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Results: 4

1.
Figure 2

Figure 2. Tat–beclin 1 peptide binds to GAPR-1, a beclin 1-interacting protein. From: Identification of a candidate therapeutic autophagy-inducing peptide.

a, HeLa cells were treated with biotin-conjugated peptides (30 μM, 3 h) and proteins bound to peptides were analysed by immunoblot with anti-GAPR-1. b-T-B, biotin–Tat–beclin 1; b-T-S, biotin–Tat-scrambled. b, Immunoprecipitation of Flag–beclin 1 with GAPR-1–Myc in HeLa cells 24 h post-transfection. c, GFP–LC3-positive dots in GAPR-1 siRNA-transfected peptide-treated HeLa/GFP–LC3 cells (20 μM, 3 h) with or without 100nM bafilomycin A1. Bars represent mean ± s.e.m. of triplicate samples (50–100 cells per sample). Similar results were observed in three independent experiments. NC, non-silencing control. d, Localization of beclin 1 and GM130 (a Golgi marker) in HeLa cells stably transduced with empty vector (left panel) or GAPR-1–Myc (middle panel) or transfected with GAPR-1 siRNA (right panel) and treated with peptide (20 μM, 1 h). Scale bar, 20 μm. e, Immunoblot of beclin 1 in post-nuclear supernatant (PNS) and Golgi-enriched fractions in HeLa cells stably transduced with empty vector or GAPR-1–Myc after peptide treatment (20 μM, 2 h). f, WIPI2 dots in cells in the experimental conditions shown in d. Bars represent mean ± s.e.m. for 100–150 cells. *P < 0.05, ***P < 0.001; t-test. †††P < 0.001; two-way ANOVA for comparison of magnitude of changes between groups.

Sanae Shoji-Kawata, et al. Nature. 2013 February 14;494(7436):201-206.
2.
Figure 1

Figure 1. Tat–beclin 1 peptide induces autophagy in vitro. From: Identification of a candidate therapeutic autophagy-inducing peptide.

a, Immunoprecipitation of Flag–beclin 1 constructs with Nef–HA in HeLa cells 24 h post-transfection. b, GFP–LC3-positive dots (autophagosomes) in MCF7 cells expressing GFP–LC3 and Flag–beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h. c, Sequences of beclin 1 amino acids 267–284, Tat–beclin 1 (T-B) and Tat-scrambled (T-S) control peptide. Red letters indicate amino acid substitutions to enhance hydrophilicity. d, Biochemical assessment of autophagy (p62 and LC3 immunoblots) in peptide-treated HeLa cells (3 h). e, f, Representative images (e) and quantification of GFP–LC3-positive dots (f) in peptide-treated HeLa/GFP–LC3 cells (30 μM, 3 h). Scale bars, 20 μm. g, GFP–LC3-positive dots in siRNA-transfected peptide-treated HeLa/GFP–LC3 cells (30 μM, 3 h). h, Model of Tat–beclin 1 peptide (left) based on corresponding elements of the beclin 1 evolutionarily conserved domain (ECD) structure (centre). Essential phenylalanine side chains, magenta; positions of solubility mutations, pink; lipid interaction site, yellow. ECD surface representation (right) illustrates exposure of corresponding peptide (cyan). i, p62 and LC3 immunoblots in peptide-treated HeLa cells (3 h). In b, f, g, bars represent mean ± s.e.m. of triplicate samples (50–100 cells per sample). Similar results were observed in three independent experiments. *P < 0.05, **P < 0.01; t-test.

Sanae Shoji-Kawata, et al. Nature. 2013 February 14;494(7436):201-206.
3.
Figure 3

Figure 3. Tat–beclin 1 peptide decreases aggregates of a polyglutamine expansion protein and has anti-infective activity. From: Identification of a candidate therapeutic autophagy-inducing peptide.

a, Percentage of cells with small htt103Q aggregates (left) and number of aggregates per cell (right) in HeLa cells expressing doxycycline (Dox)-repressible CFP–htt103Q after daily treatment with doxycycline or peptide (20 μM, 4 h per day) for 2 days. Bars represent mean ± s.e.m. of triplicate samples (60–120 cells per sample). Similar results were observed in three independent experiments. b, Filter trap assays for htt103Q large and small aggregates in HeLa/htt103Q cells. c, Viral titres in HeLa cells infected with 0.1 plaque-forming units (p.f.u.) per cell of SINV, CHIKV or WNV (strain TX02) and treated with peptide (10 μM, 4–8 h post-infection). Values represent geometric mean ± s.e.m. for triplicate samples of supernatants collected 18 h post-infection (SINV) or 24 h post-infection (CHIKV and WNV). Similar results were observed in three independent experiments. d, Bacteria colony-forming units (c.f.u.) in primary BMDMs infected with L. monocytogenes ΔactA mutant strain DPL-402927 for 30 min and treated with peptide (10 μM from 0 to 2 h post-infection). Bars represent mean ± s.e.m. of triplicate samples. Similar results were observed in three independent experiments. e, HIV-1 p24 antigen release in primary human MDMs infected with HIV-1 24 h after initiation of daily peptide treatment. Values represent mean ± s.e.m. of triplicate samples. Similar results were observed in MDMs from three independent donors. f, HIV-1 p24 antigen release in MDMs transduced with nonspecific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and treated daily with peptide (5 μM). Values represent mean ± s.e.m. of triplicate wells. Similar results were observed in MDMs from two independent donors. g, LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV-1 infection. *P<0.05; **P < 0.01; ***P < 0.001; t-test.

Sanae Shoji-Kawata, et al. Nature. 2013 February 14;494(7436):201-206.
4.
Figure 4

Figure 4. Tat–beclin 1 peptide induces autophagy and exerts antiviral activity in vivo. From: Identification of a candidate therapeutic autophagy-inducing peptide.

a, GFP–LC3-positive dots in tissues of 6-week-old GFP–LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). A minimum of ten fields was counted per tissue section. Bars represent mean ± s.e.m. for three mice. Similar results were observed in three independent experiments. b, p62 immunoblot of brains of 5-day-old GFP–LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). c, Survival curves of 5-day-old C57BL/6J mice infected with CHIKV(106 p.f.u. s.c.) and treated daily with peptide (15 mg kg−1 i.p. beginning 1 day post-infection). d–f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e), and survival curves (f) for 5-day-old C57BL/6J mice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (d-amino acid forms, 20 mg kg−1 i.p. beginning 1 day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20 μm. Bars in e represent mean ± s.e.m. TUNEL-positive cells per unit area of brain for three mice. g, Geometric mean + s.e.m. viral titres of WNV-infected mouse brains day 6 post-infection. Values represent combined data for 12–20 mice per treatment group from 10 to 12 litters. Data in c and f represent combined survival probabilities for three and four independent litters, respectively, in each group. Similar results were observed in each independent experiment. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; t-test.

Sanae Shoji-Kawata, et al. Nature. 2013 February 14;494(7436):201-206.

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