Journal: OncoTargets and Therapy

Article Title:

Mitofusin1 Is a Major Mediator in Glucose-Induced Epithelial-to-Mesenchymal Transition in Lung Adenocarcinoma Cells

doi: 10.2147/ott.s238714

Figure Lengend Snippet: Figure 5 High glucose led to the induction of autophagy in the LAD cell line through MFN1. (A and B) Expression of LC3B-II, BECN-1 and SQSTM1 in A549 cells from the NG+NC, HG+NC, and HG+siMFN1 groups. (C) Cells were transfected with the eGFP-mRFP-LC3 plasmid and exposed to different concentrations of glucose for 24 h. Yellow and red dots refer to autolysosomes and autophagosomes respectively. Scale bar = 50 µm. Data shown are mean ± SEM. *P < 0.05, **P < 0.01 vs NG+NC group. #P < 0.05, ##P < 0.01 vs HG+NC group (n = 6). Abbreviations: LAD, lung adenocarcinoma; NC, non-targeted control; NG, normal glucose; HG, high glucose. MFN1, mitofusin1; siMFN1, small interfering RNA of MFN1; LC3B, microtubule-associated proteins 1A/1B light chain 3B; BECN, beclin-1; SQSTM, sequestosome 1; eGFP, enhanced green fluorescent protein; mRFP, monomer red fluorescent protein.

Article Snippet: Materials Antibodies against SQSTM1 (PB0458, 1:400) was obtained from Boster Biological Technology Co. Ltd. Antibody against MFN1 (ab107129), LC3B (ab48394), Pink (ab23707), Parkin (ab77924) and Snail (ab53519) were purchased from Abcam.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Small Interfering RNA

Journal: Frontiers in immunology

Article Title: Activator-Mediated Pyruvate Kinase M2 Activation Contributes to Endotoxin Tolerance by Promoting Mitochondrial Biogenesis.

doi: 10.3389/fimmu.2020.595316

Figure Lengend Snippet: FIGURE 2 | Mitochondrial biogenesis is induced in PKM2-activated macrophages. (A) Mitochondrial mass was assessed by measuring the uptake of NAO by using flow cytometry (n=3). (B) The autophagy level of RAW264.7 cells after stimulation with TEPP-46 was assessed by measuring the protein expression of p62 and LC3 using Western blotting (n=3). (C) The microstructure of RAW264.7 cells stimulated with TEPP-46 was measured using transmission electron microscopy (TEM) (n=3). (D) mtDNA copy number was determined by measuring MTND1 relative to B2M using RT-PCR in RAW264.7 cells transducted with a control siRNA (con siRNA) or siPKM2 (n=4). (E) Western blot analysis of mtTFA in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 for different times (n=3). (F) The mitochondrial OCR was measured in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 by using a Seahorse XF96e Extracellular Flux analyzer. Dashed vertical lines indicate the addition of 1 mM oligomycin (Oligo), 0.5 mM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 mM rotenone plus 1 mM antimycin A (Rot/Ant) (n=3). (G–I) Quantitative analysis of basal respiration, ATP-linked respiration and maximal respiration in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 for different times (n=3). *p < 0.05.

Article Snippet: We used antibodies against PKM2 (Cell Signaling, #4053, 1:1,000), PKM1 (Cell Signaling, #7067, 1:1,000), PGC-1a (Cell Signaling, #2178, 1:1,000), PGC-1b (Abcam, ab176328, 1:1,000), p62 (Cell Signaling, #16177, 1:1,000), LC3 (Abcam, ab192890, 1:1,000), mtTFA (Abcam, ab252432, 1:1,000), NRF1 (Abcam, ab221792, 1:1,000), NRF2 (Abcam, ab137550, 1:1,000), p-AMPK (Cell Signaling, #4186, 1:500), AMPK (Cell Signaling, #4150, 1:1,000), SIRT1 (Abcam, ab189494, 1:1,000), p-Akt (Abcam, ab38449, 1:500), Akt (Abcam, ab8805, 1:500), p-PI3K (Cell Signaling, #17366, 1:1,000), PI3K (Cell Signaling, #4255, 1:1,000), and GAPDH (Santa Cruz, sc365062, 1:1,000).

Techniques: Cytometry, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: Primers

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: BGJ398 reduces tumor burden in an oncogene-driven murine model of CCA. A, FGFRs are up-regulated in a YAP-driven murine model of CCA. mRNA expression of Fgfr1, Fgfr2, Fgfr3, and Fgfr4 using qPCR and RNA sequencing of mouse tumors compared with adjacent liver. Thr dashed line represents adjacent liver, which served as the control. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, representative immunostaining images for phospho-FRS2 in vehicle (Veh)- and BGJ398-treated animals. Scale bars: 50 μm. C, liver appearance of mice after intrabiliary injection of myr-Akt and YapS127A Sleeping Beauty transposon-transposase complexes coupled with lobar bile duct ligation and daily intraperitoneal injections of IL-33 (1 μg for 3 days) with (right panel) and without (left panel) BGJ398 treatment (12.5 mg/kg/day) for 2 weeks. D, ratio of tumor weight to liver weight of the ligated lobe expressed as a percentage in vehicle (n = 9)- and BGJ398 (n = 6)-treated animals. *, p < 0.05. E, number of nodules in vehicle (n = 8)- and BGJ398 (n = 6)-treated animals with tumors. *, p < 0.05. F, representative photomicrographs of hematoxylin and eosin-stained tumor sections and adjacent liver are shown in vehicle- and BGJ398-treated animals. Scale bars: 100 μm. G, apoptotic cells were quantified by counting TUNEL-positive nuclei in five random microscopic fields (×20) using a fluorescent microscope. Shown are images (top panel) and the percentage of TUNEL-positive cells (bottom panel) in representative sections of vehicle- and BGJ398-treated animals. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. H, immunofluorescence images (top panel) and percentage of Ki67-positive cells (bottom panel) in representative sections of vehicle- and BGJ398-treated animals. Mean ± S.E. are depicted for n = 3. Representative immunofluorescence experiments included tissue sections from three mice from each group. Scale bars: 50 μm.

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, RNA Sequencing Assay, Immunostaining, Injection, Ligation, Staining, TUNEL Assay, Microscopy, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: BGJ398 inhibits YAP activation in a PDX model of CCA. A, representative immunostaining images for nuclear YAP (brown staining) in YAP-positive (PDX1) and YAP-negative (PDX2) tumors. B, mRNA expression of Yap, Ctgf, and Sox4 in PDX1 and PDX2. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. C, mRNA expression of Fgfr1, Fgfr2, Fgfr3, and Fgfr4 in PDX1 and PDX2. Mean ± S.E. are depicted for n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, tumor weight in mg of PDX1 (left panel) and PDX2 (right panel) mice treated for 2 weeks with vehicle (n = 5) or 12.5 mg/kg/day BGJ398 (n = 5). *, p < 0.05. E, representative photomicrographs of hematoxylin and eosin-stained tumors in vehicle- and BGJ398-treated PDX1 (left panel) and PDX2 (right panel) animals. Scale bars: 1 mm. F, immunofluorescence images of CK-19 staining (to outline the biliary epithelium) and YAP in tissue sections obtained from PDX1 mice treated with vehicle or 12.5 mg/kg BGJ398 for 2 weeks. G, mRNA expression of Yap, Ctgf, Sox4, and Mcl-1 in PDX1 animals treated with vehicle or 12.5 mg/kg/day BGJ398 for 2 weeks. Mean ± S.E. are depicted for n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001. H, fluorescence images (left panel) and percentage of TUNEL-positive cells (right panel) in representative sections of vehicle- and BGJ398-treated PDX1 (top panel) and PDX2 (bottom panel) animals. Apoptotic cells were quantified by counting TUNEL-positive nuclei in five random microscopic fields (×20) using a fluorescent microscope. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. I, immunofluorescence images (left panel) and percentage of Ki67-positive cells (right panel) in representative sections of vehicle- and BGJ398-treated PDX1 (top panel) and PDX2 (bottom panel) animals. Mean ± S.E. are depicted for n = 3. Representative immunofluorescence experiments included tissue sections from three mice from each group. Scale bars: 50 μm.

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Immunostaining, Staining, Expressing, Immunofluorescence, Fluorescence, TUNEL Assay, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

doi: 10.1016/j.jbc.2024.107206

Figure Lengend Snippet: The expression of DYRK3 and p62 was higher in various melanoma cells lines. A , the median expression level of the DYRK3 and p62 genes in normal and melanoma cancer tissues was analyzed by GEPIA (TPM: Transcripts per Million). According to TCGA data, the analysis indicated that the expression level of DYRK3 and p62 in melanoma cancer tissues (n = 461) was enhanced compared to normal tissues (n = 558). GEPIA, Gene Expression Profiling Interaction Analysis ( http://gepia.cancer-pku.cn ). B and C , microarray data of DYRK3 and p62 from the U.S. National Cancer Institute ( http://dtp.nci.nih.gov/mtweb/targetdata ). The expression of DYRK3 (Exp. ID: 30355; https://dtp.cancer.gov/mtweb/targetinfo?moltid=GC30356&moltnbr=30355 ) and p62 (SQSTM1, Exp. ID: 9233; https://dtp.cancer.gov/mtweb/targetinfo?moltid=GC18635&moltnbr=9233 ) is consistently higher in melanoma cell lines than in the control. D , immunoblot analyses for DYRK3 levels in three control cell lines (HeLa, RWPE-1, and HEK293) and seven melanoma cancer cells were shown. E , quantification of DYRK3 expression levels from the blots in ( D ). All data represent the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). F , immunoblot analyses for p62 levels in three control cell lines (HeLa, RWPE-1, and HEK293) and seven melanoma cancer cells were shown. G , quantification of p62 expression levels from the blots in ( D ). All data represent the mean ± standard deviation of three independent experiments (∗ p < 0.05). DYRK, dual-specificity tyrosine-phosphorylation-regulated kinase; HEK, human embryonic kidney cells; TCGA, The Cancer Genome Atlas.

Article Snippet: Rabbit phospho-S207-p62 antibodies (OAAB16350) were purchased from Aviva Systems Biology.

Techniques: Expressing, Gene Expression, Microarray, Control, Western Blot, Standard Deviation, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

doi: 10.1016/j.jbc.2024.107206

Figure Lengend Snippet: DYRK3 interacts with p62. A and B , HEK293 ( A ) or SH-SY5Y cells ( B ) were transfected for 24 h with a plasmid encoding Myc-p62 or Flag-DYRK3 alone or in combination. Cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody. Hsp90 served as a loading control. C – F , where indicated, cell lysates prepared from HEK293 ( C ), SH-SY5Y ( D ), SK-Mel-28 ( E ), or UACC257 ( F ) cells were immunoprecipitated with anti-DYRK3, anti-p62, or preimmune IgG, followed by immunoblotting with the indicated antibody. G and H , representative confocal images of immunostaining of HEK293 ( G ) or SK-Mel-28 cells ( H ) stably expressing both Flag-DYRK3 ( red ) and Myc-p62 ( green ). Nuclei were counterstained with DAPI ( blue ). The scale bar represents 20 μm. I , the diagram outlines WT p62 (p62-WT) and its deletion mutants, delineating various domains such as the N-terminal Phox1 and Bem1p (PB1) domain, zinc finger (ZZ) domain, tumor necrosis factor receptor-associated factor 6 (TRAF6)-binding (TB) motif, LC3-interacting region (LIR) domain, and the C-terminal ubiquitin-associated (UBA) domain. Summarized results from co-IP assays exploring DYRK3 and p62 interaction are displayed on the left and right , with 'x' indicating no binding and 'o' indicating binding. J , HEK293 cells were transfected for 24 h with a plasmid encoding Myc-tagged p62-WT, one of its deletion mutants, or Flag-DYRK3 alone or in combination. Cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody. Hsp90 served as a loading control. DAPI, 4′,6-diamidino-2-phenylindole; DYRK, dual-specificity tyrosine-phosphorylation-regulated kinase; HEK, human embryonic kidney cells; co-IP, coimmunoprecipitation.

Article Snippet: Rabbit phospho-S207-p62 antibodies (OAAB16350) were purchased from Aviva Systems Biology.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Immunostaining, Stable Transfection, Expressing, Binding Assay, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

doi: 10.1016/j.jbc.2024.107206

Figure Lengend Snippet: DYRK3 phosphorylates p62 at S207 and T269 residues in the TB domain. A , recombinant purified DYRK3 protein and commercial bovine serum albumin (BSA) as a control were stained with Coomassie blue dyes. B , after HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62, cell lysates (∼1000 μg of protein) were immunoprecipitated with an anti-Myc antibody. Where specified, the samples were mixed with bacterially expressed WT DYRK3 (DYRK3-WT) or its kinase-inactive mutant (DYRK3-KM), incubated for 30 min at 30 ° C with the kinase buffer and [γ- 32 P]ATP, resolved by SDS-PAGE, and analyzed by autoradiography. Proper expression of transiently expressed p62 in cell extracts was verified by immunoblotting with an anti-Myc antibody (Input). C , HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62-WT or one of its deletion mutants (D1-D7), followed by immunoprecipitation with an anti-Myc antibody. The anti-Myc immunocomplexes as a substrate were mixed with bacterially expressed DYRK3-WT, incubated for 30 min at 30 ° C with the kinase buffer and [γ- 32 P]ATP, resolved by SDS-PAGE, and analyzed by autoradiography. D , HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62-WT, Myc-p62-T269A, or Myc-p62-S207/T269A, followed by immunoprecipitation with an anti-Myc antibody. The anti-Myc immunocomplexes as a substrate were mixed with bacterially expressed DYRK3-WT, incubated for 30 min at 30 ° C with the kinase buffer and [γ- 32 P]ATP, resolved by SDS-PAGE, and analyzed by autoradiography. E , quantification of the protein band densities in ( D ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). F , HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62-WT, Myc-p62-S207/T269A, or Flag-DYRK3 alone or in combination. Cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with anti-pT269/S272-p62 or anti-pS207-p62 antibodies. G , quantification of the protein band densities in ( F ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). DYRK, dual-specificity tyrosine-phosphorylation-regulated kinase; HEK, human embryonic kidney cells; TB, TRAF6-binding.

Article Snippet: Rabbit phospho-S207-p62 antibodies (OAAB16350) were purchased from Aviva Systems Biology.

Techniques: Recombinant, Purification, Control, Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Incubation, SDS Page, Autoradiography, Expressing, Western Blot, Standard Deviation, Phospho-proteomics, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

doi: 10.1016/j.jbc.2024.107206

Figure Lengend Snippet: DYRK3-mediated phosphorylation of p62 enhances its binding to TRAF6. A , after HEK293 cells were transfected for 24 h with a plasmid encoding V5-TRAF6 or/and Flag-DYRK3, cell lysates were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. Hsp90 served as a loading control. B , HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62, V5-TRAF6, or Flag-DYRK3 alone or in combination. Cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody. C , quantification of the protein band densities in ( B ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗ p < 0.01). D , where specified, HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62, V5-TRAF6, Flag-DYRK3-WT, or Flag- DYRK3-KM alone or in combination. Cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody. E , quantification of the band densities in ( D ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001, ∗∗ p < 0.01). F , after HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62, V5-TRAF6, or Flag-DYRK3-WT alone or in combination, the cells were then left untreated or treated for 6 h with 1 μM of GSK-626616. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with the indicated antibody. G , quantification of the band densities was performed in ( F ). All data represent the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). H , after HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62-WT, Myc-p62-T269A, V5-TRAF6, or Flag-DYRK3-WT alone or in combination, cell lysates were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. I , quantification of the protein blots in ( H ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗ p < 0.01, ∗ p < 0.05). J , after HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62-WT, Myc-p62-T269E, or V5-TRAF6 alone or in combination, cell lysates were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. K , quantification of the band densities in ( J ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). DYRK, dual-specificity tyrosine-phosphorylation-regulated kinase; HEK, human embryonic kidney cells; TRAF6, tumor necrosis factor receptor–associated factor 6.

Article Snippet: Rabbit phospho-S207-p62 antibodies (OAAB16350) were purchased from Aviva Systems Biology.

Techniques: Phospho-proteomics, Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

doi: 10.1016/j.jbc.2024.107206

Figure Lengend Snippet: Upon p62 phosphorylation, TRAF6 promotes the polyubiquitination of mTOR via K63-linkage. A , after HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62 or/and Flag-DYRK3, cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody. B , after HEK293 cells were transfected for 24 h with a plasmid encoding HA-Raptor, Myc-p62, V5-TRAF6, or Flag-DYRK3-WT alone or in combination, cell lysates were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. C , quantification of the protein blots in ( B ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). D , after HEK293 cells were transfected for 24 h with a plasmid encoding Myc-mTOR, V5-TRAF6, HA-p62, or Flag-DYRK3-WT alone or in combination, cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody. E , after HEK293 cells were transfected for 24 h with a plasmid encoding Myc-mTOR, HA-Ub-WT, HA-Ub-K48, HA-Ub-K63, or Flag-DYRK3-WT alone or in combination, cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody. F , after HEK293 cells were transfected for 24 h with a plasmid encoding Flag-DYRK3-WT, followed by immunoblotting with the indicated antibody. G , quantification of the protein blots in ( F ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). H , where indicated, cells were left untreated or treated for 6 h with 1 μM GSK-626616 or/and 100 ng/ml EGF, followed by immunoblotting with the indicated antibody. I , quantification of the protein blots in ( H ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗ p < 0.01, ∗ p < 0.05). J , after HEK293 cells were transfected for 24 h with a plasmid encoding Myc-p62-WT, Myc-p62-T269A, Myc-p62-pT269E, or Flag-DYRK3-WT alone or in combination, followed by immunoblotting with the indicated antibody. K , quantification of the blots in ( J ) was performed. All data represent the mean ± standard deviation of three independent experiments (∗∗ p < 0.01, ∗ p < 0.05). DYRK, dual-specificity tyrosine-phosphorylation-regulated kinase; EGF, epidermal growth factor; HA, hemagglutinin; HEK, human embryonic kidney cells; TRAF6, tumor necrosis factor receptor–associated factor 6.

Article Snippet: Rabbit phospho-S207-p62 antibodies (OAAB16350) were purchased from Aviva Systems Biology.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

doi: 10.1016/j.jbc.2024.107206

Figure Lengend Snippet: The sequential activation of the DYRK3-p62-TRAF6-mTORC1 pathway stimulates the growth of melanoma cancer cells. A , after SK-Mel-28 cells were transfected for 24 h with a plasmid encoding scrambled control shRNA or shRNA- DYRK3 , immunoblotting was performed with the indicated antibody. B , SK-Mel-28 cell lines were transfected with either control shRNA or shRNA- DYRK3, and cell viability was counted using the CCK-8 assay. Data are represented as the mean ± standard deviation of six independent experiments (∗∗∗ p < 0.001, ∗∗ p < 0.01). C , cell lysates were prepared from SK-Mel-28 cells stably expressing mock vector (Mock) or DYRK3 and subjected to Western blotting with the indicated antibody. D , cell viability of SK-Mel-28 cell lines stably expressing mock vector (control) or DYRK3 was measured using the CCK-8 assay. Data are represented as the mean ± standard deviation of six independent experiments (∗∗∗ p < 0.001). E , cell lysates were prepared from SK-Mel-28 cells stably expressing mock vector (control), p62-WT, p62-T269A, or p62-T269E and subjected to Western blotting with the indicated antibody. F , cell viability of SK-Mel-28 cell lines stably expressing mock vector (control), p62-WT, p62-T269A, or p62-T269E was measured using the CCK-8 assay. Data are represented as the mean ± standard deviation of six independent experiments (∗∗∗ p < 0.001). CCK-8, cell counting kit-8; DYRK, dual-specificity tyrosine-phosphorylation-regulated kinase; TRAF6, tumor necrosis factor receptor–associated factor 6.

Article Snippet: Rabbit phospho-S207-p62 antibodies (OAAB16350) were purchased from Aviva Systems Biology.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Control, shRNA, Western Blot, CCK-8 Assay, Standard Deviation, Stable Transfection, Expressing, Cell Counting, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

doi: 10.1016/j.jbc.2024.107206

Figure Lengend Snippet: The activation of the DYRK3-p62-TRAF6-mTORC1 pathway enhances the progression of melanoma skin cancer. A and B , SK-Mel-28 cell lines transfected with control shRNA or shRNA- DYRK3 were cultured for 10 days, and stained with crystal violet, and their colony formation percentages were determined. Data are presented as the mean ± standard deviation of three independent experiments. C and D , SK-Mel-28 cells were transfected with control shRNA or shRNA- DYRK3 for 24 h, plated for an additional 24 h, and then subjected to a wound-healing assay by scratching the monolayers with a pipette tip. The cell migration rate was assessed after 18 h of incubation and visualized under a microscope. Data are presented as the mean ± standard deviation of three independent experiments (∗∗ p < 0.01). The scale bar represents 10 μm. E and F , SK-Mel-28 cells stably expressing mock vector (control) or DYRK3 were cultured for 10 days, and stained with crystal violet, and their colony formation percentages were determined. Data are presented as the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). G and H , SK-Mel-28 cell lines stably expressing mock vector (control) or DYRK3 were plated for 24, subjected to a wound-healing assay by scratching the monolayers with a pipette tip, and incubated for 18 h. The cell migration rate was assessed and viewed under a microscope. Data are presented as the mean ± standard deviation of three independent experiments (∗∗ p < 0.01). The scale bar represents 10 μm. I and J , SK-Mel-28 cells stably expressing mock vector (control), p62-WT, p62-T269A, or p62-T269E were plated for 24 h, subjected to a wound-healing assay, and incubated for 18 h. The cell migration rate was assessed and viewed under a microscope. Data are presented as the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001, ∗∗ p < 0.01). K and L , SK-Mel-28 cells stably expressing mock vector (control), p62-WT, p62-T269A, or p62-T269E were plated for 24 h, subjected to a wound-healing assay, and incubated for 18 h. The cell migration rate was assessed and viewed under a microscope. Data are presented as the mean ± standard deviation of three independent experiments (∗∗∗ p < 0.001). The scale bar represents 10 μm. DYRK, dual-specificity tyrosine-phosphorylation-regulated kinase; TRAF6, tumor necrosis factor receptor–associated factor 6.

Article Snippet: Rabbit phospho-S207-p62 antibodies (OAAB16350) were purchased from Aviva Systems Biology.

Techniques: Activation Assay, Transfection, Control, shRNA, Cell Culture, Staining, Standard Deviation, Wound Healing Assay, Transferring, Migration, Incubation, Microscopy, Stable Transfection, Expressing, Plasmid Preparation, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

doi: 10.1016/j.jbc.2024.107206

Figure Lengend Snippet: The activation of the DYRK3-p62-TRAF6-mTORC1 pathway promotes the progression of melanoma skin cancer in nude mice. A , SK-Mel-28 cells expressing p62 variants were subcutaneously injected into mice. Tumor growth was monitored every 3 days until the end of the study. Values represent the mean ± S.E.M. for four animals per group ( p ∗ < 0.05). B , tumor tissues harvested from each group were visualized. C , individual growth curve of each mouse in each group was monitored. D , tumors were harvested on day 21 for histological analysis. H&E staining was performed on tumor sections from each group of mice. The scale bar represents 100 μm. DYRK, dual-specificity tyrosine-phosphorylation-regulated kinase; TRAF6, tumor necrosis factor receptor–associated factor 6.

Article Snippet: Rabbit phospho-S207-p62 antibodies (OAAB16350) were purchased from Aviva Systems Biology.

Techniques: Activation Assay, Expressing, Injection, Staining, Phospho-proteomics

Journal: Autophagy

Article Title: The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

doi: 10.1080/15548627.2015.1061170

Figure Lengend Snippet: The n-3 PUFA DHA increases protein level of SQSTM1 and induces autophagy in ARPE-19 cells. ( A ) Cells were treated with DHA (70 µM) for 24 h and lysed in Triton X-100 (Tx100) buffer. Equal amounts of protein (20 µg) from T × 100 fraction were centrifugated at 10,000 x g and the pellet was dissolved in the same volume of 8 M urea buffer before loading on the gel. The membrane was immunoblotted for SQSTM1 and MAP1LC3B. β-actin (ACTB) and PCNA are used as loading controls. ( B ) The cells were treated with DHA, OA or AA (70 µM) with or without BafA1 (100 nM) for 16 h. Total cell extracts were immunoblotted for SQSTM1. ACTB and PCNA are used as loading controls. ( C ) Protein levels of SQSTM1 and MAP1LC3B determined by immunoblotting of cells treated with DHA (70 µM), BafA1 (100 nM) or a combination of DHA and BafA1 for the indicated time points. The numbers below the MAP1LC3B-II bands represent fold change relative to BafA1 for each time point normalized to PCNA intensity. ACTB and PCNA are used as loading controls. ( D ) The mRNA levels of SQSTM1, MAP1LC3B, MAP1LC3A , and GABARAPL1 relative to ACTB after DHA (70 and 140 µM) supplementation for 16 h determined by quantitative real-time PCR. qRT-PCR data displayed are representative for 2 independent experiments. Mean fold change from triplicate wells ± SD is displayed. Data shown are representative of 3 or more independent experiments, unless otherwise stated.

Article Snippet: The following antibodies were used: anti-SQSTM1/p62 (Progen, GP62-C); anti- NFE2L2/NRF2 (Santa Cruz Biotechnology, sc-13032); anti-MAP1LC3B/LC3B (Cell Signaling Technology, D11); anti-HMOX1 (Enzo, ADI-OSA-110), anti-ATG5 (Novus Biologicals, NB110-53818), anti-ACTB/β-actin (Abcam, ab6276), anti-KEAP1 (Santa Cruz Biotechnology, E20), anti-phospho-SQSTM1/p62 (Ser351; MBL, PM074), anti–mono- and polyubiquitininated conjugates (clone FK2; Biomol, PW8810), anti-TUBB/β-tubulin (Abcam, ab6046), anti-PCNA (Santa Cruz Biotechnology, sc-7907), anti-COX4I1/COX IV (Abcam, ab33985).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Autophagy

Article Title: The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

doi: 10.1080/15548627.2015.1061170

Figure Lengend Snippet: The number of SQSTM1-positive protein speckles in ARPE-19 cells increases after DHA supplementation. ( A ) Immunostaining for SQSTM1 and MAP1LC3B after DHA (70 µM) treatment for indicated time points. Nuclear DNA was stained using Draq5 (5 µM). Scale bar: 10 µm. ( B ) Cells were treated with vehicle (V) or DHA (70 µM) for 1, 3, and 6 h. The SQSTM1-positive speckles were automatically quantified using ScanR automated image acquisition. The quantification displayed are representative for 3 independent experiments from where 2 are automatically quantified for more than 1,000 cells per condition and one is manually counted. *) indicates significantly different from control, Student t test P < 0.05. ( C ) The number of SQSTM1-positive speckles per cell (upper panel) and SQSTM1 speckles positive for MAP1LC3B (lower panel) in ARPE-19 cells supplemented with vehicle (V) or DHA (70 µM) for the indicated time points. The quantification displayed was performed manually for more than 100 cells per condition from one representative experiment. This quantification is representative for 3 independent experiments.

Article Snippet: The following antibodies were used: anti-SQSTM1/p62 (Progen, GP62-C); anti- NFE2L2/NRF2 (Santa Cruz Biotechnology, sc-13032); anti-MAP1LC3B/LC3B (Cell Signaling Technology, D11); anti-HMOX1 (Enzo, ADI-OSA-110), anti-ATG5 (Novus Biologicals, NB110-53818), anti-ACTB/β-actin (Abcam, ab6276), anti-KEAP1 (Santa Cruz Biotechnology, E20), anti-phospho-SQSTM1/p62 (Ser351; MBL, PM074), anti–mono- and polyubiquitininated conjugates (clone FK2; Biomol, PW8810), anti-TUBB/β-tubulin (Abcam, ab6046), anti-PCNA (Santa Cruz Biotechnology, sc-7907), anti-COX4I1/COX IV (Abcam, ab33985).

Techniques: Immunostaining, Staining

Journal: Autophagy

Article Title: The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

doi: 10.1080/15548627.2015.1061170

Figure Lengend Snippet: DHA induces a transient increase in ROS and induce NFE2L2 cytoprotective genes. ( A ) Changes in ROS levels measured at different time points after DHA (70 and 140 µM) using a fluorescent ROS DCF probe. The data represent the mean fold change ± SD for 6 independent experiments for 3 h and 3 independent experiments for 6 h and 24 h. Each experiment was performed in duplicates where the mean intensity of 10,000 cells per well ± SD was measured. *) indicates significantly different from control, Student t test P < 0.05 and **) P < 0.01. ( B ) Where indicated the cells were pretreated with antioxidants (5 mM N-acetyl-cysteine (NAC) or 150 µM vitamin E) for 16 h before further stimulations with 140 µM DHA for 3 h. The data represent the average of 3 independent experiments for the DHA and NAC treatments and the average of 2 independent experiments for the Vitamin E treatment. Each experiment was performed in duplicates where the mean intensity of 10,000 cells per well ±SD was measured. **) indicates significantly different from DHA, Student t test P < 0.01. ( C ) Changes in ROS levels measured 3 h after DHA, OA or AA (140 µM) supplementation using a DCF fluorescent probe. The data represent the average of 3 independent experiments ±SD for DHA treated samples and 2 independent experiments ±SD for AA and OA treated samples. Each experiment was performed in duplicates where the mean intensity of 10,000 cells ±SD per well was measured. ( D ) Immunostaining of NFE2L2 after 70 µM DHA, OA, AA for 6 h. Nuclear DNA was stained using Draq5 (5 µM). Scale bar: 10 µm. The results are representative for 3 independent experiments. Nuclear NFE2L2 staining from one representative experiment was automatically quantified using ScanR automated image acquisition of more than 3,000 cells. Each experiment was performed in duplicates and the data are presented as average percentage number of cells with NFE2L2 nuclear staining ± SD. ( E ) The cells were pretreated with NAC (5 mM) for 1 h prior to further stimulation with DHA (70 µM) in combination with NAC for 6 h. After fixation, the cells were immunostained for NFE2L2. Data are representative for 2 independent experiments. The percentage of cells with NFE2L2 nuclear staining from one representative experiment was automatically quantified using ScanR automated image acquisition. Each experiment was performed in duplicate and the data displayed represent the average percentage number of cells with NFE2L2 nuclear staining ± SD. ( F ) ARPE-19 cells were pretreated with NAC (5 mM) for 1 h following stimulation with DHA (70 µM) for 6 h and BafA1 (100 nM) the last 2 h. Levels of HMOX1 was determined by immunoblotting. COX4I1 was used as loading control. ( G ) The ARPE-19 cells were treated with DHA, OA or AA (70 µM) with or without BafA1 (100 nM) for 16 h before immunoblotting for HMOX1 (100 µg protein loaded). ACTB/β-actin and PCNA were used as loading controls. ( H ) Immunoblot of NFE2L2, HMOX1, KEAP1 (100 µg protein loaded), SQSTM1, ACTB and PCNA (loaded 20 µg protein) after DHA (70 µM) with or without BafA1 (100 nM) for 16 h. Arrows represent NFE2L2 and KEAP1 bands while *) represents a nonspecific NFE2L2 band. ACTB and PCNA were used as loading controls. ( I ) Cells were treated as in ( G ) and immunoblotted for phosphorylated SQSTM1 (Ser351) and total SQSTM1 (100 µg protein loaded). ACTB and PCNA were used as loading controls. Data shown are representative of 3 or more independent experiments, unless otherwise stated.

Article Snippet: The following antibodies were used: anti-SQSTM1/p62 (Progen, GP62-C); anti- NFE2L2/NRF2 (Santa Cruz Biotechnology, sc-13032); anti-MAP1LC3B/LC3B (Cell Signaling Technology, D11); anti-HMOX1 (Enzo, ADI-OSA-110), anti-ATG5 (Novus Biologicals, NB110-53818), anti-ACTB/β-actin (Abcam, ab6276), anti-KEAP1 (Santa Cruz Biotechnology, E20), anti-phospho-SQSTM1/p62 (Ser351; MBL, PM074), anti–mono- and polyubiquitininated conjugates (clone FK2; Biomol, PW8810), anti-TUBB/β-tubulin (Abcam, ab6046), anti-PCNA (Santa Cruz Biotechnology, sc-7907), anti-COX4I1/COX IV (Abcam, ab33985).

Techniques: Immunostaining, Staining, Western Blot

Journal: Autophagy

Article Title: The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

doi: 10.1080/15548627.2015.1061170

Figure Lengend Snippet: ATG5 is important in the cellular responses to DHA. ( A ) ARPE-19 cells were transfected with control siRNA or ATG5 siRNA (100 nM) and left for 24 h before reseeding. Following incubation for 24 h, the cells were added DHA (70 μM) or BafA1 (100 nM) for 24 h and immunoblotted for ATG5, SQSTM1, and MAP1LC3B. ACTB and PCNA were used as loading controls. ( B ) The cells were siRNA-transfected as in ( A ). After DHA (140 µM) treatment for 3 h changes in ROS levels were measured using a fluorescent ROS DCF probe. The results are representative for 2 independent experiments. Each experiment was performed in duplicates where the mean intensity ±SD of 10,000 cells per well was measured. The control is normalized to one and the relative fold changes are shown. ( C ) Relative cell index after transfection with control or ATG5 siRNA (100 nM) after vehicle or DHA (140 μM) based on real-time monitoring using the xCELLigence instrument. The cell index for each treatment was normalized to one at the start of the experiment. For each time point the cell index of control samples (Control siRNA + vehicle and ATG5 siRNA + vehicle) was normalized to 1. The effect of DHA treatment after transfection with either Control siRNA or ATG5 siRNA is shown relative to the corresponding controls. Mean normalized cell index with standard deviation of triplicate wells of vehicle or DHA treated cells is displayed. Data shown are representative for 2 independent experiments.

Article Snippet: The following antibodies were used: anti-SQSTM1/p62 (Progen, GP62-C); anti- NFE2L2/NRF2 (Santa Cruz Biotechnology, sc-13032); anti-MAP1LC3B/LC3B (Cell Signaling Technology, D11); anti-HMOX1 (Enzo, ADI-OSA-110), anti-ATG5 (Novus Biologicals, NB110-53818), anti-ACTB/β-actin (Abcam, ab6276), anti-KEAP1 (Santa Cruz Biotechnology, E20), anti-phospho-SQSTM1/p62 (Ser351; MBL, PM074), anti–mono- and polyubiquitininated conjugates (clone FK2; Biomol, PW8810), anti-TUBB/β-tubulin (Abcam, ab6046), anti-PCNA (Santa Cruz Biotechnology, sc-7907), anti-COX4I1/COX IV (Abcam, ab33985).

Techniques: Transfection, Incubation, Standard Deviation

Journal: Autophagy

Article Title: The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

doi: 10.1080/15548627.2015.1061170

Figure Lengend Snippet: The atg5 knockout MEFs are more sensitive to DHA compared to wild-type MEFs. ( A ) The levels of ROS were measured in wild-type (WT) and atg5 −/− MEFs after 3 h DHA treatment (70 and 140 µM) using the fluorescent DCF probe. The data from one representative experiment of 3 independent experiments are displayed. Each experiment was performed in triplicate wells where the mean intensity ±SD of 10,000 cells per well was measured. ( B ) The levels of NFE2L2 and SQSTM1 after vehicle or DHA (70 μM, 16 h) treatment in wild-type and atg5 −/− MEFs (85 μg protein loaded). TUBB/β-tubulin was used as loading control. The immunoblot is representative for 3 independent experiments. ( C ) Wild-type and atg5 −/− MEFs were exposed to DHA (75 μM) and cellular responses observed over time using the xCELLigence real-time monitoring system. The cell index was normalized to one at the start of the experiment. Mean normalized cell index ±SD of triplicate wells of vehicle or DHA treated cells are displayed. The results are representative for 5 independent growth experiments scored by cell index using xCELLigence.

Article Snippet: The following antibodies were used: anti-SQSTM1/p62 (Progen, GP62-C); anti- NFE2L2/NRF2 (Santa Cruz Biotechnology, sc-13032); anti-MAP1LC3B/LC3B (Cell Signaling Technology, D11); anti-HMOX1 (Enzo, ADI-OSA-110), anti-ATG5 (Novus Biologicals, NB110-53818), anti-ACTB/β-actin (Abcam, ab6276), anti-KEAP1 (Santa Cruz Biotechnology, E20), anti-phospho-SQSTM1/p62 (Ser351; MBL, PM074), anti–mono- and polyubiquitininated conjugates (clone FK2; Biomol, PW8810), anti-TUBB/β-tubulin (Abcam, ab6046), anti-PCNA (Santa Cruz Biotechnology, sc-7907), anti-COX4I1/COX IV (Abcam, ab33985).

Techniques: Knock-Out, Western Blot

Journal: Autophagy

Article Title: The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

doi: 10.1080/15548627.2015.1061170

Figure Lengend Snippet: NFE2L2 and SQSTM1 are important in the cellular responses to DHA in ARPE-19 cells. ( A ) Cells were transfected with control, NFE2L2 and SQSTM1 siRNA (25 nM) and left for 24 h before reseeding. Following incubation for 24 h, the cells were added DHA (70 μM) or BafA1 (100 nM) for 24 h. Immunoblot for SQSTM1 and MAP1LC3B. ACTB/β-actin and PCNA were used as loading controls. ( B ) The cells were siRNA-transfected as in ( A ). After vehicle (V) and DHA (70 and 140 µM) treatment for 3 h changes in ROS levels were measured using a fluorescent ROS DCF probe. The data are representative for 2 independent experiments both performed in duplicates. The data represent the mean intensity ±SD of 10,000 cells per well and is displayed as relative DCF intensity. ( C ) Relative cell index after transfection with control, NFE2L2 or SQSTM1 siRNA (25 nM) after vehicle and DHA treatment (70 μM) based on real-time monitoring using the xCELLigence instrument. The cell index was normalized to one at the start of the experiment. Mean normalized cell index with standard deviation of triplicate wells of vehicle and DHA treated cells is displayed. Data shown are representative for 3 independent experiments.

Article Snippet: The following antibodies were used: anti-SQSTM1/p62 (Progen, GP62-C); anti- NFE2L2/NRF2 (Santa Cruz Biotechnology, sc-13032); anti-MAP1LC3B/LC3B (Cell Signaling Technology, D11); anti-HMOX1 (Enzo, ADI-OSA-110), anti-ATG5 (Novus Biologicals, NB110-53818), anti-ACTB/β-actin (Abcam, ab6276), anti-KEAP1 (Santa Cruz Biotechnology, E20), anti-phospho-SQSTM1/p62 (Ser351; MBL, PM074), anti–mono- and polyubiquitininated conjugates (clone FK2; Biomol, PW8810), anti-TUBB/β-tubulin (Abcam, ab6046), anti-PCNA (Santa Cruz Biotechnology, sc-7907), anti-COX4I1/COX IV (Abcam, ab33985).

Techniques: Transfection, Incubation, Western Blot, Standard Deviation

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: The expression of genes was determined in HT29 intestinal cells using the Human GE 4x44K v2 Microarray Kit (Agilent Technologies) after a 72 h transduction with lentiviral particles expressing the CdtB of H . hepaticus strain 3B1 versus the tdTomato fluorescent protein (TFP) as previously described . The relative expression of genes in response to CdtB is reported as a fold change versus the value for cells cultured with lentiviral particles expressing the TFP. Results are presented as the mean of 4 replicates as 4 independent transduction experiments were performed. The data presented for ATG5, HIF1A and NPC1 are the results of 40 replicates as 10 probes for each mRNA of these genes were included on the Microarray Kit. The list of genes related in autophagy to be checked in the Microarray data was first extracted from the Human Autophagy Database (HADb, http://autophagy.lu/clustering/ ). Then a subsequent selection based on their autophagic status annotation available in The Human Gene Database was applied in order to select the major genes involved in autophagy. The discontinuous line shows the basal rate in cells expressing TFP. Asterisks denote significant results. P1 and P2 represent the 2 probe names used for mRNA quantification. Details are presented in (name and sequence of the probes, the corresponding gene name, the genbank accession number, the locus and the transcript variant). Abbreviations: AMBRA1, Autophagy and Beclin-1 Regulator 1; ATG, Autophagy Related Gene; ATG16L, Autophagy Related 16 Like Gene; BAG3, BAG Cochaperone 3;BECN1/Vps30/ATG6, Beclin 1; CALCOCO2, Calcium Binding And Coiled-Coil Domain 2; DRAM1, DNA-damage Regulated Autophagy Modulator 1; eiF2AK3, Eukaryotic Translation Initiation Factor 2-Alpha Kinase 3; FOXO1, Forkhead Box O1; GABARAPL, GABA(A) Receptor-Associated Protein Like; HIF1A, Hypoxia Inducible Factor 1 Subunit Alpha; ITPR1, Inositol 1,4,5-Triphosphate Receptor, type 1; KIAA0226/RUBCN, Rubicon Autophagy Regulator; LAMP1, Lysosomal Associated Membrane Protein 1; LAMP2, Lysosomal Associated Membrane Protein 2; MAP1LC3A, Microtubule Associated Protein 1 Light Chain 3 Alpha; MAP1LC3B, Microtubule Associated Protein 1 Light Chain 3 Beta; MLST8, MTOR (Mechanistic Target of Rapamycin Kinase) Associated Protein, LST8 Homolog (mammalian lethal with Sec13 protein); MTMR14, MyoTubularin Related Protein 14; NPC1, NPC Intracellular Cholesterol Transporter 1 (Niemann-Pick disease, type C1); PARK2/PARKN, Parkin RBR E3 Ubiquitin Protein Ligase; PEX3, Peroxisomal Biogenesis Factor 3; PIK3C3//Vps34, Phosphoinositide-3-Kinase, class 3; PIK3R4/Vps15, Phosphoinositide-3-Kinase, Regulatory subunit 4; PINK1; Phosphatase and Tensin Homolog (PTEN) Induced Kinase 1; RAB1A, Member RAS Oncogene Family; RAB7A, Member RAS Oncogene Family; RAB24, member RAS oncogene family; RB1CC1/FIP200, RB1 (RetinoBlastoma Transcriptional Corepressor 1) Inducible Coiled-Coil 1; RGS19, Regulator of G-protein Signaling 19; RPS6KB1, Ribosomal Protein S6 Kinase B1; RPTOR, Regulatory Associated Protein Of MTOR Complex 1; SESN2, Sestrin 2; SH3GLB1/BIF1, SH3 Domain Containing Growth Factor Receptor Bound Protein 2 (GRB2) Like, Endophilin B1; SIRT2, Sirtuin 2; SPNS1, Sphingolipid Transporter 1 (Putative); P62/SQSTM1, Sequestosome 1; STK11; Serine/Threonine Kinase 11; TMEM49/VMP1, Vacuole Membrane Protein 1; TP53INP2, Tumor Protein P53 Inducible Nuclear Protein 2; ULK, unc-51-like kinase; WD, tryptophan-aspartic acid; WDR45/WIPI4, WD (tryptophan-aspartic acid) Repeat Domain 45; WIPI, WD Repeat Domain, Phosphoinositide Interacting; ZFYVE1/DFCP1, Zinc Finger FYVE-Type Containing 1.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Expressing, Microarray, Transduction, Cell Culture, Selection, Sequencing, Variant Assay, Binding Assay

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: Transgenic HT29 and Hep3B cells were cultivated with doxycycline for 72 h to induce the expression of the control Red Fluorescent Protein (RFP), the CdtB of H . hepaticus strain 3B1 or the CdtB of H . hepaticus strain 3B1 with the H265L mutation which has no catalytic activity . Then, cells were processed for western blot analysis or fluorescent staining with primary antibodies generated against P62/SQSTM1 associated with fluorescent labeled-secondary antibodies (green) and DAPI to counterstain the nuclei (blue) . The number of fluorescent P62/SQSTM1 bodies was quantified using the "Find Maxima" function of ImageJ. The results are presented as the mean in one representative experiment (performed in triplicate) out of three. A minimum of 500 cells were measured. A) Time-course western blot analysis of the protein expression level of LC3 and P62/SQSTM1 in response to the CdtB of H . hepaticus in HT29 cells. B) The protein expression level and phosphorylation status of P62/SQSTM1 and AMPK in response to RFP, CdtB and H265L were analyzed by western blot in transgenic HT29 cells. (C) Quantification of P62/SQSTM1 bodies in transgenic HT29 cells. (D) Quantification of P62/SQSTM1 bodies in transgenic Hep3B cells. E) Confocal image of Hep3B transgenic cells expressing the CdtB of H . hepaticus strain 3B1 (72 h) processed for P62/SQSTM1 fluorescent staining (green) and DAPI (blue). Scale bar, 10 μm. *p<0.05, **p< 0.01, ***p< 0.001. Abbreviations: AMPK, AMP-activated protein kinase; CdtB, CdtB of H . hepaticus strain 3B1; DAPI, 4′, 6′-diamidino-2-phenylindol; H265L, H . hepaticus CdtB with the mutation His→Leu at residue 265 involved in catalytic activity; P-AMPK, phosphorylated AMP-activated protein kinase; P62, P62/SQSTM1; P-P62, phosphorylated P62/SQSTM1; RFP, Red fluorescent protein; Tub., tubulin.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Transgenic Assay, Expressing, Mutagenesis, Activity Assay, Western Blot, Staining, Generated, Labeling

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: HT29- and Hep3B-transgenic cell lines were engrafted into immunodeficient mice as previously reported . Three μm-tissue sections of the xenograft-derived tumors were prepared from formalin-fixed paraffin-embedded tissues and submitted to standard hematoxylin staining and immunostaining raised against LC3 (A) and P62/SQSTM1 (B) . Boxes correspond to enlargement. The yellow, black, green and red arrows represent the murine infiltrates, the giant cells, the mucins (HT29) and the LC3 puncta or P62/SQSTM1 bodies, respectively. Quantification was performed by using the ‘Threshold’ function of ImageJ (v. 1.52n). Scale bar, 50 μm. **p< 0.01. Scale bar, 50 μm. Abbreviations: CdtB of H . hepaticus strain 3B1; P62, P62/SQSTM1; RFP, Red fluorescent protein.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Transgenic Assay, Derivative Assay, Formalin-fixed Paraffin-Embedded, Staining, Immunostaining

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: Wide field image of Hep3B transgenic cells expressing the CdtB of H . hepaticus strain 3B1 processed for the nuclear lamina (red) and P62/SQSTM1 fluorescent staining (green), and DAPI to counterstain the nuclei (blue). Yellow arrows indicate DAPI-lacking nucleoplasmic reticulum enclosing P62/SQSTM1 bodies. Boxes correspond to enlargement. Scale bar, 10 μm. Abbreviations: DAPI, 4′, 6′-diamidino-2-phenylindol; P62, P62/SQSTM1.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Transgenic Assay, Expressing, Staining

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: As previously demonstrated, NR formation is primarily observed in response to CDT intoxication, via its active CdtB subunit . Thus, images of non-infected cells are not presented below. (A) Hep3B and (B) SW480 Transgenic cells were cultivated with doxycycline for 72 h to induce the expression of the CdtB of H . hepaticus strain 3B1 . Then cells were processed for staining with primary and fluorescent secondary antibodies: P62/SQSTM1 (red), UNR (green) and DAPI to counterstain the nuclei (blue). Subsequent quantification of P62/SQSTM1 in nucleoplasm, cytoplasm and foci were performed using capture of fluorescent staining (confocal imaging) by measuring the pixel intensity with the “Plot Profile” function of ImageJ (v. 1.52n), each count being performed on 100 NRs. The relative expression rate of P62/SQSTM1 in NR in response to the CdtB was reported as fold increase versus the expression in the cytosol. Scale bar, 20 μm. ***p<0.0001. Abbreviations: Cyto., cytoplasm; DAPI, 4′, 6′-diamidino-2-phenylindol; NR, nucleoplasmic reticulum; Nuc., nucleus; P62, P62/SQSTM1.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Infection, Transgenic Assay, Expressing, Staining, Imaging

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: HT29 and Hep3B cells were infected for 3 days with CDT-secreting H . hepaticus or colibactin-secreting extra-intestinal pathogenic E . coli . Then, cells were processed for fluorescent staining with primary antibodies generated against γH2AX (red), P62/SQSTM1 (green), associated with fluorescent-labeled secondary antibodies and DAPI to counterstain the nuclei (blue). Fluorescent staining was observed using wide field fluorescence imaging. (A) Images of HT29 cells: P62/SQSTM1 (green), DAPI (blue). (B) Images of HT29 cells: γH2AX (red), P62/SQSTM1 (green), DAPI (blue). (C) Images of Hep3B cells: γH2AX (red), P62/SQSTM1 (green), DAPI (blue). Scale bars, 20 μm. Yellow arrowheads indicate extra-nuclear structures containing chromatin and P62/SQSTM1 aggregates. Yellow and pink boxes correspond to enlargement in which the DAPI and γH2AX were overexposed, respectively, in order to see the micronucleus-like structures. White boxes on the right correspond to enlargement of the merge. Abbreviations: DAPI, 4′, 6′-diamidino-2-phenylindol.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Infection, Staining, Generated, Labeling, Fluorescence, Imaging

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: Hep3B cells were processed as in . Fluorescent staining was observed using wide field fluorescence imaging. (A) Images of Hep3B cells: γH2AX (red), LC3 (green), DAPI (blue). (B) Images of Hep3B cells: nuclear lamina (red), P62/SQSTM1 (green), DAPI (blue). Scale bars, 20 μm. Yellow arrowheads indicate extra-nuclear structures containing chromatin. Blue arrowheads indicate P62/SQSTM1 aggregates tightly connected all along the nuclear membrane. Yellow boxes correspond to enlargement of micronucleus-like structures with DAPI. White boxes on the right correspond to enlargement of the merge. Abbreviations: DAPI, 4′, 6′-diamidino-2-phenylindol; NR, nucleoplasmic reticulum.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Staining, Fluorescence, Imaging

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: A) CdtB-transgenic Hep3B cell line cultivated with doxycycline for 72 h to induce the expression of the CdtB of H . hepaticus strain 3B1. Cells were also treated with bafilomycin A1 (30 nM) or chloroquine (30 μM) 48 h and 24 h after doxycycline induction for a duration of 24 h and 48 h, respectively. Then cells were processed for fluorescent staining with primary antibodies generated against UNR (red) and P62/SQSTM1 (green) associated with fluorescent labeled-secondary antibodies and DAPI to counterstain the nuclei (blue). Quantification of UNR-NR positive cells (%) was performed on a minimum of 500 nuclei. The results are presented as the mean in one representative experiment (performed in triplicate) out of three. B) to G) Mock-KO, ATG5-KO and ATG7-KO Hep3B cells were infected for 3 days with H . hepaticus and its corresponding ΔCDT mutant strain. Then, the medium was removed, new medium was added and incubation continued until 8 days. These cells were processed daily for fluorescent staining with DAPI to detect the nucleus and fluorescent primary and secondary antibodies targeting γH2AX, UNR, cleaved caspase-3, and P62/SQSTM1. Fluorescent staining was observed using wide field fluorescence imaging . The results are presented as the mean in one representative experiment (performed in triplicate) out of three. A minimum of 500 cells were measured. (B) Nucleus surface (area) was quantified by isolating the DAPI fluorescence for each nucleus by using the ‘Threshold’ function of ImageJ (v. 1.52n). Nucleus size was measured in viable and early apoptotic cells. (C) γH2AX foci quantification was performed in viable and early apoptotic cells by measuring the pixel intensity with the “Integrated density” measure function of ImageJ (v. 1.52n). (D) The percentage of cells presenting UNR-NR was determined by manually counting the number of nuclei displaying UNR spots in the nucleoplasm. (E) Cell number quantification was performed manually. (F) Caspase-3-positive cells were quantified by counting the caspase 3 positive cells on 10 fields. *p<0.05, **p<0.01, ***p<0.001. Abbreviations: AU, arbitrary unit; Baf., bafilomycin A1; CQ, chloroquine; ΔCDT, CDT isogenic mutant of H . hepaticus strain 3B1; DAPI, 4′, 6′-diamidino-2-phenylindol; KO, Knock-Out, NR, nucleoplasmic reticulum; P62, P62/SQSTM1.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Transgenic Assay, Expressing, Staining, Generated, Labeling, Infection, Mutagenesis, Incubation, Fluorescence, Imaging, Knock-Out

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: ( continued) Mock-KO, ATG5-KO and ATG7-KO Hep3B cells were processed as in . Cells were stained for nuclei (DAPI), as well as for γH2AX, UNR, cleaved caspase-3, and P62/SQSTM1 (red), along with DAPI to counterstain the nuclei (blue). Wide field images of cocultures experiment at days 4 are presented. Scale bars: 50 μm for cell number (DAPI only), 100 μm for γH2AX and DAPI double staining, and 20 μm for other double staining (UNR, cleaved caspase-3, and P62/SQSTM1 with DAPI). *p<0.05, **p<0.01, ***p<0.001. Abbreviations: ΔCDT, CDT isogenic mutant of H . hepaticus strain 3B1; DAPI, 4′, 6′-diamidino-2-phenylindol; KO, Knock-Out, P62, P62/SQSTM1.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Staining, Double Staining, Mutagenesis, Knock-Out

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: Then, cells were processed for fluorescent staining with primary antibodies generated against UNR, cleaved Caspase-3 or P62/SQSTM1 or γH2AX associated with fluorescent labeled-secondary antibodies and DAPI to counterstain the nuclei. (A) The percentage of cells presenting UNR-NR was determined by manually counting the number of nuclei displaying UNR spots in the nucleoplasm. (B) Cell number quantification was performed manually. (C) Caspase-3-positive cells were quantified by counting the caspase 3 positive cells on 10 fields. (D) P62/SQSTM1 bodies were quantified using the "Find Maxima" function of ImageJ. The results are presented as the mean in one representative experiment (performed in triplicate) out of three. (E) Quantification of micronucleus-like structures in Hep3B cells was performed manually. (F) Quantification of Hep3B cells with colocalized γH2AX-foci and P62/SQSTM1-bodies was performed manually. A minimum of 500 cells were measured. *p<0.05, **p<0.01, ***p<0.001. Abbreviations: AU, arbitrary unit; DAPI, 4′, 6′-diamidino-2-phenylindol; KO, Knock-Out, NR, nucleoplasmic reticulum; P62, P62/SQSTM1; pks - , bacterial artificial chromosome vector; pks + , bacterial artificial chromosome vector with pks island encoding colibactin.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Staining, Generated, Labeling, Knock-Out, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1

doi: 10.1371/journal.ppat.1009320

Figure Lengend Snippet: Hep3B cells were cultivated in the presence of etoposide (5 μM) or steptozocin (10 mM) for a duration of 24 h. Then, the medium was removed and incubation was continued for 48 h. The cells were then stained with fluorescent primary and secondary antibodies targeting LC3 (red)/γH2AX (green), P62/SQSTM1 (red)/UNR (green) and DAPI to counterstain the nuclei (blue). Fluorescent staining was observed using wide field fluorescence imaging. γH2AX foci quantification was performed by measuring the pixel intensity with the “Integrated density” measure function of ImageJ (v. 1.52n). LC3 puncta and P62/SQSTM1 bodies were quantified using the "Find Maxima" function of ImageJ. The percentage of cells presenting UNR-NR was determined by manually counting the number of nuclei displaying UNR spots in the nucleoplasm. Quantification was performed on a minimum of 500 cells. The results are presented as the mean in one representative experiment (performed in triplicate) out of three. ***p<0.001. Scale bar, 20 μm. White arrow indicates DAPI-lacking large aggregate positive for LC3 and γH2AX foci. Yellow arrow indicates DAPI-lacking nucleoplasmic reticulum enclosing UNR and P62/SQSTM1 bodies. Abbreviations: AU, arbitrary unit; DAPI, 4′, 6′-diamidino-2-phenylindol; ETP, etoposide; NR, nucleoplasmic reticulum; P62, P62/SQSTM1; STZ, steptozocin.

Article Snippet: Antibodies used for western blot analyses were: mouse monoclonal anti-LC3 (clone 4E12) (#M152-3, MBL Life science) distributed by CliniSciences (Nanterre, France); mouse monoclonal anti-P62/SQSTM1 (Clone 3/P62 Ick ligand) (#610832) from BD Biosciences (San Jose, CA); rat monoclonal anti-phospho-P62/SQSTM1 (Ser403) (clone 4F6) (#MABC186, Merck Chimie Fontenay sous Bois), rabbit polyclonal anti-UNR/CSDE1 (#HPA018846) from Sigma Aldrich and mouse monoclonal anti-α-tubulin (clone DM1A) (#05–829) from Sigma Aldrich; rabbit polyclonal anti-AMPKα1 (#2795) and rabbit monoclonal anti-phospho-AMPKα (Thr172) (clone 40H9) (#2535) from Cell Signaling Technology (distributed by Ozyme, Saint-Cyr-L’École, France).

Techniques: Incubation, Staining, Fluorescence, Imaging

Journal: Autophagy

Article Title: Cytosolic PINK1 promotes the targeting of ubiquitinated proteins to the aggresome-autophagy pathway during proteasomal stress

doi: 10.1080/15548627.2016.1147667

Figure Lengend Snippet: PINK1-s phosphorylates SQSTM1 at Ser28 during proteasomal stress. (A) Immunoprecipitation and western blot analysis of PINK1-SQSTM1 interaction. AD293 cells were transfected with the indicated plasmids. The FLAG-tagged PINK1 proteins were immunoprecipitated with FLAG antibody and the associated EGFP-SQSTM1 was detected with EGFP antibody. (B) Western blot analysis of SQSTM1-bound, K48-ubiquitinated proteins in control cells and cells expressing MYC-PINK1-s or MYC-PINK1-s(KinD). Cells were transfected with the indicated plasmids and immunoprecipitated with HA antibody. HA-SQSTM1-bound ubiquitinated proteins were detected with FLAG antibody. Quantification results are shown as mean±SEM of 3 independent experiments. *, P < 0.05; NS, non-significant. (C) In vitro phosphorylation assay of the wild-type (WT) and S28A mutant GST-SQSTM1 by PINK1-s. GST-SQSTM1(WT) and GST-SQSTM1S28A were expressed in bacteria and affinity purified with glutathione beads. FLAG-PINK1-s was expressed in AD293 cells and immunopurified with FLAG antibody. GST-SQSTM1(WT) or GST-SQSTM1S28A was incubated with FLAG-PINK1-s in the presence of γ-32P-ATP to label the phosphorylated proteins. GST-SQSTM1 and FLAG-PINK1-s in the reactions were detected by western blot analysis with GST and FLAG antibodies, respectively. (D) Western blot analysis of SQSTM1 Ser28 phosphorylation in the soluble and insoluble fractions of PINK1+/+, PINK1−/− and rescue cells after a 12-h treatment with DMSO or MG132 (1 μM). Eight percent of the soluble and 20% of the insoluble fractions of each sample were loaded for SDS-PAGE and western blot analysis. Ser28 phosphorylation was detected with a custom-made antibody. Soluble ACTB levels were detected as the loading control. Quantification results are shown as mean±SEM of 3 independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Detection of Ser28 phosphorylation by western blot analysis Rabbit polyclonal antibody for phosphorylated Ser28 of SQSTM1 was generated and purified by PhosphoSolutions Inc. To analyze Ser28 phosphorylation during proteasomal inhibition, AD293 PINK1 +/+ , PINK1 −/− and its rescue cells were grown on 6-well plates and treated with DMSO or 1 μM MG132 for 12 h. They were next incubated on ice with 200 μl/well NP40 Alternative-containing cell lysis buffer for 20 min.

Techniques: Immunoprecipitation, Western Blot, Transfection, Control, Expressing, In Vitro, Phospho-proteomics, Mutagenesis, Bacteria, Affinity Purification, Incubation, SDS Page

Journal: Autophagy

Article Title: Cytosolic PINK1 promotes the targeting of ubiquitinated proteins to the aggresome-autophagy pathway during proteasomal stress

doi: 10.1080/15548627.2016.1147667

Figure Lengend Snippet: Ser28 phosphorylation in SQSTM1 was required for efficient aggresome-targeting of ubiquitinated proteins during proteasomal inhibition. (A) Coimmunoprecipitation and western blot analysis of the K48-ubiquitinated proteins bound by the WT and Ser28 mutant SQSTM1. AD293 cells were transfected with the indicated plasmids, immunoprecipitated with HA antibody and the associated ubiquitinated proteins were detected with FLAG antibody. The D69A mutation was introduced into SQSTM1 to reduce self-oligomerization and thus increase its solubility. Soluble TUBB (tubulin β class I) was detected as the loading control. Quantification results are shown as mean ± SEM of 3 independent experiments. *, P < 0.05; NS, nonsignificant. (B) Aggregate formation in AD293 cells expressing the WT or the S28E or S28A mutant SQSTM1. The overexpressed SQSTM1 and ubiquitinated proteins were detected by immunofluorescence staining with FLAG and UB(K48) antibodies, respectively. Scale bar: 20 μm. Quantification results are shown as mean ± SEM of 3 independent experiments. **, P < 0.01; NS, non-significant. (C) Western blot analysis of the soluble and insoluble K48-ubiquitinated proteins in AD293 cells expressing the WT or mutant SQSTM1. Cells were transfected with the indicated plasmids. Sixteen percent of the soluble proteins and 50% of the insoluble proteins of each sample were loaded for SDS-PAGE. UB(K48) antibody was used to detect the soluble and insoluble ubiquitinated proteins. HA antibody was used to detect the WT and mutant HA-SQSTM1. TUBB was used as the loading control. Quantification results are shown as mean ± SEM of 3 independent experiments. *, P < 0.05; NS, nonsignificant. (D) Aggresome formation in SQSTM1S28A,flox/− (SQSTM1−/−), SQSTM1+/− and SQSTM1S28A/− cells after a 14-h treatment with DMSO or MG132 (1.5 μM). Ubiquitinated proteins were detected by immunofluorescence staining with UB(K48) antibody. The nuclei were visualized by DAPI staining. Aggresomes are indicated by arrowheads. Scale bar: 20 μm. Quantification results are shown as mean ± SEM of 3 independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Detection of Ser28 phosphorylation by western blot analysis Rabbit polyclonal antibody for phosphorylated Ser28 of SQSTM1 was generated and purified by PhosphoSolutions Inc. To analyze Ser28 phosphorylation during proteasomal inhibition, AD293 PINK1 +/+ , PINK1 −/− and its rescue cells were grown on 6-well plates and treated with DMSO or 1 μM MG132 for 12 h. They were next incubated on ice with 200 μl/well NP40 Alternative-containing cell lysis buffer for 20 min.

Techniques: Phospho-proteomics, Inhibition, Western Blot, Mutagenesis, Transfection, Immunoprecipitation, Solubility, Control, Expressing, Immunofluorescence, Staining, SDS Page