1. 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, Guido Kroemer 
Molecular Cell 
Volume 48, Issue 5, Pages (December 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 Chemical STAT3 Inhibitors Are Potent Inducers of Autophagy
(A) A high-content screening identified JSI-124 as an autophagy inducer in human osteosarcoma U2OS cells stably expressing GFP-LC3. Z scores of cytoplasmic GFP-LC3+ dot counts, propidium iodide (PI) intake, and nuclear area (as monitored by Hoechst staining) are plotted, in decreasing order of cytoplasmic GFP-LC3+ dots.
(B) Representative images of U2OS cells stably expressing GFP-LC3 upon exposure to three distinct STAT3 inhibitors (0.5 μM JSI-124, 10 μM Stattic, 10 μM WP1066) for 6 hr. Scale bar, 10 μm.
(C and D) Dependency of Stattic-induced autophagy on ATG5. U2OS cells were transfected with a control siRNA (siUNR) or with an ATG5-targeting siRNA (siATG5) for 48 hr, followed by the administration of 10 μM Stattic for 6 hr. Representative images are shown in (C) (scale bar, 5 μm) and the percentage of cells bearing cytoplasmic GFP-LC3+ dots (GFP-LC3VAC cells) in (D). The inset depicts the efficiency of siRNA-mediated ATG5 downregulation, as determined by immunoblotting. Mean ± SD, n = 3; ∗∗p < 0.01, Student's t test, as compared to equally treated siUNR-transfected cells. See also Figure S1.
(E) Induction of FYVE-RFP+ puncta by STAT3 inhibitors. U2OS cells stably expressing a FYVE-RFP construct were treated as in (B), followed by the quantification of cells exhibiting FYVE-RFP+ dots. Representative images (scale bar, 5 μm) and quantitative results (mean ± SD, n = 3; ∗∗p < 0.01, Student's t test, compared to untreated cells) are shown.
(F) Ultrastructural evidence of autophagosome induction by Stattic in wild-type U2OS cells. Scale bar, 1 or 2 μm, as indicated.
(G and H) Effect of Stattic on the autophagic flux. Wild-type (G) or GFP-LC3-expressing (H) U2OS cells were treated with 10 μM Stattic and/or 50 nM bafilomycin A1 (BafA1) for 6 hr (or the indicated time), followed by immunoblotting for the detection of LC3 lipidation and p62 degradation, or fluorescence microscopy for the quantification of cells exhibiting cytoplasmic GFP-LC3+ dots. Numbers illustrate normalized LC3-II and p62 abundance. Mean ± SD, n = 3; ∗∗p < 0.01, ANOVA test, compared to the administration of BafA1 alone. See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Autophagy Inhibition by STAT3 In Vitro
(A–F) Autophagy induction by STAT3 knockdown and autophagy inhibition by STAT3 overexpression. Wild-type (WT) (A and D) or GFP-LC3-expressing (B, C, E, and F) human osteosarcoma U2OS cells were transfected with control (siUNR) or STAT3-specific (siSTAT3) siRNAs (A–C) for 48 hr. Alternatively, U2OS cells were transfected with an empty vector (pBabe) or with a construct encoding WT STAT3 (D–F) for 24 hr. Where indicated, cells were then treated with 1 μM rapamycin (Rapa) or 1 μM thapsigargin (Thaps) or kept in nutrient-free (NF) conditions for 4 hr. STAT3 levels, LC3 lipidation, and p70S6K phosphorylation were then determined by immunoblotting (A and D), and the proportion of cells exhibiting cytoplasmic GFP-LC3+ dots (GFP-LC3vac) was quantified (B, D–F). Numbers illustrate normalized p70S6K phosphorylation or LC3-II abundance. Representative images (A, B, D, and E; scale bar, 5 μm) and quantitative results (C and F, mean ± SD, n = 3; ∗p < 0.05, Student's t test, compared to equally treated siUNR- or pBabe-transfected cells) are shown. Co, control conditions.
(G and H) Effects of STAT3 depletion and overexpression on FYVE-RFP+ puncta. U2OS cells stably expressing FYVE-RFP were treated with Rapa or Thaps or kept in NF conditions as in (A)–(F). Representative images (G; scale bar, 5 μm) and quantitative data (H, mean ± SD, n = 3; ∗p < 0.05, ∗∗p < 0.01, Student's t test, compared to pBabe-transfected cells) are shown.
(I and J) Inhibition of autophagy by cytoplasmic STAT3. U2OS cells were cotransfected with an RFP-LC3-encoding plasmid and with a control GFP-encoding vector (pcDNA3-GFP) or with constructs coding for NES-STAT3-GFP or NLS-STAT3-GFP chimeras for 24 hr and then treated as in (A)–(F). Representative images (I; scale bar, 10 μm) and quantitative results (J, mean ± SD, n = 3; ∗∗p < 0.01, Student's t test, compared to untreated RFP-LC3-expressing cells) are shown. Co, control conditions. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 STAT3-Mediated Inhibition of Autophagy In Vivo
(A) Immunoblotting for the detection of LC3 lipidation and p62 degradation in Stat3FL/FL or Stat3Δ/Δ mouse embryonic fibroblasts (MEFs) transfected or not with an empty vector only (pBabe) or with wild-type (WT) STAT3- or STAT3Y705F-encoding constructs and exposed or not to nutrient-free (NF) conditions for 15 hr. Numbers illustrate normalized LC3-II or p62 abundance.
(B) Immunoblotting for the detection of LC3 lipidation, p62 degradation, and p70S6K, JNK, and eIF2α phosphorylation in lysates from the indicated tissues of Stat3FL/FL and Stat3hepΔ/Δ mice. Results are representative of three experiments. Numbers illustrate normalized phosphorylation status or protein (LC3-II or p62) abundance.
(C–F) Histopathological quantification of autophagy in tissues from Stat3FL/FL and Stat3hepΔ/Δ mice. The indicated tissues were subjected to immunofluorescence detection of Lc3b (C and D) or p62 (E and F), followed by the quantification of cells exhibiting cytoplasmic Lc3b+ dots (Lc3bvac) or p62 aggregation (p62vac). Representative microphotographs (C and E; scale bar, 10 μm) and quantitative data (D and F; mean ± SD, n = 3; ∗p < 0.05, Student's t test, compared to the corresponding Stat3FL/FL tissue) are shown.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Interaction of STAT3 with PKR
(A) Autophagy-relevant interactome of STAT3. Based on bioinformatic data mining, STAT3 interactors were compared with core autophagy-modulatory proteins. Overlapping proteins are highlighted in the rectangle. See also Figure S4.
(B) STAT3 inhibitors impact on different signaling pathways. WT human osteosarcoma U2OS cells were exposed to the indicated agents for 2 hr and then subjected to immunoblotting for the detection of p70S6K, eIF2α, and STAT3 phosphorylation or LC3 lipidation. Results are representative of three experiments. See also Figure S5.
(C) Induction of PKR phosphorylation by STAT3 inhibitors. WT U2OS cells were treated with 10 μM Stattic or 10 μM WP1066 for 15 hr, and cell lysates were processed for the immunoblotting-assisted detection of PKR, as such, or following treatment with λ-phosphatase. Numbers illustrate the normalized phosphorylation status of PKR.
(D) Coimmunoprecipitation of STAT3 with PKR. U2OS cells were maintained in control conditions, administered with 10 μM Stattic, or kept in nutrient-free (NF) conditions for 15 hr, followed by lysis, immunoprecipitation with a STAT3-specific antibody, and revelation of PKR by immunoblotting. Numbers illustrate normalized PKR abundance. See also Figure S6.
(E) Molecular model of the STAT3-PKR interaction. The upper and lower panels show surface models and ribbon diagrams, respectively. The insets highlight residues that may contribute to the interaction surface. See also Movie S1.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Interaction between the SH2 Domain of STAT3 and the C Terminus of PKR
(A) The C terminus of PKR interacts with STAT3. Flagged variants of full-length (FL) PKR, its C terminus (Cter), or its N terminus (Nter) were transfected into human osteosarcoma U2OS cells, followed by immunoprecipitation with an anti-Flag antibody and detection of endogenous STAT3 (upper panel) or flagged proteins (lower panel).
(B) Identification of the STAT3 domain that interacts with PKR. Myc-tagged full-length (FL) STAT3 or Myc-tagged STAT3 variants corresponding to the indicated residues were transfected alone or together with flagged PKR into U2OS cells, followed by immunoprecipitation of flagged proteins and detection of Myc-tagged or flagged proteins. Asterisks identify specific bands. See also Figure S7.
(C) Effects of single amino acid substitutions on the STAT3-PKR interaction. Myc-tagged STAT3 variants bearing the indicated point mutations were cotransfected with flagged PKR into U2OS cells, followed by immunoprecipitation of flagged proteins and detection of Myc-tagged proteins by immunoblotting. E680A and W623A (red) decrease the interaction between STAT3 and PKR. Numbers illustrate normalized STAT3 abundance within immunoprecipitates.
(D) Effects of dual amino acid exchanges on the STAT3-PKR interaction. U2OS cells were cotransfected with flagged PKR plus distinct Myc-tagged STAT3 variants (FL, full-length; #1, K679A+W623A; #2, K658A+E680A; #3, K658A+K679A; #4, E680A+W623A), followed by immunoprecipitation and immunoblotting as in (C). Numbers illustrate normalized STAT3 abundance within immunoprecipitates.
(E and F) Inhibition of autophagy by STAT3 mutants. Constructs encoding the STAT3 mutants described in (B) and (D) were transfected into U2OS cells stably expressing GFP-LC3 together with a plasmid coding for RFP (to visualize transfected cells) for 24 hr, followed by a 4 hr long culture in control or nutrient-free (NF) conditions. Thereafter, the number of GFP-LC3+ dots per transfected cell was quantified by fluorescence microscopy. Mean ± SD, n = 3; ∗p < 0.05, ∗∗p < 0.01, Student's t test, compared to cells transfected with an empty vector (EV) and subjected to NF conditions. The inset in (F) depicts p62 degradation as monitored by immunoblotting. Numbers illustrate normalized p62 abundance.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Essential Role of PKR in STAT3 Inhibition-Induced Autophagy
(A–D) Human osteosarcoma U2OS cells stably expressing a GFP-LC3 (A and B) or FYVE-RFP (C and D) chimera were transfected with control (siUNR) or PKR-specific (siPKR) siRNAs for 48 hr, followed by the administration of 10 μM Stattic or 10 μM WP1006 for 6 hr and fluorescence microscopy for the quantification of cells exhibiting GFP-LC3+ puncta (GFP-LC3VAC cells) and the abundance of FYVE-RFP+ dots. Representative images (A and D; scale bar, 10 μm) and quantitative data (B and D, mean ± SD, n = 3; ∗∗p < 0.01, Student's t test, compared to equally stimulated siUNR-transfected cells) are shown.
(E–G) U2OS cells were transfected with siUNR or siPKR for 48 hr, followed by treatment with 1 μM thapsigargin (Thaps) or 10 μM Stattic for 2 hr and immunoblotting (E) for the determination of eIF2α phosphorylation and PKR levels or immunofluorescence coupled to single-cell profiling (F and G). Numbers in (E) illustrate normalized eIF2α phosphorylation. In (G), the percentage of cells displaying eIF2α phosphorylation is indicated. See also Figure S8.
(H and I) Wild-type (WT) mouse embryonic fibroblasts (MEFs) or MEFs bearing the S51A eIF2α mutation were transfected with a GFP-LC3-encoding construct for 24 hr, followed by administration of 10 μM Stattic for 6 hr and fluorescence microscopy for the quantification of GFP-LC3+ dots. Representative images (H; scale bar, 10 μm) and quantitative data (I, mean ± SD, n = 3; ∗p < 0.05, Student's t test, compared to equally treated WT MEFs) are reported.
(J) U2OS cells were transfected with siUNR or with a siRNA targeting eIF2α (sieIF2α) and then left untreated or treated with 10 μM Stattic for 6 hr, followed by immunoblotting for the detection of eIF2α levels, p62 degradation, and LC3 lipidation. Numbers illustrate normalized LC3-II or p62 abundance.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
PKR-Mediated eIF2α Phosphorylation in STAT3 Inhibitor-Induced Autophagy
(A) Inhibition of eIF2α phosphorylation by STAT3Y570F overexpression. Human osteosarcoma U2OS cells were transfected with constructs encoding wild-type (WT) STAT3 or the STAT3 Y570F mutant for 24 hr, followed by the administration of 1 μM thapsigargin (Thaps) or culture in nutrient-free (NF) conditions for 2 hr. STAT3 levels and the phosphorylation of eIF2α were then detected by immunoblotting. Numbers illustrate the normalized phosphorylation status of eIF2α.
(B) Inhibition of eIF2α phosphorylation by reintroduction of STAT3 into STAT3-deficient cells. Stat3Δ/Δ mouse embryonic fibroblasts (MEFs) were transfected with a control vector (pBabe) or with plasmid encoding WT STAT3 for 24 hr, followed by administration of Thaps or culture in NF conditions as in (A). Finally, STAT3 levels and eIF2α phosphorylation were detected by immunoblotting. Numbers illustrate the normalized phosphorylation status of eIF2α.
(C and D) Identification of palmitate (PAL) as a PKR-dependent inducer of autophagy. Quantile-quantile (Q-Q) plots of autophagy-inducing compounds evaluated for their ability to induce GFP-LC3 aggregation (C) and eIF2α phosphorylation (D) in U2OS cells stably expressing GFP-LC3 or in WT U2OS cells, respectively, in both cases upon transfection with control (siUNR) or PKR-targeting (siPKR) siRNAs. Cells were treated with autophagy-inducing chemicals for 16 (C) or 2 hr (D), followed by automated imaging. Q-Q plots were generated with the R software package. See also Spreadsheet S1.
(E) Palmitate-induced eIF2α and PKR phosphorylation in vivo. c57BL/6 mice were fed with 1 mL 35% albumin (negative control) or palm oil, or were starved for the indicated time. Then, mice were sacrificed and their livers were rapidly frozen in liquid nitrogen. Liver lysates were subjected to immunoblotting for the detection of LC3 lipidation and phosphorylation of eIF2α and PKR. Numbers illustrate the normalized phosphorylation status of eIF2α and PKR or normalized LC3-II abundance.
(F) Palmitate-induced dissociation of PKR from STAT3. U2OS cells transfected with control (siUNR) or PKR-targeting (siPKR) siRNAs for 48 hr were treated with 0.5 mM PAL or 10 μM Stattic or maintained in NF conditions for 2 hr, and then processed for the immunoblotting-assisted detection of PKR total levels, LC3 lipidation, and JNK phosphorylation. Numbers illustrate normalized JNK phosphorylation and LC3-II abundance.
(G) Palmitate-induced dissociation of PKR from STAT3. U2OS cells were treated with 0.5 mM PAL or 1 μM Stattic for 15 hr followed by immunoprecipitation with anti-STAT3 antibodies and immunoblotting-assisted detection of PKR. Numbers illustrate normalized PKR abundance within immunoprecipitates.
(H) STAT3-mediated inhibition of palmitate-induced autophagy. U2OS cells were transiently transfected with an empty vector (pBabe) or with plasmids encoding STAT3 or the STAT3Y705F mutant for 24 hr, followed by the administration of 0.5 mM PAL for 2 hr. Finally, cell lysates were analyzed by immunoblotting for STAT3 levels or PKR and eIF2α phosphorylation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions