Klein et al.

SUPPLEMENTARY MATERIAL

MATERIAL AND METHODS

Cell culture and reagents

HeLa cells were cultured in DMEM (1X), and MRC5, U251 MG, and A549 cells were cultured in DMEM/F-12 50:50 medium (Invitrogen). U87 MG cells were cultured in MEM with 10% FBS and 1% essential amino acids. Wild-type and JNK1/2-/- MEFs were provided by Roger Davis (University of Massachusetts Medical School, MA). JNK1-/- and JNK2-/- MEFs were described previously [34]. c-Jun−/− and c-Jun+/+ 3T3 fibroblasts were obtained from Ann MacLaren (The Scripps Research Institute, CA). Wild-type and knock-out AMPKa1 MEFs (AMPK-/-) were obtained from Keith Laderoute (SRI International, San Francisco, CA). Wild-type and knock-out Atg5 MEFs (Atg5-/-) are a generous gift from Noboru Mizushima (Tokyo Medical and Dental University, Tokyo, Japan). Wild-type and knock-out p62 MEFs (p62-/-) were a generous gift from Jorge Moscat (Sanford-Burnham Medical Research Institute, La Jolla, CA). MEFs were cultured in DMEM/F-12 50:50 medium. Cells were cultured with 10% FBS at 37°C in 5% CO2 in air.

Reagents

The JNK kinase inhibitor SP600125 and anisomycin were purchased from Sigma-Aldrich. Antibodies to phospho-AKT (S473), AKT, AMPKa1, TSC2, LC3, phospho-JNK (T183/Y185, T221/Y223), phospho-c-Jun (S63), c-Jun, Beclin 1 were purchased from Cell Signaling Technologies. Anti-phospho-Bcl-2 and anti-JNK2 antibodies were purchased from Abcam. Anti-JNK1, anti-JNK3, anti-panJNK, anti-E1A, anti-p62, anti-actin, and anti-Bcl-2 antibodies were purchased from Santa Cruz Biotechnology. Anti-adenoviral hexon was purchased from Novus Biologicals, and anti-adenoviral fiber from ThermoScientific. Allophycocyanin (APC)-conjugated anti-mouse secondary antibodies and fluorescein isothiocyanate (FITC)-conjugated secondary antibody were purchased from Santa Cruz Biotechnology.

Animal protocol for tissue immunofluorescence

All animal studies were performed in the veterinary facilities at The University of Texas MD Anderson Cancer Center in accordance with institutional guidelines and ethical guidelines for experimental animal care and approved by the MD Anderson IACUC. U87 MG human glioma cells (5 x 105) were engrafted into the caudate nuclei of athymic mice using a guide-crew system as described previously [20]. Three days after cell implantation, the animals were given 5 mL intratumoral injections of Delta-24-RGD (106 pfu/mouse) or PBS. Animals exhibiting generalized or local signs of disease were euthanized using a CO2 chamber (~28-35 days after cell implantation). Their brains were then removed, fixed in 4% formalin for 48 h, and embedded in paraffin, and 5-mm slices were mounted on slides. Paraffin embedded brain sections were heated (60°C), and the tissues were rehydrated with xylene and ethanol following conventional procedures. Hematoxylin- and eosin-stained coronal sections were evaluated for evidence of tumors. Heat-mediated antigen retrieval was performed on the tissues by boiling them in 10 mM citrate buffered to pH 6.0 in a pressure cooker for 20 min and then cooling them for 20 min at room temperature. The tissues were blocked using appropriate IgGs and a streptavidin/biotin blocking kit (Vector Labs). Anti-p-JNK (1:100) or anti-p-Bcl-2 serine 87 (1:100) and anti-hexon (1:500) primary antibodies were applied, and then the tissues were incubated with their respective biotin-conjugated secondary antibodies (1:100, Vector Labs). Streptavidin-conjugated anti-biotin antibodies (1:100, Vector Labs) were used to visualize the co-localization of infected cells (i.e., hexon) and p-JNK or p-Bcl-2. DAPI was used for nuclear staining. Images were captured on a Zeiss Axiovert Zoom fluorescence microscope (Carl Zeiss, Inc.) equipped with AxioCam MRM camera and 40x 1.3 oil EC Plam-NEOFLUAR objective, using Immersol (n = 1.518) at room temperature. The acquisition software utilized was AxioVision Release 4.7.1 (Carl Zeiss, Inc).

Western blot analysis

Cells were lysed with RIPA lysis buffer. Protein concentrations were determined using a Bio-Rad protein assay. Protein samples (25 µg) in 1X SDS loading buffer were boiled and loaded into Tris-glycine SDS-PAGE gels (Invitrogen). Samples were separated using electrophoresis at a constant voltage of 100 V (~2 h) in running buffer. Gels were then transferred to polyvinylidene fluoride membranes via western blotting (25 V for 40 min via a BioRad semi-dry transfer apparatus). The membranes were blocked using 5% non-fat milk in 1X TBS-T (0.025% Tween) for 1 h at room temperature and then incubated overnight at 4°C in a primary antibody at appropriate dilutions. Membranes were washed three times with TBS-T (0.05% Tween) and then incubated at room temperature for 1 h with appropriate secondary antibodies (Santa Cruz Biotechnology; 1:4000) prepared in 1% non-fat milk in TBS-T. SuperSignal West Femto chemiluminescent substrate (ThermoScientific) and HyBlot CL autoradiography film (Denville Scientific Inc.) were used to visualize protein bands.

Co-immunoprecipitation

Cells were infected with AdWT or Delta-24-RGD for up to 48 h. Floating and attached cells were collected and lysed with immunoprecipitation (IP) lysis buffer (100 mM NaCl, 50 mM Tris-HCl [pH 7.4], 2 mM EDTA, 10% glycerol, 1% IgePal CA-630). The protein samples were pre-cleared with protein A and protein G agarose beads. Anti-Bcl-2, anti-adenoviral fiber, or anti-p62 antibodies were added to the lysates at a dilution of 1:100, and complexes of proteins were immobilized with protein A and protein G (1:1) agarose beads. Immunoprecipitated samples, pre-cleared beads, and 5% input were analyzed via western blotting. IP detection secondary antibody was used to detect proteins bound by all anti-rabbit and anti-mouse primary IgGs.

Electron microscopy

Cells (>1,000,000) were fixed in a solution that comprised 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3). The samples were treated with 0.1% cacodylate-buffered tannic acid and then fixed for an additional 1 h with 1% buffered osmium tetroxide. They were then stained with 1% uranyl acetate. All reagents were obtained from Sigma-Aldrich. The samples were dehydrated in ethanol at increasing concentrations, infiltrated, and then embedded in Spurr's low-viscosity medium. They were polymerized for 2 days at 70°C. Ultrathin sections were cut in a Leica Ultracutmicrotome, stained with uranyl acetate and lead citrate in a Leica EM stainer, and visualized using a JEM-1010 transmission electron microscope (JEOL USA Inc.) at an accelerating voltage of 80 kV. Digital images were obtained using an imaging system from Advanced Microscopy Techniques.

Statistical analysis

A two-tailed Student’s t-test was used to determine the statistical significance of treated and infected samples relative to control samples. P values less than 0.05 were accepted as statistically significant.

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1. Analysis of phospho-proteins after adenoviral infection using a phospho-kinase antibody array. MRC5 lung fibroblasts (ATCC) were infected with AdWT at an MOI of 50 for 0h, 16h, 32h or 48h. Protein extracts were analyzed, as recommended by the manufacturer, using the human phospho-kinase antibody array from R&D Systems. The entire phospho-protein array of proteins is displayed.

Supplementary Figure 2. Kinetics of c-Jun and individual JNK isoform phosphorylation during adenoviral infection. (a) Whole-cell lysates from MRC5 lung fibroblasts infected with AdWT at an MOI of 50 for 0h, 16h, 32h or 48h were analyzed using a human phospho-kinase antibody array. Phosphorylated c-Jun protein expression levels were quantified by densitometry (top panel). The values represent the average of two dots (bottom panel). (b) U-87 MG cells were mock-infected with UVi or active AdWT or Delta-24-RGD adenoviruses at dose of 50 MOI for 24h. Protein extracts were analyzed by Western blot for the expression of total and phosphorylated JNK and c-Jun. Anisomycin treatment is showed as control for phosphorylated JNK. Actin is shown as a loading control. UVi, inactivated adenovirus. (c) Whole-cell lysates from MRC5 lung fibroblasts infected with AdWT at an MOI of 50 for 0h, 16h, 32h or 48h were analyzed using a human phospho-kinase antibody array. Phosphorylated JNK1, JNK2, and JNK3 protein expression levels were quantified by densitometry (top panel). The values represent the average of two dots (bottom panel). (d) MRC5 lung fibroblasts were infected with AdWT at an MOI of 50 for 0h, 16h, 32h or 48h. Protein extracts were analyzed using the human phospho-MAPK antibody array from R&D Systems. The entire phospho-protein array of proteins is displayed.

Supplementary Figure 3. Phosphorylation of Bcl-2 and JNK occurs concomitantly in time with autophagy. (a) A549 cells transiently transfected to over-express Bcl-2 were infected with UVi or active AdWT at an MOI of 25 for 24h or 48h and whole cell lysates were analyzed by Western blot for phosphorylated Bcl-2 and JNK. (b) Cell lysates from HeLa cells infected with UVi or active AdWT at an MOI of 25 for 24h or 48h were prepared in IP lysis buffer, and both Bcl-2-IP and input (5%) samples were analyzed by Western blotting. Actin is shown as a loading control. UVi, inactivated adenovirus.

Supplementary Figure 4. Knock-out of both JNK1 and JNK2 is required to inhibit autophagosome formation. (a and b) Representative electron micrographs of wild-type JNK, JNK1-/-, JNK2-/-, and JNK1/2-/- MEF cells mock-infected or infected with AdWT at an MOI of 100. (a) The cytoplasm of wild type JNK or cells with deletion of either JNK1 or JNK2 infected with adenovirus show morphological changes indicative of autophagy. (b) Representative images of double-membrane bound vacuoles containing cytoplasmic content are indicated (black arrows).

Membrane Coordinates / Kinase/Control / Phosphorylation Site
A-A1, A2 / Reference Spot / ___
A-A3, A4 / p38α / T180/Y182
A-A5, A6 / ERK1/2 / T202/Y204, T185/Y187
A-A7, A8 / JNK / pan T183/Y185,T221/Y223
A-A9, A10 / GSK-3α/β / S21/S9
B-A13, A14 / p53 / S392
B-A17, A18 / Reference Spot / ___
A-B3, B4 / MEK1/2 / S218/S222, S222/S226
A-B5, B6 / MSK1/2 / S376/S360
A-B7, B8 / AMPKα1 / T174
A-B9, B10 / Akt / S473
B-B11, B12 / Akt / T308
B-B13, B14 / p53 / S46
A-C1, C2 / TOR / S2448
A-C3, C4 / CREB / S133
A-C5, C6 / HSP27 / S78/S82
A-C7, C8 / AMPKα2 / T172
A-C9, C10 / β-Catenin / ___
B-C11, C12 / p70 S6 kinase / T389
B-C13, C14 / p53 / S15
B-C15, C16 / p27 / T198
B-C17, C18 / Paxillin / Y118
A-D1, D2 / Src / Y419
A-D3, D4 / Lyn / Y397
A-D5, D6 / Lck / Y394
A-D7, D8 / STAT2 / Y689
A-D9, D10 / STAT5a / Y694
B-D11, D12 / p70 S6 kinase / T421/S424
B-D13, D14 / RSK1/2/3 / S380/S386/S377
B-D15, D16 / p27 / T157
B-D17, D18 / PLCγ-1 / Y783
A-E1, E2 / Fyn / Y420
A-E3, E4 / Yes / Y426
A-E5, E6 / Fgr / Y412
A-E7, E8 / STAT3 / Y705
A-E9, E10 / STAT5b / Y699
B-E11, E12 / p70 S6 kinase / T229
B-E13, E14 / RSK1/2 / S221/S227
B-E15, E16 / c-Jun / S63
B-E17, E17 / Pyk2 / Y402
A-F1, F2 / Hck / Y411
A-F3, F4 / Chk-2 / T68
A-F5, F6 / FAK / Y397
A-F7, F8 / STAT6 / Y641
A-F9, F10 / STAT5a/b / Y694/Y699
B-F11, F12 / STAT1 / Y701
B-F13, F14 / STAT4 / Y693
B-F15, F16 / eNOS / S1177
B-F17, F18 / PBS / (Negative Control)
A-G1, G2 / Reference Spot / ___
A-G5, G6 / PBS / (Negative Control)

Table 1.

Table 2.

Membrane Coordinates / Kinase/Control / Phosphorylation Site
A1, A2 / Reference Spot / ___
A21, A22 / Reference Spot / ___
B3, B4 / Akt1 / S473
B5, B6 / Akt2 / S474
B7, B8 / Akt3 / S472
B9, B10 / Akt / pan S473, S474, S472
B11, B12 / CREB / S133
B13, B14 / ERK1 / T202/Y204
B15, B16 / ERK2 / T185/Y187
B17, B18 / GSK-3α/β / S21/S9
B19, B20 / GSK-3β / S9
C3, C4 / HSP27 / S78/S82
C5, C6 / JNK1 / T183/Y185
C7, C8 / JNK2 / T183/Y185
C9, C10 / JNK3 / T221/Y223
C11, C12 / JNK / pan T183/Y185, T221/Y223
C13, C14 / MKK3 / S218/T222
C15, C16 / MKK6 / S207/T211
C17, C18 / MSK2 / S360
D3, D4 / p38α / T180/Y182
D5, D6 / p38β / T180/Y182
D7, D8 / p38δ / T180/Y182
D9, D10 / p38γ / T183/Y185
D11, D12 / p53 / S46
D13, D14 / p70 S6 Kinase / T421/S424
D15, D16 / RSK1 / S380
D17, D18 / RSK2 / S386
D19, D20 / TOR / S2448
F1,F2 / Reference Spot / ___

SUPPLEMENTARY TABLE LEGENDS

Supplementary Table 1. Phospho-kinase antibody array description. The location of each phospho-protein (in duplicate) as well as the site of phosphorylation is described as in the manufacturer’s instructions and shown for clarity. The kinases analyzed in this study are highlighted in orange.

Supplementary Table 2. Phospho-MAPK antibody array description. The location of each phospho-protein (in duplicate) as well as the site of phosphorylation is described as in the manufacturer’s instructions and shown for clarity. The kinases analyzed in this study are highlighted in orange.

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