Molecular Cell, Volume 48

Supplemental Information

Cytoplasmic STAT3 Represses Autophagy by Inhibiting PKR Activity

Shensi Shen, Mireia Niso-Santano, Sandy Adjemian, Tetsuo Takehara, Shoaib Ahmad Malik, Hervé Minoux, Sylvie Souquere, Guillermo Mariño, Sylvie Lachkar, Laura Senovilla, Lorenzo Galluzzi, Oliver Kepp, Gérard Pierron, Maria Chiara Maiuri, Hayato Hikita, Romano Kroemer, and Guido Kroemer

Supplemental Information - Inventory

Supplemental Figures

Figure S1. Specific inhibitors of STAT3 stimulate ATG7- and Beclin 1-dependent autophagy

(related to Figure 1c,d)

Figure S2. STAT3 inhibitors stimulate the autophagic flux (related to Figure 1g,h)

Figure S3. Cytoplasmic, not nuclear, STAT3 inhibits autophagy, irrespective of phosphorylation

on Y705 (related to Figure 2i,j)

Figure S4. STAT3 inhibitors induce autophagy independent of HSP90 (related to Figure 4a)

Figure S5. STAT3 inhibitors induce the dissociation of polyribosomes and the appearance of stress

granules (related to Figure 4b)

Figure S6. Interaction between STAT family members and eIF2α kinases (related to Figure 4d)

Figure S7. Characterization of the interaction between STAT3 and PKR (related to Figure 5b)

Figure S8. The autophagic response to STAT3 inhibitors requires PKR and eIF2α but not PERK,

GCN2 and HRI (related to Figure 6e-g)

Supplemental Tables

Table S1. Primers employed for site-directed mutagenesis (related to Experimental Procedures)

Legends to Additional Supplementary Items

Movie S1. Molecular model of the STAT3-PKR interaction

Spreadsheet S1. Panel of autophagy inducers employed in this study (related to Figure 7c,d)

Supplemental Experimental Procedures (related to Experimental Procedures)

Supplemental References

Supplemental Information - Supplemental Figures

Figure S1. Specific inhibitors of STAT3 induce ATG7- and Beclin 1-dependent autophagy.

(a) Chemical structure of JSI-124, Stattic and WP1066.

(b,c) Human osteosarcoma U2OS cells were left untreated or incubated with 10 μM Stattic or 10 μM

WP1066 for 1 h, followed or not by the administration of 100 ng/mL recombinant interleukin-6 (IL-6)

for additional 30 min. Finally, cells were processed for the immunoblotting-assisted detection of

STAT3 (b) or STAT1 (c) phosphorylation. β actin was monitored as a loading control. Numbers

illustrate band intensity upon normalization to the STAT3 (b) or STAT1 (c) total protein levels.

(d,e) U2OS cells stably expressing a GFP-LC3 chimera were transfected with a control siRNA

(siUNR), or with siRNAs targeting ATG7 (siATG7) and Beclin 1 (siBeclin1) for 48h, followed by the

administration of 10 μM Stattic for 6 h. Representative images are shown in (d), quantitative data in

(e). Scale bar = 5 μm. Columns report the percentage of cells exhibiting GFP-LC3 dots in the

cytoplasm (GFP-LC3vac cells, Mean ± SEM, n = 3; **p<0.01, Student’s t test, compared to equally

treated siUNR-transfected cells). The inset in (e) depicts the efficacy of siRNA-mediated ATG7 and

Beclin 1 downregulation, as assessed by immunoblotting. β actin levels were monitored to ensure

equal loading of lanes.

Figure S2. STAT3 inhibitors stimulate the autophagic flux.

(a) Human osteosarcoma U2OS cells transiently expressing a RFP-GFP-LC3 chimera were

maintained in control (Co) or nutrient-free (NF) conditions, or administered with 10 μM Stattic, in the

absence or in the presence of 50 nM bafilomycin A1 (BafA1) for the indicated time, followed by the

quantification of the number of RFP+GFP+ or RFP+GFP- dots per cell by fluorescence microscopy

(Mean ± SEM, n = 3; *p<0.05, **p<0.01 Student’s t test, RFP+GFP- dots compared to untreated cells,

#p<0.05, ##p<0.01, RFP+GFP+ dots compared to cells treated with the same inducer of autophagy in

the absence of BafA1).

(b) U2OS cells were treated as in (a), followed by immunoblotting for the assessment of LC3

lipidation and p62 levels. β actin was monitored as a loading control. Numbers illustrate the

abundance of LC3-II and p62 upon normalization to β actin levels.

(c,d) U2OS cells were treated with 1 μM JSI-124 (c) or 10 μM WP1066 (d), alone or in combination

with 50 nM BafA1 for 6 h, followed by the immunoblotting-assisted determination of LC3 lipidation

and p62 levels. β actin was monitored as a loading control. Numbers illustrate the abundance of LC3-

II and p62 upon normalization to β actin levels.

(e,f) U2OS cells stably expressing a GFP-LC3 fusion protein were treated as in (d), followed by

immunostaining with a LAMP2a-specific antibody and confocal fluorescence microscopy. Columns

in (e) indicate the percentage of cells in which GFP-LC3 and LAMP2a colocalized (Mean ± SEM, n =

3; **p<0.01, Student’s t test, compared to untreated cells, #p<0.05, compared to Stattic-treated cells).

In (f) representative images are reported together with co-localization profiles as determined by the

ImageJ software along the indicated α--ω axis.

Figure S3. Cytoplasmic, not nuclear, STAT3 inhibits autophagy, irrespective of phosphorylation

on Y705.

(a,c) Full version of Figure 2i. Human osteosarcoma U2OS cells were co-transfected with a plasmid

coding for a RFP-LC3 chimera and either a GFP-coding control vector (pcDNA3-GFP, a) or

constructs coding for merely cytoplasmic (NES, b) or merely nuclear (NLS, c) GFP-tagged STAT3

variants for 24 h. Then, cells were maintained in control (Co) or nutrient-free (NF) conditions, or

administered with 1 μM thapsigargin (Thaps) or 10 μM Stattic, for 6 h, followed by nuclear

counterstaining with Hoechst 33342 (blue) and confocal fluorescence microscopy. Representative

images are reported. Scale bar = 5 μm.

(d) Stat3Δ/Δ mouse embryonic fibroblasts (MEFs) were transiently transfected with an empty plasmid

(pBabe) or with constructs encoding wild-type (WT) STAT3 or a non-phosphorylatable STAT3

variant (STAT3Y705F). Twenty-four h later, MEFs were either maintained in Co conditions or

stimulated with 100 ng/mL recombinant interleukin-6 (IL-6) for 30 min and processed for the

immunoblotting-assisted determination of STAT3 phosphorylation. GAPDH abundance was

monitored as a loading control. Numbers illustrate STAT3 phosphorylation normalized to total

STAT3 levels.

(e) U2OS cells stably expressing a GFP-LC3 fusion protein were transiently transfected with an

empty plasmid (pBabe) or with constructs encoding WT STAT3 orSTAT3Y705F. Twenty-four h later,

cells were maintained in Co or NF conditions, or administered with 1 μM rapamycin (Rapa) or 1 μM

Thaps for 15 h, and then subjected to fluorescence microscopy for the quantification of cells

exhibiting GFP-LC3 dots in the cytoplasm (GFP-LC3vac cells, Mean ± SEM, n = 3; **p<0.01,

Student’s t test, compared to equally treated pBabe-transfected cells).

Figure S4. STAT3 inhibitors induce autophagy independent of HSP90.

(a,b) Human osteosarcoma U2OS cells stably expressing a GFP-LC3 fusion protein were maintained

in control (Co) or nutrient-free (NF) conditions, or treated with 1 μM rapamycin (Rapa), 10 μM Stattic

or 10 μM WP1066 for 6 h, alone or combined with 1 μM 17-allylamino-17-demethoxygeldanamycin

(17-AAG). Representative images are shown in (a), quantitative data in (b). Scale bar = 5 μm.

Columns report the percentage of cells exhibiting GFP-LC3 dots in the cytoplasm (GFP-LC3vac cells,

Mean ± SEM, n = 3; *p<0.05, Student’s t test, compared to cells stimulated to undergo autophagy by

the same inducer in the absence of 17-AAG).

Figure S5. STAT3 inhibitors induce the dissociation of polyribosomes and the appearance of

stress granules.

(a) Human osteosarcoma U2OS cells were left untreated (Co) or incubated with 10 μM Stattic for 12

h, and then processed for transmission electron microscopy. Representative images of ribosomes

(dissociated by Stattic) and stress granules (induced by Stattic, indicated by asterisks) are reported in

upper and lower panels respectively. Scale bars are provided.

(b,c) U2OS cells stably expressing a G3BP-GFP chimera were maintained in Co or nutrient-free (NF)

conditions, or treated with 1 μM thapsigargin (Thaps), 10 μM Stattic or 10 μM WP1066 for 6 h,

followed by immunostaining with an anti-PKR antibody and confocal fluorescence microscopy.

Representative images are shown in (d), quantitative data in (e). Scale bar = 5 μm. Columns report the

number of G3BP-GFP+ dots/cell (Mean ± SEM, n = 3; *p<0.05, **p<0.01, Student’s t test, compared

to untreated cells).

Figure S6. Interaction between STAT family members and eIF2α kinases.

(a) Human osteosarcoma U2OS cells were maintained in control (Co) of nutrient-free (NF)

conditions, or treated with 10 μM Stattic, 10 μM WP1066, 1 μM rapamycin (Rapa) or 0.5 mM

palmitate (PAL) for 15h. Thereafter, cells were lysed and lysates were immunoprecipitated with a

STAT3-specific antibodies, followed by the immunoblotting-assisted detection of the indicated eIF2α

kinases. Numbers illustrate PKR abundance within immunoprecipitates (normalized to PKR levels in

input lysates).

(b) U2OS cells were treated as in (a), followed by lysis, immunoprecipitation with a PKR-specific

antibody and immunoblotting-assisted determination of STAT1 and STAT3 levels. Numbers illustrate

STAT3 abundance within immunoprecipitates (normalized to STAT3 levels in input lysates).

(c) Superimposition of the crystal structures of eIF2α (in pink the relevant domain) and STAT3 (in

light blue the relevant domain). The panel on the right focuses on STAT3 SH2 domain, folding as a α-

helix that mimics the C-terminal of eIF2α.

(d) Diagram of the STAT3 mutants employed in this study. CC, coiled-coil domain; DBD, DNA-

binding domain; LD, linker domain; N-ter: N-terminal domain; SH2, SH2 domain; TAD,

transactivation domain.

(e) U2OS cells were co-transfected with a construct encoding Flag-PKR and plasmids for the

expression of Myc-tagged full-length (FL) STAT3 or Myc-tagged STAT3 variants bearing the

indicated point mutations for 24 h, followed by immunoprecipitation with an anti-Flag antibody and

immunoblotting with an antibody specifically recognizing Myc. β actin levels were monitored as a

loading control. Numbers illustrate STAT3 abundance within immunoprecipitates (normalized to

STAT3 levels in input lysates).

Figure S7. Characterization of the interaction between STAT3 and PKR.

(a) Recombinant GST-tagged STAT3 variants, including wild-type (WT) STAT3 and STAT3 mutants

bearing the point mutations K658A+E680A (#2) or E680A+W623A (#4), were immobilized on GST-

binding beads, followed by overnight incubation with recombinant full-length PKR. Eventually, PKR

pull-down as well as PKR levels in the input solution were evaluated by immunoblotting.

(b) Recombinant GST-tagged WT STAT3 was immobilized on GST-binding beads, followed by

incubation with DMSO (control conditions), 5 or 10 μg/mL polyinosinic:polycytidylic acid (polyI:C),

160 μM Stattic, 16 μM WP1066 for 2 h. Thereafter, recombinant full-length PKR was added and the

incubation was protracted overnight. Eventually, PKR pull-down as well as PKR levels in the input

solution were evaluated by immunoblotting.

(c) U2OS cells were maintained in Co conditions, mock-transfected or transfected with either an

empty plasmid (pBabe) or with a construct encoding WT STAT3, alone or in combination with 2.5 μg

polyI:C for 24 h, followed by immunoblotting for the assessment of STAT3 levels, PKR

phosphorylation and eIF2α phosphorylation. β actin levels were monitored as a loading control.

Numbers illustrate the phosphorylation status of PKR and eIF2α (normalized to total PKR and eIF2α

levels, respectively).

Figure S8. The autophagic response to STAT3 inhibitors requires PKR and eIF2α but not

PERK, GCN2 and HRI.

(a,b) Human osteosarcoma U2OS cells stably expressing a GFP-LC3 chimera were transfected with a

control siRNA (siUNR) or with siRNAs targeting PKR (siPKR), PERK (siPERK), GCN2 (siGCN2)

or HRI (siHRI) for 48 h, and then kept in control (Co) conditions or exposed to 1 μM JSI-124, 10 μM

Stattic or 10 μM WP1066 for additional 6 h. Finally, cells were fixed and analyzed by confocal

fluorescence microscopy for the prevalence of cells exhibiting GFP-LC3 dots in the cytoplasm (GFP-

LC3vac cells). Representative images are shown in (a), quantitative data (Mean ± SEM, n = 3;

**p<0.01, Student’s t test, compared to siUNR-transfected cells treated with the same STAT3

inhibitor) in (b). Scale bar = 5 μm.

(c,d) Mouse embryonic fibroblasts (MEFs) of the indicated genotype were maintained in control or

nutrient-free (NF) conditions, or treated with 1 μM thapsigargin (Thaps), 1 μM JSI-124, 10 μM Stattic

or 10 μM WP1066 for 6 h, and then processed for the immunofluorescence-assisted detection of

phosphorylated eIF2α. Representative images are shown in (c), quantitative data (Mean ± SEM, n = 3;

*p<0.05, **p<0.01, Student’s t test, compared to equally treated wild-type MEFs) in (d). Scale bar = 5

μm.

(e,f) U2OS cells were maintained in Co conditions or treated with 0.5 mM palmitate (PAL), 0.5 mM

oleate (OL), 0.5 mM pentedecanoic acid (PA) and 0.5 mM heptadecanoic acid (HA) for 15 h,

followed by the immunoblotting-assisted determination of eIF2α phosphorylation and LC3 lipidation

(e). β actin levels were monitored as a loading control. Numbers illustrate eIF2α phosphorylation and

LC3-II abundance (normalized to total eIF2 and β actin levels, respectively). Alternatively, lysates

were immunoprecipitated with a STAT3-specific antibody and then subjected to immunoblotting for

the detection of PKR and STAT3 (f). Numbers illustrate PKR abundance within immunoprecipitates

(normalized to PKR levels in input lysates).

(g,h) U2OS cells were transfected with siUNR or with siRNAs targeting PKR, PERK or GCN2 for 48

h, and maintained in Co or nutrient-free (NF) conditions, or treated with 10 μM Stattic, 10 μM

WP1066 or 0.5 mM PAL for 15 h. Thereafter, cells were subjected to immunoblotting for the

assessment of PKR, PERK and GCN2 levels (g) or eIF2α and IRS1 phosphorylation (h). β actin levels

were monitored as a loading control.

Supplemental Information - Table S1

Primers employed for site-directed mutagenesis.

Name Sequence Tm K488R_fwd 5'-gctgaccaataaccccaggaacgtgaacttcttca-3'

78.10 °C K488R_rev 5'-tgaagaagttcacgttcctggggttattggtcagc-3'

K495R_fwd 5'-caagaacgtgaacttcttcactaggccgccaattgg-3' 79.33 °C

K495R_rev 5'-ccaattggcggcctagtgaagaagttcacgttcttg-3'

K517R_fwd 5'-tctcgtccaccaccaggcgggggc-3' 78.25 °C

K517R_rev 5'-gcccccgcctggtggtggacgaga-3'

K531R_fwd 5'-ctgacaacgctggctgagaggctcctaggg-3' 80.27 °C

K531R_rev 5'-ccctaggagcctctcagccagcgttgtcag-3'

K551R_fwd 5'-cacatgggctaaattctgcagagaaaacatggctgg-3' 78.19 °C

K551R_rev 5'-ccagccatgttttctctgcagaatttagcccatgtg-3'

K557R_fwd 5'-gcaaagaaaacatggctggcaggggcttctcct-3' 79.14 °C

K557R_rev 5'-aggagaagcccctgccagccatgttttctttgc-3'

K573R_fwd 5'-acaatatcatcgaccttgtgagaaagtatatcttggccctttg-3' 78.73 °C

K573R_rev 5'-caaagggccaagatatactttctcacaaggtcgatgatattgt-3'

K574R_fwd 5'-atcatcgaccttgtgaaaaggtatatcttggccctttgg-3' 78.45 °C

K574R_rev 5'-ccaaagggccaagatataccttttcacaaggtcgatgat-3'

K591R_fwd 5'-tcatgggtttcatcagcagggagcgggagc-3' 78.90 °C

K591R_rev 5'-gctcccgctccctgctgatgaaacccatga-3'

K615R_fwd 5'-ttcagcgagagcagcagagaaggaggggtc-3' 78.90 °C

K615R_rev 5'-gacccctccttctctgctgctctcgctgaa-3'

K626R_fwd 5'-actttcacttgggtggaaagggacatcagtggcaa-3' 78.10 °C

K626R_rev 5'-ttgccactgatgtccctttccacccaagtgaaagt-3'

K631R_fwd 5'-aggacatcagtggcaggacccagatccagtc-3' 78.98 °C

K631R_rev 5'-gactggatctgggtcctgccactgatgtcct-3'

K642R_fwd 5'-ctgtagagccatacaccaggcagcagctgaacaa-3' 79.21 °C

K642R_rev 5'-ttgttcagctgctgcctggtgtatggctctacag-3'

K658R_fwd 5'-gaaatcatcatgggctataggatcatggatgcgaccaa-3' 78.37 °C

K658R_rev 5'-ttggtcgcatccatgatcctatagcccatgatgatttc-3'

K679R_fwd 5'-tctaccccgacattcccagggaggaggca-3' 78.81 °C

K679R_rev 5'-tgcctcctccctgggaatgtcggggtaga-3'

K685R_fwd 5'-gaggaggcatttggaaggtactgtaggcccgag-3' 80.38 °C

K685R_rev 5'-ctcgggcctacagtaccttccaaatgcctcctc-3'

K707R_fwd 5'-ctgccccgtacctgaggaccaagttcatctg-3' 78.98 °C

K707R_rev 5'-cagatgaacttggtcctcaggtacggggcag-3'

K709R_fwd 5'-cccgtacctgaagaccaggttcatctgtgtgacac-3' 80.44 °C

K709R_rev 5'-gtgtcacacagatgaacctggtcttcaggtacggg-3'

Δ585-688_fwd 5'-tggaatgaagggtaccccgagagccaggag-3' 80.27 °C

Δ585-688_rev 5'-ctcctggctctcggggtacccttcattcca-3'

K679A_fwd 5'-ctaccccgacattcccgcggaggaggcatttgga-3' 78.68 °C

K679A_rev 5'-tccaaatgcctcctccgcgggaatgtcggggtag-3'

K658A_fwd 5'-ctgaaatcatcatgggctatgcgatcatggatgcgaccaac-3' 78.16 °C

K658A_rev 5'-gttggtcgcatccatgatcgcatagcccatgatgatttcag-3'

W623A_fwd 5'-caaagaaggaggggtcactttcactgcggtggaaaaggac-3' 80.13 °C

W623A_rev 5'-gtccttttccaccgcagtgaaagtgacccctccttctttg-3'

D661A_fwd 5'-gggctataagatcatggctgcgaccaacatcctgg-3' 80.44 °C

D661A_rev 5'-ccaggatgttggtcgcagccatgatcttatagccc-3'

E680A_fwd 5'-cccgacattcccaaggcggaggcatttggaaag-3' 80.38 °C

E680A_rev 5'-ctttccaaatgcctccgccttgggaatgtcggg-3'

Y674A_fwd 5'-ctccacttgtctacctcgcccccgacattcccaagg-3' 79.97 °C

Y674A_rev 5'-ccttgggaatgtcgggggcgaggtagacaagtggag-3'

M586A_fwd 5'-ttggaatgaagggtacatcgcgggtttcatcagcaaggag-3' 78.08 °C

M586A_rev 5'-ctccttgctgatgaaacccgcgatgtacccttcattccaa-3'

Abbreviations: Tm, melting temperature.

Supplemental Information - Legends to Additional Supplementary Items

Movie S1. Molecular model of the STAT3-PKR interaction.

The core domain of STAT3 (aa 127-688, green) and the catalytic domain of PKR (aa 258-551, red)

are illustrated as mixed surface models and ribbon diagrams. The residues of STAT3 (W623, K658

and E680) and PKR (E375, K380 and F489) that are predicted to be essential for the STAT3-PKR

interaction are depicted in yellow and blue, respectively.

Spreadsheet S1. Panel of autophagy inducers employed in this study.

All inducers of autophagy used in this study are listed, together with molecular weight (MW,

expressed in Da), official International Union of Pure and Applied Chemistry (IUPAC) nomenclature,

Simplified Molecular Input Line Entry Specification (SMILES) notation and chemical structure.

Supplemental Information – Supplemental Experimental Procedures

Chemicals, cell lines and culture conditions. Unless otherwise indicated, chemicals were purchased

from Sigma-Aldrich (St Louis, USA), media and supplements for cell culture from Gibco-Invitrogen

(Carlsbad, USA) and plasticware from Corning Life Sciences (Lowell, USA). Stattic was obtained

from Tocris (Minneapolis, USA). Fatty acids (palmitate, oleate, pentadecanoic acid and hexadecanoic

acid) were obtained by Larodan Fine Chemical (Malmö, Sweden). Human non-small cell lung

carcinoma A549 cells were cultured in DMEM/F12 medium supplemented with 10% fetal calf serum

and 10 mM HEPES buffer, wild-type human osteosarcoma U2OS cells, their GFP-LC3- and FYVE-

RFP-expressing derivatives and MEFs were maintained in DMEM supplemented as above + 1 mM

sodium pyruvate. All cells were maintained in standard conditions (37°C, 5% CO2). Usually, negative

control conditions were generated by administering cells with an equivalent volume of solvent (i.e.,

PBS or DMSO). To achieve serum and nutrient deprivation, cells were cultured in serum-free Earle’s

balanced salt solution (EBSS) (Boya et al., 2005). In this latter case, negative control conditions were

provided by cells maintained in complete culture medium.

siRNAs, plasmids and transfections. siRNAs were reversed transfected by means of the Hiperfect

reagent (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Custom-deisgned

siRNAs were purchased from Sigma-Proligo: siATG5, 5’- UUUCUUCUUAGGCCAAAGGdTdT-3’;

siATG7 , 5’-UUGAGAUUUAGAUCCACUGAdTdT-3’; siBeclin1, 5’-

UUCCGUAAGGAACAAGUCGGdT-3’; siSTAT3, 5’-UUGAAUUCUGCAGAGAGGCTGdTdT-3’;

siPKR, 5’-UAAUAACGUAAGUCUUUCCGdTdT-3’. siRNAs targeting GCN2 and HRI were

purchased from Santa-Cruz Biotechnology (Santa Cruz, USA). An irrelevant siRNA (siUNR) was

employed as a negative control (de La Motte Rouge et al., 2007). Plasmid transfections were carried

out by means of LipofectamineTM 2000 (Invitrogen, Eugene, USA) or jetPEI® (Polyplus Transfection,

New York, USA), as recommended by the manufacturers. Constructs encoding Myc-tagged full-

length (FL) STAT3, and different truncated mutants, including Myc-STAT3(1-722), Myc-STAT3(1-

130), Myc-STAT3(1-465), Myc-STAT3(1-585), Myc-STAT3(585-770) were a generous gift of Dr.

Eugene Chin (Brown University, Rhode Island, USA) (Yuan et al., 2005); the FYVE-RFP-encoding

construct was a kind gift from Prof. Junying Yuan (Harvard Medical School, Boston, USA) (Zhang et

al., 2007); the RFP-LC3 construct was purchased from Addgene (Cambridge, USA); the plasmids

coding for NES-STAT3-GFP and NLS-STAT3-GFP were kind gifts from Prof. Nancy Reich (Stony

Brook University, New York, USA) (Liu et al., 2005); the G3BP-GFP-encoding construct was a kind

gift from Prof. Jamal Tazi (Institut de Génétique Moléculaire de Montpellier, France) (Tourriere et al.,

2003). Point mutations were introduced into the wild-type STAT3 coding sequence, using the primers

reported in Table S1, by means of the QuikChange® site-directed mutagenesis kit (Stratagene, La

Jolla, USA) according to the manufacturer’s instructions.

Immunoblotting and immunoprecipitation. For immunoblotting, approximately 5x105 cells were

washed with cold PBS and lysed following standard procedures (Galluzzi et al., 2010). Forty to fifty

g of proteins were separated according to molecular weight on NuPAGE Novex Bis-Tris 10% pre-

cast gels (Invitrogen) and electrotransferred to PVDF membranes (Bio-Rad, Hercules, USA).

Unspecific binding sites were blocked with 5% non-fat milk in 0.1% Tween20 for 1 h, followed by

overnight incubation at 4 C with primary antibodies specific for the following proteins: actin

(Millipore, Temecula, USA), ATG7 (Sigma-Aldrich), Beclin 1 (Santa-Cruz Biotechnology), P-

eIF2α(Cell Signaling Technology), eIF2α(Cell Signaling Technology), Flag (Sigma-Aldrich),

GAPDH (Millipore), GCN2 (Cell Signaling Technology), HRI (Abcam), P-IRS1 (Cell Signaling

Technology), IRS1 (Cell Signaling Technology), P-JNK (Cell Signaling Technology), JNK (Cell

Signaling Technology), Lc3b (Cell Signaling Technology), Myc (Cell Signaling Technology), p62

(BD Biosciences, San José, USA), P-p70S6K (Cell Signaling Technology), p70S6K (Cell Signaling

Technology), PERK (Cell Signaling Technology); P-PKR (Abcam), PKR (Santa-Cruz

Biotechnology), P-STAT1 (Cell Signaling Technology), STAT1 (Cell Signaling Technology), P-

STAT3 (Cell Signaling Technology) and STAT3 (Cell Signaling Technology). Primary antibodies

were detected with the appropriate horseradish peroxidase-labeled secondary antibodies (Southern

Biotechnologies Associates, Birmingham, UK) and revealed with the SuperSignal West Pico

chemoluminescent substrate (Thermo Fisher Scientific, Rockford, USA) or ECL Plus Western

blotting detection system (GE Healthcare Life Science, Piscataway, USA). The levels of actin or

GAPDH were monitored to ensure equal loading of lanes.

For immunoprecipitation, 1 x 106 cells were lysed as previously described (Criollo et al., 2007).

Proteins were then pre-cleared for 1 h with sepharose G beads (Amersham Pharmacia Biotech,

Uppsala, Sweden) and then incubated overnight at 4 C with antibodies specific for Flag (Sigma-

Aldrich), Myc (Cell Signaling Technology) and STAT3 (Cell Signaling Technology). Lysates were

then incubated with sepharose G beads for additional 2 h at room temperature, followed by extensive

washing and immunoblotting as described above.

Pull-down experiments with recombinant proteins. Recombinant full-length PKR, full-length

STAT3 and two STAT3 variants bearing the point mutations K658A+E680A (#2) and

E680A+W623A (#4) were generated as GST-tagged proteins using the PGEX 6P1 vector (GE

Healthcare Life Science) and optionally liberated from GST using the PreScission Protease (GE

Healthcare Life Science), following the manufacturer’s instructions. Glutathione SepharoseTM 4B

beads (GE Healthcare Life Science) were washed in interaction buffer (100 mM Tris pH 7.4, 300 mM

NaCl, 0.5% NP40, 1% glycerol and Complete™ Protease Inhibitor Cocktail Tablet, from Roche

Applied Science, Indianapolis, USA) and then GST-tagged recombinant proteins (25-100 μg) were

immobilized on 30 μL beads for 2.5 hours at 4°C. Thereafter, beads were washed three times and

diluted 1:10 in interaction buffer (final volume = 500 μL) followed by incubation for 1.5 hours at 4°C

on a roller with 50 μg of untagged recombinant proteins, either in control conditions or in the presence

of Stattic, WP1066 or polyinosinic:polycytidylic acid (polyI:C). Finally, beads were washed three

times and proteins were eluted at 95°C for 5 min in the presence of SDS. Five μL of eluates were

analyzed by immunoblotting.

Immunofluorescence microscopy. Cells were fixed with 4% PFA for 15 min at room temperature,

and permeabilized with 0.1% TritonX-100 for 10 min (Boehrer et al., 2008). Non-specific binding

sites were blocked with 5% bovine serum in PBS, followed by staining with primary antibodies

recognizing Lc3b (MBL International, Woburn, USA), P-eIF2α(Cell Signaling Technology), p62

(BD Biosciences) or PKR (Santa-Cruz Biotechnology) overnight at 4 C. Revelation was carried out

with appropriate AlexaFluorTM conjugates (Molecular Probes-Invitrogen). Ten μM Hoechst 33342

(Molecular Probes-Invitrogen) was employed for nuclear counterstaining. Fluorescence and confocal

fluorescence microscopy assessments were performed on an IRE2 microscope (Leica Microsystems)

equipped with a DC300F camera and with an LSM 510 microscope (Carl Zeiss, Jena, Germany),

respectively. The percentage of co-localization was quantified by means of ImageJ.

Electron microscopy. Cells were fixed for 1 h at 4 C in 1.6% glutaraldehyde in 0.1 M Sörensen

phosphate buffer (pH 7.3), washed and fixed in aqueous 2% osmium tetroxide, stained in 2% uranyl

acetate in 30% methanol and finally embedded in Epon. Electron microscopy was performed by

means of a Tecnai G2 spirit electron microscope (FEI, Eindhoven, Netherlands), at 80kV, on ultrathin

sections (80 nm) stained with lead citrate and uranyl acetate.

Automated high-content microscopy. Eight x 103 U2OS cells stably expressing GFP-LC3 or their

wild-type counterparts were seeded in 96-well imaging plates (BD Falcon, Sparks, USA) 24 h before

stimulation. Cells were treated with the agents from the ICCB known bioactive library (Harvard,

USA), the NCI mechanistic set library and the autophagy inducers library (Tocris, USA) for 15 h or 2

h. Subsequently, cells were fixed with 4% PFA and - when required - immunostained with antibodies

recognizing P-eIF2α(Cell Signaling Technology). Ten μM Hoechst 33342 was employed for nuclear

counterstaining. Images were acquired using a BD pathway 855 automated microscope (BD Imaging

Systems, San José, USA) equipped with a 40X objective (Olympus, Center Valley, USA) coupled to a

robotized Twister II plate handler (Caliper Life Sciences, Hopkinton, USA). Images were analyzed for

the presence of GFP-LC3 puncta in the cytoplasm by means of the BD Attovision software (BD

Imaging Systems), or for eIF2α phosphorylation with ImageRail (Semantic Biology, USA), as

previously described (Millard et al., 2011). Statistical analysis were implemented on the R software

(http://www.r-project.org/).

Docking model and mutagenesis. Two x-ray structures of PKR complexed with eIF2α (PDB codes

2A1A and 2A19) (Dar et al., 2005) and two monomers of STAT3 (PDB code 3CWG) (Ren et al.,

2008) were isolated to run 4 docking experiments. Dockings were performed using ClusPro (Comeau

et al., 2004). Top-ranked solutions were visually inspected and filtered to select best complexes. The

top-scored docking model was visualized in a static fashion or in the form of a 3D movie by means of

the PyMol software (freely available at http://www.pymol.org).

Stat3 conditional knockout mice and in vivo experiments. c57BL/6 male mice were maintained in

pathogen-free conditions, and all experiments were carried out (upon approval by local ethical

committees) in compliance with Directive EU 63/2010 and following the Federation of European

Laboratory Animal Science Association (FELASA) guidelines. Hepatocyte-specific Stat3 knockout

mice have previously been described (Sakamori et al., 2007). Mice were housed with 12 h light/dark

cycles and normally had access to food and water ad libitum. Upon sacrifice, organs were removed

and immediately frozen in liquid nitrogen. For immunoblotting, tissues were lysed in 1% NP-40, 0.5%

sodium deoxycholate, 0.1% SDS supplemented with protease and phosphatase inhibitors (both from

Nacalai Tesque, Kyoto, Japan). Palm oil (1 mL) or 35% albumin in PBS (negative control) were

administered by oral gavage to 6-week old animals. Alternatively, mice were starved for the indicated

time.

Autophagy quantification by fluorescence microscopy. Two different methods for quantifying

autophagy-associated markers by fluorescence microscopy were used in this study, depending on the

stability of GFP-LC3 or FYVE-RFP expression. In the case of cells stably transfected with GFP-LC3-

or FYVE-RFP-encoding constructs (which, upon selection, result in the homogenous expression of

GFP-tagged LC3 or RFP-tagged FYVE by all cells), the percentage of cells exhibiting GFP-LC3+ dots

was quantified. Conversely, in the case of cells transiently transfected with GFP-LC3- or FYVE-RFP-

encoding plasmids, the average number of cytoplasmic GFP-LC3+ or FYVE-RFP+ dots/cell among

fluorescent cells was calculated. Thus, the number of GFP-LC3+ or FYVE-RFP+ dots in the whole

microscopic field was quantified and then divided by the total number of fluorescent cells in the same

field. This approach allowed us to avoid an underestimation of autophagic responses due to non-

transfected cells and/or to transfected cells that would fail to express the fluorescent reporters. In this

case, the average number of GFP-LC3+ or FYVE-RFP+ dots observed in cells that had been

maintained in control conditions (5-10 dots) was used as a threshold setting, representing baseline

levels of autophagy.

Densitometry. Densitometry was performed on n = 3 independent immunoblots by means of the

open-source software ImageJ (available at http://rsbweb.nih.gov/ij/download.html). Densitometry data

were normalized: to the total levels of the protein of interest for phospho-neoepitope-specific

antibody-based immunoblotting, to the levels of β actin or GADPH (monitored as a loading control)

for standard immunoblotting, or to the total levels of the protein of interest in input lysates, for

immunoprecipitation-related immunoblotting.

Statistical procedures. Unless otherwise mentioned, experiments were performed in triplicate and

repeated at least twice. Data were analyzed using the GraphPad Prism 5 software and statistical

significance was assessed by means of two-tailed Student’s t or ANOVA tests, appropriate.

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