Materials and Methods

The QuikChange site-directed mutagenesis kit was from Stratagene. BCA protein assay reagent kit and ImmunoPure® Immobilized Protein A were purchased from Pierce. Mouse anti-p62 antibody was from BD Transduction Laboratories.

Mouse anti-GFP (B-2) antibody, rabbit anti-GAPDH (FL-335) antibody, rabbit anti-calnexin (H-70) antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, HRP-conjugated anti-rat IgG, and HRP-conjugated anti-mouse IgG were from Santa Cruz Biotechnology, Inc. Mouse anti-Htt antibody and Immobilon Western Chemiluminescent HRP Substrate were from Millipore Corporation. Earle’s balanced salt solution, saponin, poly-L-lysine, mouse anti-GFP antibody (clone GFP-20) (for

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immunoprecipitation), and dimethyl pimelimidate dihydrochloride (DMP) were purchased from Sigma-Aldrich, Inc. Rapamycin was from CalBiochem.

Lipofectamine 2000 transfection reagent, DMEM, Alexa Fluor® 633 goat anti-mouse IgG (H+L), and 4´,6-diamidino-2-phenylindole, dihydrochloride (DAPI) were from Invitrogen. Fetal bovine serum (FBS) was from Biological Industries Ltd (Kibbutz Beit Haemek, Israel). All other reagents were at least reagent grade and obtained from standard suppliers.

Site-directed mutagenesis of GFP-LC3 mutants

The pEGFP-C1-LC3 construct encoding wild-type LC3 was generated as previously described. Mutagenized pEGFP-C1-LC3 constructs encoding mutant LC3s (LC3-K30D, LC3-K51A, and LC3-K53A) were generated by QuikChange site-directed mutagenesis kit according to the manufacturer's instructions. Primers

were designed by Primer-X website

(http://www.bioinformatics.org/primerx/index.htm). The sequences of paired primers for each mutant were as follows: K30D-forward (5'- GAG CAG CAC CCC ACC GAC ATC CCA GTG ATT ATA G-3') and K30D-reverse (5'-C TAT AAT CAC TGG GAT GTC GGT GGG GTG CTG CTC-3'); K51A-forward (5'-GTC CTG GAC AAG ACC GCC TTC CTT GTA CCT GAT C-3') and K51A-reverse (5'-G ATC AGG TAC

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AAG GAA GGC GGT CTT GTC CAG GAC-3'); K53A-forward (5'-G GAC AAG ACC AAG TTC GCC GTA CCT GAT CAC GTG-3') and K53A-reverse (5'-CAC GTG ATC AGG TAC GGC GAA CTT GGT CTT GTC C-3'). The underlined nucleotides denote the base changes made to incorporate the desired missense mutations. The mutagenized sequences were confirmed by DNA sequencing.

To generate wild-type and mutant DsRed-tagged LC3 constructs, LC3 sequences were excised from pEGFP-C1-LC3 constructs by Eco RI and Bam HI. Purified DNA inserts were ligated with and subcloned into Eco RI/Bam HI-digested

pDsRed-Monomer-C1 vector (Clontech Laboratories, Inc. ).

Cell culture

Human embryonic kidney cells (HEK293) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. Cells were incubated in a humidified incubator at 37 ºC in 5% CO₂.

Transient transfection and induction of autophagy

Transient transfection of LC3 constructs into HEK293 cells was performed using Lipofectamine 2000 tansfection reagent as described by the manufacturers.

HEK293 cells were seeded onto 6-well microplates and transfected with 1 µg of

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wildt-type or mutant pEGFP-LC3 constructs for 24 or 48 h to allow protein expression. To induce autophagy, following the removal of growth medium, transfected cells were washed by Earle’s buffered salt saline (EBSS) once and incubated in EBSS for 2 h. Cells incubated in fresh culture medium were included as controls. Alternatively, HEK293 cells transfected with different GFP-LC3s were treated with 0.2 µg/ml of rapamycin for various intervals as specified to induce autophagy. Clarified lysates derived from transfected cells were analyzed by SDS-PAGE and Western blotting.

To determine the autophagy-mediated clearance of Htt aggregates, HEK293 cells in 10-cm dish were transfected with 5 µg of pcDNA3.1-Htt-(Q)109-hrGFP (a generous gift from Dr. Yijuang Chern at Academia Sinica [193]) for 24 h.

Htt-(Q)109-hrGFP-transfected cells were subcultured onto 6-well microplates and

allowed to adhere overnight. Htt-(Q)109-hrGFP-expressing cells were then transfected with 0.5 µg/well of wild-type or mutant DsRed-LC3 constructs for 5h later.

Following the removal of transfection mixtures, cells co-transfected with Htt-(Q)109-hrGFP and DsRed-LC3 were incubated with culture medium in the presence of 0.1% DMSO or 0.2 µg/ml rapamycin for additional 48 h to induce autophagy.

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The Generation of a tetracycline-inducible cell line that is stably transfected with YFP-tagged neomycin phosphotransferase II fusion construct

To generate an inducible neomycin phosphotransferase II-YFP (NeoR-YFP) construct, a marker protein for constitutive autophagy degradation [194], the coding sequence of NeoR was first subcloned into pEYFP-C1 (Clontech) in-frame with YFP tag. The NeoR-YFP cDNA sequence was subsequently subcloned into pcDNA5 plasmid (Invitrogen), and the expression of NeoR-YFP fusion protein would be under the control of tetracycline operator sequences. The NeoR-YFP construct was transfected into T-REx293 cells by lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. Transfected cells were cultured in DMEM supplemented with 10% FBS, 200 µg/ml hygromycin and 5 µg/ml blasticidin, and single colonies resistant to antibiotic selection were isolated individually. Each of independent cell lines was screened for the tetracycline-inducible expression of NeoR-YFP. Cell lines in which the accumulation of NeoR-YFP can be suppressed by rapamycin (an autophagy activator) and enhanced by pepstatin A/E64D (lysosomal protease inhibitors) were retained, and the one (T-REx-NeoRY) with maximal induction of NeoR-YFP expression that is responsive to rapamycin or pepstatin A/E64D treatment was chosen for subsequent experiments.

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SDS-polyacrylamide gel electrophoresis and Western blot analysis

Transfected cells were lysed by 1% Triton X-100 lysis buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 1 mM β-glycerophosphate, 2 mM EDTA, 50 mM β-glycerophosphate, 1 mM Na3VO4, and the Complete® protease inhibitor cocktail) on ice for 30min. Following removal of cell debris by centrifugation, protein concentrations of clarified lysates were determined by the BCA protein assay reagent kit.

Cell lysates containing equal amounts of proteins were mixed with 6x sample-loading buffer, and boiled at 100 °C for 10 min. Proteins were separated by 10% Tris-glycine polyacrylamide gels, then transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (Pall). Membranes were blocked by 5% BSA in TBST (blocking buffer) at room temperature for 1 h, followed by incubation with appropriated first antibody in blocking buffer (1:5000) at 4 °C overnight. After being washed with TBST for three times, membranes were incubated with HRP-conjugated secondary antibody in TBST (1:5000) at room temperature for 1 h. Following extensive washes with TBST, antibody-reactive proteins were visualized by Immobilon Western Chemiluminescent HRP Substrate. Images were captured and processed with ChemiGenius2 (Syngene).

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Co-immunoprecipitation of GFP-LC3s and endogenous p62

Anti-GFP (GFP-20) antibody (1.5 µl) and Immobilized Protein A (20 µl of 50%

slur per well for lysates from 6-well microplates) were mixed in 1 ml PBS at 4 °C overnight. Following removal of unbound antibodies by PBS washes, the Protein A and bound antibodies were cross-linked in 1 ml borate buffer (0.2 M sodium borate, pH 9.0) containing 20 mM DMP at room temperature for 30 min. The coupling reaction was stopped by addition of 0.2 M ethanolamine (pH 8.0), and the antibody-protein A conjugates were further incubated in 1 ml of 0.2 M ethanolamine for 1 h. Clarified lysates containing equivalent amounts of proteins (about 200 µg) were applied to the coupled antibody-protein A complexes and mixed at 4 °C overnight to pull down target proteins. Following removal of unbound proteins and extensive wash by PBS, immunoprecipitated proteins were eluted by boiling at 100

°C in 3x sample loading buffer and analyzed by SDS-PAGE and Western blotting.

Immunofluorescence staining and confocal image analysis

To observe the starvation-induced formation of autophagsomes in GFP-LC3s transfected cells, cells were seeded on poly-L-lysine-coated coverslips overnight.

Cells were incubated with EBSS for 24 h and fixed with 4% paraformaldehyde in PBS at room temperature for 1 h, followed by washes with PBS prior to mounting.

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Approximately 150 cells in each group from four independent experiments were analyzed, and the numbers and sizes of GFP-LC3 punctas (i.e. autophagosomes) per cell were measured.

To visualize colocalization of GFP-LC3 and endogenous p62, cells were fixed at 48 h post-transfection. Following washes with PBS, transfected cells were permeablized by 10% formaldehyde in PBS containing 0.5% Triton X-100 at room temperature for 10 min, and blocked with PBS containing 2% BSA at room temperature for 1 h. After removal of blocking buffer, cells were stained with a mouse-anti p62 antibody in PBS-0.1% BSA at 4 °C overnight. Following PBS washes, cells were incubated with Alexa Fluor 633 goat anti-mouse IgG in PBS at room temperature for 1 h. GFP and Alexa Fluor 633 fluorescence images were captured separately by sequential scanning. Approximately, 100 cells in each group from three independent experiments were analyzed. The overlapped areas between GFP puncta and p62 signals were measured and shown as the percentage of the total number of GFP puncta.

To assess the clearance of huntingtin aggregates, cells co-transfected with Htt-(Q)109-hrGFP and DsRed-LC3 were fixed after 48 h rapamycin treatment. Nuclei were stained with DAPI for 5min. The coverslips were washed with PBS and mounted as described above. In 12~13 viewing areas from two independent experiments, total

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numbers of Htt-(Q)109-hrGFP-expressing cells and Htt aggregate-containing cells were counted. Data were shown as the percentage of cells with aggregates.

All the immunofluorescence images were obtained by using a Leica TCS SP5 spectral confocal microscope. Quantitative analyses were processed by using the Metamorph 6.2 software package (Molecular Devices).

Quantitative densitometry and statistical analysis

The density of the immunoreactive bands on Western blots was quantitated by the TotalLab v2.01 program after acquisition of the blot image with ChemiGenius2 (Syngene). Results were expressed as the mean (±S.D.) of three to four independent experiments. Statistical analyses were done by a two-tailed Student’s t-test. A value of p＜0.05 was considered significant.