. (A) Internalization of radiolabeled RNase A by fluid-phase endocytosis was compared in WT and LAMP-2A(-) mouse fibroblasts. (Left) Radiolabeled RNase A was added to the medium, and the amount of remaining protein in the medium was analyzed at the indicated times. Values are expressed as percentage of the protein present in the medium at time 0 and are mean + SE of two different experiments with triplicate samples. (Right) Degradation of internalized RNase A in the same cells. Cells were incubated for 10 h with radiolabeled RNase A and, after extensive washing, placed in a radioactivity-free medium. Rates of degradation of the internalized proteins were measured by analyzing the amount of acid-soluble radioactivity (amino acids and peptides) released into the medium at the indicated times. Values are expressed as percentage of the internalized radioactivity and are mean + SE of two different experiments with triplicate samples. (B) Internalization of Texas red-labeled transferrin in the same cells. Cells maintained in the presence or absence of serum for 10 h were incubated with Texas red-transferrin, and, at the indicated times, cells were washed, mounted in DAPI-containing mounting medium, and observed under a fluorescence microscope.
Fig. 9. Growth and viability of cells stably RNA interfered for LAMP-2A. (A) Growth rates of WT or LAMP-2A(-) mouse fibroblasts were measured by the tetrazolium salt assay as described in Supporting Methods. Values are the mean + SE of two different experiments and are expressed as absorbance units at 560 nm. (B) Changes in levels of LC3-II (recognized with the antibody that preferentially labels the LC3-II form; see Fig. 4A) in LAMP-2A(-) mouse fibroblasts 6 h after exposure to the indicted stressors. "Inhibitors" refers to cells treated with the pepstatin A/E-64 inhibitory combination to prevent degradation of LC3-II in lysosomes. Values are mean + SE of the densitometric quantification of immunoblots corresponding to three different experiments. This method to measure macroautophagy was selected to avoid the variability that the large number of cell deaths occurring during the course of the experiment introduced in the metabolic studies. In this case, an equal amount of protein (100 mg) was loaded per lane. Heat shock was the only condition for which a blockage in autophagic vacuole clearance (measured as LC3-II turnover) was observed. (C) Time course of cellular death (annexin V and 7-aminoactinomycin D positive) in cells treated with 100 mM paraquat for 6 h. Cells were labeled and analyzed as in Fig. 5D. Values are the mean + SE of triplicates.
Supporting Methods
Intracellular Protein Turnover.
Rates of protein synthesis were measured in confluent cells as the incorporation of [3H]leucine [10 mCi/ml (1 Ci = 37 GBq)] into acid-insoluble material in the presence of an excess (2.8 mM) of unlabeled leucine in the medium (to minimize differences due to alteration of amino acid transport and/or intracellular amino acid pool sizes). To measure degradation of long-lived proteins, confluent cells were labeled with [3H]leucine (2 mCi/ml) for 48 h at 37°C and then extensively washed and maintained in complete (10% newborn calf serum) or serum-deprived medium containing an excess of unlabeled leucine. Aliquots of the medium taken at different times were precipitated with trichloroacetic acid, and proteolysis was measured as above (1). Total radioactivity incorporated into cellular proteins was determined as the amount of acid-precipitable radioactivity in labeled cells immediately after washing. Degradation rates of short half-life proteins were determined by the same procedure but after a labeling period of 30 min at 37°C.Endocytosis of Proteins by Cultured Cells.
Fluid-phase endocytosis was measured in confluent cells incubated with [14C]RNase A as the decrease at different times of acid-precipitable radioactivity in the medium (2). To measure the degradation rates of endocytosed proteins once in the lysosomal compartment, cells were allowed to endocytose [14C]RNase A for 12 h, and then, after extensive washing, fresh medium was added, and the amount of acid-soluble radioactivity in the medium at different times was determined as above. Receptor-mediated endocytosis was analyzed by fluorescence microscopy as the incorporation of transferrin conjugated to Texas red (Molecular Probes) into cells incubated for different times with the fluorescent protein (100 mg/ml).Fluorescence and Immunocytochemical Staining.
Cells grown on coverslips were fixed with a 3% formaldehyde solution, blocked, and then incubated with the primary and corresponding FITC- or Cy5-conjugated secondary antibodies (2). Mounting medium contained DAPI to highlight the cellular nucleus. For visualization of the acid compartment, cells attached to coverslips were washed once with warm PBS and incubated with 50 mM monodansylcadaverine (Sigma) for increasing periods of time. a-Actin was stained by incubation with 165 nM Alexa Fluor 594 phalloidin (Molecular Probes) for 30 min. Filipin staining was done after fixation and blocking by incubation with 0.5 mg/ml Filipin (Sigma) for 1 h at room temperature to detect unesterified cholesterol. Mitochondria were stained with Mitotracker (Molecular Probes), following the manufacturer’s instructions.mRNA Quantification.
Total RNA (1 mg), extracted from culture cells by using the RNAeasy Protect Mini Kit (Qiagen, Valencia, CA), was used to synthesize the first-strand cDNA with the SuperScript II RNase H Reverse Transcriptase (Life Technologies) and oligo(dT)12-18 primer. A region of exon 8 of LAMP-2A, -2B, and -2C and of actin was amplified with specific primers (LAMP-2A, 5'-GCAGTGCAGATGAAGACAAC-3', 5'-AGTATGATGGCGCTTGAGAC-3'; LAMP-2B, 5'-GGTGCTGGTCTTTCAGGCTTGATT-3', 5'-ACCACCCAATCTAAGAGCAGGACT-3'; LAMP-2C, 5'-ATGTGCTGCTGACTCTGACCTCAA-3', 5'-TGGAAGCACGAGACTGGCTTGATT-3'; actin, 5'-AAGGACTCCTATAGTGGGTGACGA-3', 5'-ATCTTCTCCATGTCGTCCCAGTTG-3') by using the SYBR green PCR kit (PE Biosystems, Warrington, U.K.). Amplification of the DNA products was measured in real time in a light cycler (SmartCycler, Cepheid, Sunnyvale, CA).Viability and Apoptosis Measurement.
Cell viability was determined by incubating cells with 1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in DMEM for 1 h at 37°C. The formazan product formed was solubilized with n-propyl alcohol, and its absorbance was measured at 560 nm. Cell viability after various treatments was calculated as the percentage of the absorbance in untreated cells. Apoptosis was determined by evaluation of phosphatidylserine cell surface expression by flow cytometry using a dual staining annexin V-phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD) commercially available kit (Pharmingen). After exposing cells for 15 min to the two labeling compounds, the stained cells were analyzed by flow cytometry in a Becton Dickinson FACS II scanner. Early apoptotic cells were distinguished by annexin V-PE-positive/7-AAD-negative staining, whereas end stage apoptotic cells or apoptotic dead cells were positive for both labels. TUNEL assays were carried out by using the In Situ Cell Death Detection Kit (Roche Applied Science), following the manufacturer’s instructions, and the stained cells were analyzed by FACS.1. Auteri, J. S., Okada, A., Bochaki, V. & Dice, J. F. (1983) J. Cell. Physiol. 115, 167-174.
2. Cuervo, A. M. & Dice, J. F. (1996) Science 273, 501-503.