Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: Schematic representation of strategy for identification of mitochondrial proteins by mass spectrometry. Rank order of protein signals in mitochondrial proteome of T47D cells exposed to hypoxia (1% O 2 for 24 h) versus normoxia. 2‐oxoglutarate‐dependent dioxygenases were highlighted. Red, upregulated; blue, downregulated; gray, not significant. Immunoblots of extracts from whole cell (WCE), mitochondria (Mito), and cytosol (Cyto) of T47D cells treated with hypoxia (H, 1% O 2 for 24 h) or normoxia (N). Immunoblots of extracts from whole cell (WCE), mitochondria (Mito), and cytosol (Cyto) as indicated of MDA‐MB‐231 and 293T cells treated with hypoxia (H, 1% O 2 for 24 h) or normoxia (N). Immunoblots of hypoxic (1% O 2 for 24 h) 293T mitochondrial extract (Mito) treated with indicated concentration of proteinase K for 1 h. Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) of T47D infected with EglN1‐Flag followed by treatment with hypoxia (1% O 2 for 24 h) or normoxia. Immunofluorescence of T47D cells infected with EglN1‐GFP (green) followed by treatment with normoxia or hypoxia (1% O 2 for 24 h) and with MitoTracker Red staining for 15 min. Nuclei were stained with DAPI (blue) (scale bar = 10 μm). Quantification of each cells' area overlap ratio for co‐localization of EglN1‐GFP and mitochondria from (G) ( N = 6 images in total). Immunoblots of extracts from mitochondria (Mito) as indicated of T47D cells treated with hypoxia (1% O 2 ) for 0, 12, 24 and 48 h, respectively. Immunoblots of extracts from mitochondria (Mito) as indicated of T47D cells treated with normoxia or hypoxia (1% O 2 )‐reoxygenation (H‐ReO 2 ) for 0, 3, and 6 h, respectively. EglN1 expression in breast cancer and normal subtypes in METABRIC cohort ( n = 1,139). Wilcoxon rank‐sum test was used for statistical analysis of these two groups. EglN1 expression in different oxygen levels in METABRIC cohort ( n = 1,139). The hypoxia score of METABRIC breast cancer cohort was calculated by using mRNA‐based signatures. Kruskal–Wallis test was used for the statistical analysis of these three groups. EglN1 expression in different breast cancer subtypes in METABRIC cohort ( n = 1,139). METABRIC breast cancer cohort was categorized into five subtypes according to Pam50 gene expression subtype classification (Basal‐like, Claudin‐low, Her2, Luminal A, and Luminal B). Kruskal–Wallis test was used for the statistical analysis of these multiple groups. Representative immunofluorescence of EglN1 and TOM20 with tumor tissues from breast cancer patients. The right panel showed the quantification of fluorescence intensity of TOM20 and EglN1 along the line in merged image. Box plot showing the co‐localization statistics of EglN1 with TOM20 in these six breast cancer patient samples ( n = 6). Y‐axis indicates the co‐location coefficient of EglN1 and TOM20. Representative immunofluorescence of HIF high and HIF low breast cancer tumors from a human breast cancer microarray, and their corresponding colocalization images of EglN1 with TOM20 from a human breast cancer microarray. Nuclei were stained with DAPI (blue) (scale bar = 10 μm). Scatterplots showing the correlation between co‐localization of EglN1 with TOM20 and the intensity of HIF1α in different breast cancer tumors ( n = 41) from a human breast cancer microarray. X‐axis indicates the mean fluorescence intensity of HIF1α, and Y‐axis indicates the Pearson coefficient of co‐localization of EglN1 and TOM20. Data information: Error bars in (H) represent ± SEM, *** denote P value of < 0.005 (unpaired t ‐test). Also See Fig . Source data are available online for this figure.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Mass Spectrometry, Western Blot, Concentration Assay, Infection, Immunofluorescence, Staining, Expressing, Gene Expression, Fluorescence, Microarray

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: A Partial least squares‐discriminant analysis (PLS‐DA) of those mitochondrial proteomes from T47D cells exposed to hypoxia (1% O 2 for 24 h) versus normoxia. B Volcano plots of mitochondrial proteomes from T47D cells exposed to hypoxia (1% O 2 for 24 h) versus normoxia showing upregulated and downregulated proteins in mitochondria under hypoxia. Red, upregulated; blue, downregulated; gray, not significant. C Heatmap showing the upregulated and downregulated proteins as identified in Fig . D Network showing the relationships between the significantly enriched oxygen signaling pathways and relevant proteins. E, F Immunoblots of mitochondrial extracts (Mito) and whole cell extracts (WCE) from 293T transfected with EglN2 (E) or EglN3 (F) followed by normoxic (N) or hypoxic (H, 1% O 2 for 24 h) treatment. G Immunoblots of mitochondrial extracts (Mito) and whole cell extracts (WCE) from T47D followed by normoxic (N) or hypoxic (H, 1% O 2 for 24 h) treatment. H Immunofluorescence of EglN1 and TOM20 with tumor tissues from breast cancer patients. Their right panels showed the quantification of fluorescence intensity of TOM20 and EglN1 along each line in merged image.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Protein-Protein interactions, Western Blot, Transfection, Immunofluorescence, Fluorescence

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: A Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) as indicated of 293T cells transfected with EglN1‐Flag WT or P317R mutant followed by treatment with normoxia (N) or hypoxia (H, 1% O 2 for 24 h). B Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) as indicated as indicated of T47D cells treated with or without IOX4 (50 μM) for 24 h. C Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) as indicated of T47D cells treated with normoxia (−), hypoxia (H, 1% O 2 for 24 h), DMOG (2 mM, for 24 h), or DFO (200 μM, for 12 h). D, E Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) of 293T cells transfected with EglN1‐Flag (D) or T47D (E) cells followed by treatment with normoxia (−), hypoxia (H, 1% O 2 for 24 h), DMOG (2 mM, for 24 h), or DFO (200 μM, for 12 h). F Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) of T47D cells infected with EglN1‐Flag followed by another infection with control sgRNA (−) or VHL sgRNA (sgVHL) under treatment with normoxia (N) or hypoxia (H, 1% O 2 for 24 h). G Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) as indicated of 786‐O cells treated with normoxia (N) or hypoxia (H, 1% O 2 for 24 h). H Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) as indicated of 786‐O cells infected with control vector (−) or HA‐VHL. I A schematic illustration of EglN1 β2β3 loop (241–251) for substrate binding. J Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) as indicated of T47D cells infected with control EglN1‐WT‐Flag or EglN1‐▵β2β3‐Flag followed by infection with EglN1 sh1045 with or without hypoxia (1% O 2 ) treatment for 24 h. K Immunofluorescence of T47D cells infected with EglN1‐GFP or EglN1‐▵β2β3‐GFP followed by treatment with hypoxia (1% O 2 for 24 h) and with MitoTracker Red staining for 15 min. Nuclei were stained with DAPI (blue) (scale bar = 10 μm). L Quantification data of each cells' area overlap ratio for co‐localization of EglN1‐GFP and mitochondria from (K) ( N = 6 images in total). Data information: Error bars in (L) represent ± SEM, *** denote P value of 0.005 (unpaired t ‐test). Also See Fig . Source data are available online for this figure.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Western Blot, Transfection, Mutagenesis, Infection, Control, Plasmid Preparation, Binding Assay, Immunofluorescence, Staining

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: Immunoblots of extracts from whole cell (WCE) of T47D cells infected with control sgRNA (−) or VHL sgRNA (sgVHL). Immunoblots of extracts from cytosol (Cyto) and Nuclei as indicated of T47D cells infected with control EglN1‐WT‐Flag or EglN1‐▵β2β3‐Flag followed by infection with EglN1 sh1045 with or without hypoxia (1% O 2 ) treatment for 24 h. Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) as indicated as indicated of T47D cells infected with control shRNA (−) or HIF1β sh1770 followed by treatment with normoxia (N) or hypoxia (H, 1% O 2 for 24 h). Immunoblots of extracts from whole cell (WCE) and mitochondria (Mito) as indicated of 786‐O cells treated with or without PT2399 (2 μM) for 24 h.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Western Blot, Infection, Control, shRNA

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: A–D Immunoblots of cell lysates (A, C), and MTT assays (B, D) of T47D and MDA‐MB‐231 cells infected with control shRNA (shCtrl), EglN1 sh1042, or EglN1 sh1045 under hypoxic condition (1% O 2 ). E, F MTT assays of T47D (E) and MDA‐MB‐231 (F) cells infected with control shRNA (shCtrl), EglN1 sh1042, or EglN1 sh1045 under normoxia. G–J Mouse xenograft experiments were performed with the MDA‐MD‐MB231 cells generated as indicated in (G). Tumor growth curves (H) and tumor weights (I) were calculated, and gross tumors (J) were presented ( n = 6 mice per group). K–N Mouse xenograft experiments were performed with the cells generated as indicated in (K). Tumor growth curves (L) and tumor weights (M) were calculated, and gross tumors (N) were presented ( n = 6 mice per group). O MTT assays of T47D and MDA‐MB‐231 cells infected with control vector (Ctrl), EglN1‐WT‐Flag, or EglN1‐▵β2β3‐Flag followed by infection with control shRNA (shCtrl) or EglN1 sh1045 (EglN1 shRNA) under normoxic condition. P Immunoblots of extracts from whole cell (WCE), cytosol (Cyto), mitochondria (Mito), and nucleus (Nuc) of 293T cells infected with TOM20‐EglN1‐Flag. Q–T Immunoblots of cell lysates (Q, S) and MTT assays (R, T) of T47D and MDA‐MB‐231 cells infected with TOM20 followed by treatment with hypoxia (1% O 2 ). U MTT assays of T47D and MDA‐MB‐231 cells infected with control vector (Ctrl), EglN1‐WT‐Flag, or TOM20‐EglN1‐Flag followed by infection with control shRNA (shCtrl) or EglN1 sh1045 (EglN1 shRNA) under normoxic condition. V MTT assays of MDA‐MB‐231 cells generated from Fig treated with or without PT2399 (4 μM). W MTT assays of MDA‐MB‐231 cells generated from Fig treated with or without PT2399 (4 μM). Data information: Error bars represent ± SEM, *, ** and *** denote P value of < 0.05, 0.01, and 0.005, respectively, and ns denotes not significant (unpaired t ‐test). n = 3 independent technical replicate experiments for MTT assays.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Western Blot, Infection, Control, shRNA, Generated, Plasmid Preparation

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: A–F Immunoblots of cell lysates (A, D), MTT assays (B, E), and 2D colony formation assays (C, F) from T47D or MDA‐MB‐231 cell lines infected with control vector (Ctrl), EglN1‐WT‐Flag, or EglN1‐▵β2β3‐Flag followed by infection with control shRNA (shCtrl) or EglN1 sh1045 (EglN1 shRNA) under hypoxic (1% O 2 ) condition. G–I Mouse xenograft experiments were performed with the cells generated in (D). Tumor growth curves (G) and tumor weights (H) were calculated, and gross tumors (I) were presented ( n = 6 mice per group). J–O Immunoblots of cell lysates (J, M), MTT assays (K, N), and 2D colony formation assays (L, O) from T47D or MDA‐MB‐231 cell lines infected with control vector (Ctrl), EglN1‐Flag, or TOM20‐EglN1‐Flag followed by infection with control shRNA (shCtrl) or EglN1 sh1045 (EglN1 shRNA) under hypoxic (1% O 2 ) condition. P–R Mouse xenograft experiments were performed with the cells generated in (m). Tumor growth curves (P) and tumor weights (Q) were calculated, and gross tumors (R) were presented ( n = 6 mice per group). Data information: Error bars in (B, E, G, H, K, N, P, Q) represent ± SEM, ** and *** denote P value of < 0.01, and 0.005, respectively, and ns denotes not significant (unpaired t ‐test). n = 3 independent technical replicate experiments for MTT assays. Also See Fig . Source data are available online for this figure.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Western Blot, Infection, Control, Plasmid Preparation, shRNA, Generated

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: Schematic representation of strategy for identification of EglN1‐interacting proteins in T47D cells exposed to hypoxia (1% O2 for 24 h) versus normoxia by mass spectrometry. Sequence coverage values of EglN1, AMPKα1, and HIF1α from mass spectrometry analysis. Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of T47D cells infected with control vector, AMPKα1‐Flag, or AMPKα2‐Flag followed by treatment with normoxia or hypoxia (1% O 2 ) for 24 h. Immunoblots (IB) of proteins from in vitro translation or recombinant protein purification (input). In vitro immunoprecipitation (IP) analyses for protein interactions between recombinant GST‐EglN1 and AMPKα1‐Flag or AMPKα2‐Flag, respectively. Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of T47D cells infected with control vector or EglN1‐Flag followed by treatment with normoxia or hypoxia (1% O 2 ) for 24 h. Immunoblots of extracts from cytosol (Cyto), mitochondria (Mito) and nucleus, and their respective immunoprecipitations (IP) from T47D mitochondrial extraction generated in Fig . Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of T47D cells infected with control vector or EglN1‐Flag followed by treatment with or without compound C (Comp C, 10 μM) for 24 h under hypoxic condition. Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of T47D cells infected with control vector, EglN1‐WT‐Flag, or EglN1‐▵β2β3‐Flag. A schematic illustration of highly conserved sequences for prolyl hydroxylation within kinase domains (KD) of AMPKα1 and AMPKα2. Those prolines for hydroxylation were highlighted in red. Immunoblots of lysates from T47D and 786‐O cells treated with or without hypoxia (1% O 2 ) for 24 h. Immunoblots of lysates from T47D and 786‐O cells treated with or without DMOG (2 mM) for 24 h. Immunoblots of lysates from 293T cells transfected with AMPKα1‐WT/P188A‐Flag or AMPKα2‐WT/P177A‐Flag, respectively. Immunoblots of lysates from T47D and 786‐O cells infected with control shRNA or EglN1 shRNA. Immunoblots of lysates from T47D cells infected with control vector (−), EglN1‐WT, or EglN1‐P317R followed by infection with control shRNA (−) or EglN1 sh1045 (EglN1 shRNA). Immunoblots of lysates from T47D cells infected with control vector (−), EglN1‐WT, or EglN1‐▵β2β3 followed by infection with control shRNA (−) or EglN1 sh1045 (EglN1 shRNA). In vitro hydroxylation assays were performed through purified GST‐EglN1 WT or P317R mutant incubated with AMPKα1‐biotinylated synthetic peptides followed by dot immunoblot analyses with anti‐AMPKα‐Pro188‐OH antibody. Indicated peptides were incubated with whole cell lysates from 293T cells transfected with HA‐VHL, and precipitated with streptavidin. Immunoblot assays of those whole cell lysates and precipitated proteins with HA antibody, dot blot assays of the indicated peptides with biotin. Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of 786‐O cells infected with control vector or HA‐VHL followed by treatment with or without DMOG (2 mM) for 24 h. Immunoblots of lysates from 786‐O cells infected with control vector or HA‐VHL followed by treatment with or without DMOG (2 mM) for 24 h. Immunoblots of lysates from 786‐O cells expressing HA‐VHL infected with control vector (−), EglN1‐WT, or EglN1‐P317R followed by infection with control shRNA (−) or EglN1 sh1045 (EglN1 shRNA). A proposed model depicting the regulatory mechanism of mitochondrial EglN1 under normoxia. Data information: Also See Fig . Source data are available online for this figure.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Mass Spectrometry, Sequencing, Western Blot, Infection, Control, Plasmid Preparation, In Vitro, Recombinant, Protein Purification, Immunoprecipitation, Extraction, Generated, Transfection, shRNA, Purification, Mutagenesis, Incubation, Dot Blot, Expressing

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: A, B Immunoblots of lysates from T47D (A) or MDA‐MB‐231 (B) cells treated with normoxia or hypoxia (1% O 2 ) for 24 h. C Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of MDA‐MB‐231 cells infected with control vector or EglN1‐Flag followed by treatment with normoxia or hypoxia (1% O 2 ) for 24 h. D Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of T47D cells infected with control vector or EglN1‐Flag followed by infection with control sgRNA (−) or VHL sgRNA (sgVHL). E Immunoblots of lysates from T47D cells infected with control sgRNA (−) or VHL sgRNA (sgVHL). F Schematic representation of AMPKα1/2 prolyl hydroxylation identification strategy by mass spectrometry. G Intensity values of potential prolyl hydroxylation sites identified for AMPKα1 and AMPKα2, respectively, in mass spectrometry analysis. H, I MS/MS spectrum for identified hydroxylated AMPKα1 and AMPKα2 peptides at Pro188 (H) and Pro177 (I), respectively. J Immunoblots of lysates from 786O cells infected with control shRNA (−) or AMPKα1 shRNA (shAMPKα1) to verify AMPKα‐OH antibody. K Immunoblots (IB) and immunoprecipitations (IP) of 293T cells transfected with control vector, AMPKα1 WT, or its T183A mutant plasmids. L Schematic representation of the WT and prolyl‐hydroxylated biotinylated synthetic AMPKα peptides used in (Fig ). Proline site for hydroxylation was highlighted in red.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Western Blot, Infection, Control, Plasmid Preparation, Mass Spectrometry, Tandem Mass Spectroscopy, shRNA, Transfection, Mutagenesis

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: A Immunoblots of mitochondrial extracts (Mito) and whole cell extracts (WCE) from T47D cells under normoxic (N) or hypoxic (H, 1% O 2 for 24 h) conditions. B Immunofluorescence of p‐AMPKα and TOM20 with tumor tissues from breast cancer patients. Their right panels showed the quantification of fluorescence intensity of TOM20 and p‐AMPKα along the each line in merged image. C Immunoblots assays of MDA‐MB‐231 xenograft tumors from Fig . D Immunoblots of lysates from MDA‐MB‐231 cells infected with control shRNA (−) or EglN1 sh1045 (EglN1 shRNA) followed by treatment with or without CQ (25 μM) under hypoxia (1% O 2 for 24 h). E, F Immunoblots of cell lysates (E) and MTT assays (F) from MDA‐MB‐231 cells infected with control vector (Ctrl), or AMPKα2 T172D‐Flag lentivirus under normoxic or hypoxic conditions. G, H Immunoblots of cell lysates (G) and MTT assays (H) from MDA‐MB‐231 cells infected with control shRNA (shCtrl), or AMPKα1 shRNA (690, 831) lentivirus under normoxic or hypoxic condition. I, J Immunoblots of cell lysates (I) and MTT assays (J) from MDA‐MB‐231 cells infected with control shRNA (shCtrl), or AMPKα2 shRNA (171, 523) lentivirus under normoxic or hypoxic condition. K MTT assays from MDA‐MB‐231 cells treated with or without compound C (2 μM) under normoxic or hypoxic conditions. L MTT assays from MDA‐MB‐231 cells generated in Fig under normoxia. Data information: Error bars represent ± SEM, * and *** denote P value of < 0.05 and 0.005, respectively (unpaired t ‐test). n = 3 independent technical replicate experiments for MTT assays.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Western Blot, Immunofluorescence, Fluorescence, Infection, Control, shRNA, Plasmid Preparation, Generated

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: A Immunoblots of mitochondrial extracts (Mito) treated with or without Proteinase K (2 μg/ml) and whole cell extracts (WCE) from T47D cells under normoxic (N) or hypoxic (H, 1% O 2 for 24 h) conditions. B Immunoblots of mitochondrial extracts (Mito) and whole cell extracts (WCE) from T47D treated with or without compound C (Comp C, 10 μM for 24 h) under hypoxia. C Representative immunofluorescence of p‐AMPKα and TOM20 with tumor tissues from breast cancer patients. The right panel showed the quantification of fluorescence intensity of TOM20 and p‐AMPKα along the line in merged image. D Immunoblots of mitochondrial extracts (Mito) and whole cell extracts (WCE) from T47D cells infected with control vector (Ctrl), EglN1‐WT‐Flag, or EglN1‐▵β2β3‐Flag followed by infection with control shRNA (shCtrl) or EglN1 sh1045 (EglN1 shRNA) under hypoxic (1% O 2 ) condition for 24 h. E Immunoblots of cell lysates from T47D or MDA‐MB‐231 cell lines infected with control vector (Ctrl), EglN1‐WT‐Flag, or EglN1‐▵β2β3‐Flag followed by infection with control shRNA (shCtrl) or EglN1 sh1045 (EglN1 shRNA) under hypoxic (1% O 2 ) condition for 24 h. F Immunoblots assays of MDA‐MB‐231 xenograft tumors from Fig (EglN1 WT and EglN1 Δβ2β3). G Immunoblots of cell lysates from MDA‐MB‐231 cell lines generated in (E). H, I Representative fluorescence imaging ( N = 30 images in total) (H) and corresponding quantification data (I) in GFP‐LC3 stably expressed MDA‐MB‐231 cell lines generated in (E) treated with hypoxia (1% O 2 for 24 h). (scale bar = 10 μm). J, K Representative fluorescence imaging ( N = 6 images in total) of lipid droplets stained with Nile Red (J) and corresponding quantification data (K) in MDA‐MB‐231 cell lines generated in (E) treated with hypoxia (1% O 2 for 24 h). Nuclei were stained with DAPI (blue) (scale bar = 10 μm). L Immunoblots of cell lysates from MDA‐MB‐231 cells infected with control vector (Ctrl), EglN1‐WT‐Flag, or TOM20‐EglN1‐WT‐Flag followed by infection with control shRNA (shCtrl) or EglN1 sh1045 (EglN1 shRNA) under hypoxic (1% O 2 ) condition for 24 h. M, N Representative fluorescence imaging ( N = 30 images in total) (M) and corresponding quantification data (N) in GFP‐LC3 stably expressed MDA‐MB‐231 cell lines generated in (L) treated with hypoxia (1% O 2 for 24 h). (scale bar = 10 μm). O, P Representative fluorescence imaging ( N = 6 images in total) of lipid droplets stained with Nile Red (O) and corresponding quantification data (P) in MBA‐MB‐231 cell lines generated in (L) treated with hypoxia (1% O 2 for 24 h). Nuclei were stained with DAPI (blue) (scale bar = 20 μm). Data information: Error bars in (I, K, N, P) represent ± SEM, *** denotes P value of 0.005 and ns denotes not significant (unpaired t ‐test). Also See Fig . Source data are available online for this figure.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Western Blot, Immunofluorescence, Fluorescence, Infection, Control, Plasmid Preparation, shRNA, Generated, Imaging, Stable Transfection, Staining

Journal: The EMBO Journal

Article Title: A mitochondrial EglN1‐AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress

doi: 10.15252/embj.2023113743

Figure Lengend Snippet: A Immunoblots of mitochondrial extracts (Mito) and immunoprecipitations (IP) generated in (Fig ). B Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of T47D cells infected with control shRNA (−) or EglN1 sh1045 (EglN1 shRNA) with or without hypoxia treatment (1% O 2 for 24 h). C, D Immunoblots (C) and MTT assays (D) of T47D (left panel) and MDA‐MB‐231 (right panel) cells infected with control vector (Ctrl) or AMPKα‐T172D‐Flag followed by infection with control shRNA (shCtrl) or EglN1 sh1045 (EglN1 shRNA) under hypoxic (1% O 2 ) condition. E–G Mouse xenograft experiments were performed with the MDA‐MD‐MB231 cells generated in (C). Tumor growth curves (E) and tumor weights (F) were calculated, and gross tumors (G) were presented ( n = 6 mice per group). H A proposed model depicting the regulatory mechanism of mitochondrial EglN1 under hypoxia. Data information: Error bars in (D–F) represent ± SEM, *** denotes P value of 0.005 (unpaired t ‐test). n = 3 independent technical replicate experiments for MTT assays. Also See Fig . Source data are available online for this figure.

Article Snippet: T47D (ATCC HTB‐133) was maintained in RPMI (C11875500BT) medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Western Blot, Generated, Infection, Control, shRNA, Plasmid Preparation

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV envelope protein (ZIKV-E, green) and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h post-infection (p.i.) with ZIKV. Scale bar, 50 μm. (B) Quantification of infection efficiency in four GSC and NPC lines 48 h p.i. with ZIKV. (C) Quantification of ZIKV+ cells in a panel of human GSCs and NPCs. (D) Kinetics of viral RNA copies p.i. with ZIKV by measuring viral RNA copies by qRT-PCR in NPC C4–7 and GSC3565. (E) ZIKV infection efficiency of GSCs and NPCs was measured by direct measurement of viral RNA copies. (F) Representative bright-field images 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. Scale bars, 50 μm. (G) Cell viability normalized to day 5 mock, as measured 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. (H) GSCs (GSC3565), differentiated GSCs, NPCs (NPC C4–7), and differentiated NPCs were assayed for cell viability 72 h p.i. with ZIKV. (I) Apoptosis of GSCs (387, 3565) and primary (NPC194, fetal human [fh] NPC) or iPSC-derived NPCs (WT83, C4–7) p.i. with ZIKV was measured by cleaved caspase-3 (CC3) staining. (J) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (K) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 72 h p.i. with ZIKV. Scale bars, 50 μm. (L) Quantification of the percentage of CC3+ cells in DAPI+ cells for GSCs and NPCs 72 h p.i. with ZIKV. (M) Cell viability of patient-derived cultures from GBM (387 and 3565), pontine glioma (3752 and 007), meningioma (CH-157MN, IOMM-LEE), ependymoma (EP1), and medulloblastoma cell lines (DAOY, D283, HDMB03, D341) 72 h after ZIKV infection. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. NS, no significance. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Quantitative RT-PCR, Derivative Assay, Staining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV-E (green), SOX2 (red), and DAPI (blue) of GSCs and forebrain-specific hiPSC-derived NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (B) Quantification of the percentage of SOX2+ cells in DAPI+ cells for GSCs and NPCs 48 h p.i. with ZIKV. (C) Representative immunostaining for ZIKV-E (green), SOX2 or AXL (red), and DAPI (blue) of GSCs (GSC3565) without transduction (shRNA) or transduced with control shRNA (shCONT), AXL shRNA (shAXL.2), or SOX2 shRNA (shSOX2.53) for 72 h and then 48 h with ZIKV infection. Scale bars, 100 μm. (D) Quantification of the percentage of ZIKV+ cells in DAPI+ cells in GSCs 1517 and 3565 under conditions for (C), with a range of ZIKV infection. (E) Viral copy number by qRT-PCR of GSCs (GSC3565 or GSC1517) or NPC C4–7 transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53) for 72 h and then either exposed to mock conditions or infected with ZIKV for another 72 h. All comparisons are versus shCONT. (F) Gene set enrichment (GSE) bubble plots showing pathways positively (top, r > 0.4) or negatively (bottom, r < −0.4) correlated with SOX2 expression in the TCGA GBM HG-U133A microarray dataset. Each circle represents an enriched pathway, with the border color indicating the false discovery rate (FDR)-corrected p value. (G) GSE graph showing the top pathway enrichments positively or negatively correlated with SOX2 as described in (F). (H) Correlation of mRNA levels of SOX2 with IFNAR1, IRF1, promyelocytic leukemia (PML), and IFITIM1 from the TCGA GBM HG-U133A microarray dataset. (I) Correlation between SOX2 with ISGs from the TCGA GBM HG-U133A microarray dataset. The size and color of the dots indicate the degree of correlation (p < 0.001). Blank cells indicate a non-significant correlation. (J) qPCR of ISGs (IFNAR-1, ISH20, IRF1, IFITM1, TLR3, and OAS2) in GSCs (GSC3565) transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53). Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. **p < 0.001, ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Derivative Assay, Transduction, shRNA, Control, Infection, Quantitative RT-PCR, Expressing, Microarray

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative images of mock- or ZIKV-infected BCOs stained with neuronal markers (CTIP2 and NeuN), a neural progenitor cell marker (SOX2), and DAPI. Scale bars, 100 μm. (B) Quantification of BCO size p.i. with ZIKV. Significance was assessed by two-tailed Student’s t test, and experiments were performed in two batches with 12 organoids per group per batch. (C) BCO size fold change of ZIKV- and mock-treated groups over a period of 1 month. (D) Quantification of SOX2+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (E) Quantification of CC3+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (F) Quantification of SATB2+ cells within MAP2+ cells in ZIKV- versus mock-infected groups. **p < 0.01 by two-tailed Student’s t test. (G) Quantification of GFAP+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (H) Quantification of NeuN+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (I) Quantification of CTIP2+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (J) Bright-field images of engraftment of two patient-derived GSCs (387 and 3565) transduced with GFP into human BCOs over a time course. Scale bars, 1 mm. (K) Engrafted GSCs (GFP+) with normal BCO immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 200 μm. (L) Quantification of integrin αvβ5+ cells in normal BCOs or GSC-BCOs. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (M) Representative images of GFP-labeled GSC-BCOs immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (N) Representative images of GFP-labeled GSC-BCOs immunostained for SOX2 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (O) Images of GFP-labeled GSC-GFP BCOs 13 days p.i. with ZIKV. Scale bars, 1 mm. (P) Representative images of residual GSCs (green) and DAPI staining (blue) of GFP-labeled GSC-GFP BCOs cultured under mock conditions or with ZIKV for 2–4 weeks. Scale bars, 200 μm. The percentage of GFP+ cells among DAPI+ cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-way ANOVA. (Q) Representative immunostaining for integrin αvβ5 (red), GFP (green), ZIKV-E (white), and DAPI (blue) of GFP-labeled GSC-GFP BCOs mock- or ZIKV-infected for 2–4 weeks. Scale bars, 200 μm (left) and 100 μm (center). The percentage of ZIKV-E+ cells among integrin αvβ5 cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (R) Representative images of 387 and 3565 GSC-BCOs with or without ZIKV, respectively, stained with SOX2, ZIKV-E, and DAPI. GFP shows the presence of GSCs (scale bars, 50 μm). ZIKV-E+, GFP+, and ZIKV-E+ cells among GFP+ cells were quantified by counting (two GSCs cell lines, two repeats, n = 12 organoids/group); *p < 0.05 by two-tailed Student’s t test. (S) Schematic of the experiment design. (T) Volcano plot showing differences between GSC-BCO ZIKV versus GSC-BCO mock. 113 genes were differentially expressed (greater than 1.5-fold) between these two groups (*p < 0.05). (U) Network analysis of genes differentially expressed upon ZIKV infection, represented as a bubble plot.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Infection, Staining, Marker, Two Tailed Test, Derivative Assay, Transduction, Labeling, Cell Culture, Immunostaining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Immunostaining of the subventricular zone (SVZ) of mice 72 h following ZIKV infection ZIKV-E (green), SOX2 (red, top panels), and integrin αvβ5 (red, bottom panels). Scale bars, 50 μm. (B) Higher magnification of images from (A), demonstrating ZIKV infection of SOX2+ (top panels) and integrin αvβ5+ cells. Scale bars, 10 μm. (C) Survival of ZIKV-infected NSG mice from (A) was plotted by the Kaplan-Meier method. (D) ZIKV-infected brains from the mice in (A) were collected upon death, and histology was assessed by H&E staining. Scale bars, 20 μm. (E) Survival of NSG mice following implantation of GSCs treated with isotype control, P1F6 antibody, ZIKV, combined P1F6 and ZIKV, combined CRISPR knockout (KO) of integrin β5 (sgRNA1 sgRNA2) with ZIKV inoculation, analyzed by log rank test; p < 0.01. (F) H&E staining of tumor-bearing brains from (E). Scale bars, 50 μm. (G) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection (10e3 FFU). Slices then underwent immunofluorescence staining for ZIKV-E (green), integrin αvβ5 (red), and DAPI (blue). Scale bars, 10 μm. (H) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection. Slices then underwent a viral RNA copy assay by qRT-PCR. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Staining, Control, CRISPR, Knock-Out, Incubation, Blocking Assay, Immunofluorescence, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Heatmap generated with qPCR data of DTFs and various genes related to three biological alterations. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black, as generated by Cluster 3.0. b. IHC of proteins related to normal renal function (KNG1, AQP2, SCNN1B). c. Immune function (TLR2, CXCR4). d. Metabolic function (ENO2, CYP2J2,ALDOB). This pattern of expression is in accord with the microarray findings. Sum scores are shown with n as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Generated, Expressing, Microarray

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. A microarray heatmap showing significant downregulation of four developmental transcriptional factors in ccRCC and a table showing fold changes of DTFs and their known renal developmental function. b. IHC validation of decreased expression of TFAP2B, DMRT2, and TFCP2L1. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match. c. Microarray heatmap showing downregulation of TFCP2L1 and its regulated genes in ccRCC. S–Stage, N–normal, T–tumor. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in yellow, as generated by Genetree.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Microarray, Biomarker Discovery, Expressing, Generated

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Heatmap showing adipogenic gene expression signature in ccRCC. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black. b. IHC showing lipid-laden clear cell morphology of ccRCC, increased expression of ADFP, and decreased expression of GATA2 in ccRCC. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Cellular differentiation experiments showing that KIJ-308 and KIJ-265 ccRCC cells are capable of adipogenic differentiation and become lipid-laden in adipogenic media, as indicated by Oil Red ‘O’ staining. Normal patient-matched cells were unable to differentiate. b. A498 ccRCC cells are also capable of adipogenic differentiation, as indicated by Oil Red ‘O’ staining, unlike normal renal canine MDCK cells. c. Under adipogenic media conditions, ccRCC also produces glycogen, as shown by a PASH stain.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Cell Differentiation, Staining

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Cellular differentiation experiments showing that KIJ-308 and KIJ-265 ccRCC cells are capable of osteogenic differentiation in osteogenic media by developing calcium deposits, as shown by Alizarin Red stain. Normal patient-matched cells were unable to differentiate. b. A498 ccRCC cells are also capable of osteogenic differentiation, as shown by Alizarin Red stain, unlike normal renal canine MDCK cells.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Cell Differentiation, Staining

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. A heatmap showing the increased expression of some markers associated with EMT. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black. b. IHC validation of two known markers of EMT: vimentin and N-cadherin. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Expressing, Biomarker Discovery

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: During normal renal development, mesenchymal stem cells undergo mesenchymal epithelial transition (MET) to develop into normal renal epithelial cells (NREs). In renal carcinogenesis, NREs undergo de-differentiation and epithelial mesenchymal transition (EMT), followed by adipogenic differentiation to develop into ccRCC.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques:

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Infection Causes a Significant Increase in the Protein HSp27, Accompanied by Large Spatial Accumulation of Hsp27 with the Bacteria in a Temporal Manner in Primary GECs. ( A ) Representative confocal microscopy images of P. g -infected human primary GECs at an MOI 100, at 6 h and 24 h after infection. GECs were then stained for P. g (rabbit anti-P. g; Alexa 488; green) or HSp27 (mouse anti-HSp27; Alexa 568; red). GECs were then imaged via the Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. ( Ai ) Imaris Software was used to create a zoomed image of infected GECs and was used to calculate the amount of co-localization between P. g and HSp27. HSp27 was found to readily colocalize with P. g , having an average Pearson correlation coefficient of 0.87 via the Imaris software. Scale bar is 30 µm for 63x and Zoomed Magnification. (B ) P. g was added at MOI 100 to GECs, which were incubated 6 or 12h. Cell lysates were then analyzed via western blot. ( Bi ) Quantitative ImageJ analyses of western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as significant via Two-Tailed Student T-test. *p<0.05.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Bacteria, Confocal Microscopy, Staining, Software, Incubation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Integrity of P. gingivalis (P. g) -Specific Autophagosomes is Highly Dependent on HSp27 Presence. Human primary GECs were treated with HSP27siRNA (100nM) for 48 h prior to incubation with P. g ( MOI 100) for 6 h. Autophagosomes were then isolated and analyzed via Confocal Microscopy. ( A ) Schematic autophagosomal isolation method of infected GECs. ( B ) Confocal microscopy images of autophagosomes (ThiolTracker Violet; blue) were obtained via Super Resolution Zeiss Airyscan LSM 880 at 20x. ( C ) Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal microscopy images of autophagosomes were obtained via Super Resolution Zeiss Airyscan LSM 880 at 20x. ( Ci ) Quantitative ImageJ analysis of Confocal microscopy results was then performed. Data is represented as Mean±SD, where n=25 and p<0.05 was considered as statistically significant (Student two-tailed T-test). **p<.005

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Incubation, Isolation, Confocal Microscopy, Infection, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Magnetic-Labelling Process Does Not Impact P. gingivalis (P. g) Infection in GECs. P. g was labeled with lipobiotin (5 μM) and were then incubated in MagCellect Streptavidin Ferrofluid. Human Primary GECs were then infected for 24 h. Representative confocal microscopy images of P. g- infected GECs at an MOI 100, were taken at 24 h after infection using via Zeiss LSM 880 (63x). P. g (rabbit anti-P . gingivalis ; Alexa 488; green) was detected in the GECs. Actin (red) was stained utilizing Rho-Phallodin.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Labeling, Incubation, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Intracellular P. gingivalis ( P. g ) Significantly Induces and Co-Localizes with LC3C, an Isomer of LC3, and this Specific Event is Highly Dependent on HSp27 for Successful Autophagic Survival. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated for 6 or 24 h. ( A ) GECs were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Following HSp27 depletion, P. g appeared to readily start to degrade. Representative transmission electron microscopy images of P. g -infected GECs were also taken at 80 kV and 100000x magnification. Scale bar is 800 nm. ( B ) 6 h and 24h P. g-infected GECs were also stained for P. g (rabbit anti-P . g ; Alexa 488; green) and LC3C (mouse anti-LC3C; Alexa 568; red) to examine whether LC3C characterizes P. g -specific autophagosomes. These cells were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. The range of z-stacks was kept consistent and representative images were selected from the mid-ranged sections. ( Bi ) The Imaris software was utilized to obtain a xoomed 63x Orthogonal Image of 24 h P. g infection and found heightened co-localization between P. g and LC3C. LC3C was found to readily colocalize with P. g, having an average Pearson correlation coefficient of 0.96 via the Imaris post-processing software. ( C ) Lysates of infected and HSp27-depleted GECs were also analyzed via western blotting. Non-target controls were performed and not shown. (Ci) Quantitative ImageJ analysis was performed for the western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. **p<.005.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Incubation, Labeling, Transmission Assay, Electron Microscopy, Infection, Staining, Confocal Microscopy, Software, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 Presence Permits the Prolonged Existence of LC3C-characterized, P. gingivalis Specific Autophagosomes by Hampering Canonical Autolysosomal Fusion in Primary GECs. ( A ) Human primary GECs were transfected with mCherry-eGFP-LC3C for 48 h. Select GECs were also treated with 1 uM of the autophagolysosomal fusion inhibitor Bafilomycin A1, 1 uM Pepstatin A, or 5 mM 3-MA. Others were treated with Hsp27 siRNA (100nM) for 24 h. P. g was added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs were then stained for P. g (mouse anti- P.g; Alexa 405; blue) and were mounted. GECs were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. ( Ai ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the high co-localization levels between P. g and the LC3C Reporter System. P. g localized readily to the LC3C construct, with a Pearson correlation coefficient of 0.82. (B) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Scale bar is 40 µm for 63x Magnification. ( Bi ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the co-localization levels between P. g and LAMP-1 in infected and treated GECs. While P. g infected GECs did not exhibit high co-localization with LAMP-1 (Pearson correlation coefficient of .25), their HSp27-depleted counterparts did, with an average Pearson correlation coefficient of 0.83. The Scale bar is 20 µm for 63x Magnification. ( C ) Finally, GECs treated with autophagic inhibitors were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Following HSp27 depletion, P. g appeared to readily start to degrade, however treatment with late-stage autophagic inhibitors Bafilomycin A1 or Pepstatin A appeared to rescue P. g from degradation. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Scale bar is 800 nm.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Infection, Construct, Labeling, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Depletion of LC3C via siRNA Collapses P. gingivalis (P. g) -Induced Non-Canonical Autophagosomal Integrity. Human primary GECs were treated with LC3C siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6, 12, or 24 h. ( A ) Intracellular P. g survival after LC3C siRNA depletion was determined using a standard antibiotic protection assay. In brief, any extracellular bacteria were killed via 1h gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular levels of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005. ( B ) P. g -specific autophagosomes were also selectively isolated. Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal images of P. g -specific autophagosomes at 6 h post-infection (63x) were taken utilizing the Super Resolution Zeiss Airyscan LSM 880.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Bacteria, Synthesized, Two Tailed Test, Isolation, Staining, Infection

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Δ ndk-P. gingivalis (P. g) Undergoes Canonical Degradative Autophagosomal Trafficking in Human Primary GECs. Primary GECs were infected with WT P. gingivalis (P. g) versus Δ ndk - P. g at MOI 100 for 3, 12, or 24h. ( A ) TEM analysis used Immunogold labeling for P. g was performed on WT P. g versus Δ ndk - P. g at 3 or 24h post-infection. Images were acquired at 40000x magnification utilizing a Hitachi H-7000 TEM (Hitachi High Technologies America, Inc.) affixed to a Veleta camera with iTEM. ( B ) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Co-localization analysis of P. g and LAMP-1 was additionally carried out using the Zeiss LSM 880 Confocal Software, determining that the WT P. g had a Pearsons value of .137 while the Δ ndk - P. g had a Pearsons value of .704.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Labeling, Staining, Software

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Depletion of HSp27 Abrogates the Inhibition of Oxidative Stress and Severely Impacts the Intracellular Survival of P. gingivalis (P. g) Studied in Human Primary Organotypic Cultures of Gingiva. To create the organotypic culture systems, human primary GECs and Fibroblasts Cells (FBCs) were co-cultured together upon a collagen raft. Select rafts were then treated with HSP27 siRNA (100nM) for 48 h. Select rafts were also treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h. P. g was added at MOI 100 to rafts, which were incubated for 24 h. Rafts were then collected and sectioned so that immunofluorescence could be performed. ( A ) Representative images of H&E stained raft culture systems at 20x magnification, which clearly mimic the oral gingival crevice. Scale bar is 50 µm. E: Multilayer Undifferentiated Epithelial Cells, C: Collagen Matrix; F: Fibroblasts. ( B ) Rafts were stained for P. g (rabbit anti- P. g; Alexa 488; green) or HpS27 (mouse anti-HSp27; Alexa 568; red). Rafts were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 20x and 63x. Scale bar is 50 um for 20x and 20 um for 63x and Zoomed View. The range of z-stacks was kept consistent. HSp27 was once again found to readily co-localize with P.g with a Pearson coefficient of .83 as determined via the Imaris Software. ( Bi ) A Zoomed (4x) version of the 63x magnification was created using the Imaris Software, highlighting the intracellular nature of individual P. g. ( C ) GECs were additionally treated with HSp27 siRNA (100nM) for 48 h. Select GECs were then treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h and/or eATP (3mM) for 30 min. P. g was added at MOI 100 to GECs, which were incubated 6 h. If any extracellular bacteria were present, they were killed by gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment for 1 h. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular level of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via One-Way Anova Test. ** p<0.005.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Inhibition, Cell Culture, Incubation, Immunofluorescence, Staining, Confocal Microscopy, Software, Bacteria, Synthesized

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Cross-Sectional Human in Situ Sample and Expression Analyses Support High Levels and Increased Co-localization of P. gingivalis (P. g) , HSp27, and LC3C in Periodontitis-Afflicted Oral Tissues. Publicly available mRNA expression data (GEO accession: GSE79705) was obtained from previously collected and examined periodontitis-afflicted and healthy gingival tissues. This microarray expression data was then analyzed via GEO2R and the relative levels of ( A ) HSp27 and ( B ) LC3C were obtained and compared. Data is represented as Mean±SD, where n=12 and p<0.05 was considered as statistically significant via One-Way Anova. *p<0.05. Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis-afflicted patients were also taken and examined. DAPI staining was utilized to visualize cellular DNA. ( C ) P. g (mouse anti-P . gingivalis ; Alexa 488; green) and LC3C (rabbit anti-LC3C; Alexa 594; red) were detected via dual staining. ( D ) HSp27 (mouse anti-HSp27; Alexa 488; green) and LC3C detection (rabbit anti-LC3C; Alexa 594; red) were also detected. Images were then captured using super resolution confocal laser scanning microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 10x and 63x magnification with oil immersion. Zoomed (2x) 3D versions of the 63x magnifications were obtained via the Imaris software. The range of z-stacks was kept consistent. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars = 200µm for 10x and 20 µm for 63x. Quantification of mean fluorescence intensity provided in Supplement. LC3C and HSp27 both were found to exhibit high levels of co-localization with each other and with P. g, as the Pearson coefficient was calculated to be .93 for LC3C and P. g and .85 for LC3C and HSp27 via the Imaris Software at the most severe state of disease.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: In Situ, Expressing, Microarray, Staining, Confocal Laser Scanning Microscopy, Software, Fluorescence

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 inhibits tube formation and influences osteomimicry. (A) Tube formation assay with constitutively transfected A673 (sh.control and sh. CHM 1) and transiently transfected MHH ‐ ES 1 (si.control and si. CHM 1_1) cells demonstrated CHM 1 to clearly inhibit endothelial differentiation potential (scale bar 0.5 mm). (B) Analysis of osteolysis of A673 sh. CHM 1 and negative controls (sh.control) in an orthotopic bone xenotransplantation model (five to eight mice per group). Affected bones were assessed by histology ( TRAP staining, scale bar 0.25 mm or 0.05 mm). Left panel: quantitative summary of the average number of osteoclasts (mm 2 ) in unaffected bone marrow, tumor samples, and attached to the bone in tumor tissues (bone). Data are mean ± SEM of at least two independent samples (at least 40 segments counted); t ‐test. Right panel: Representative pictures are shown. CHM 1 knockdown significantly enhanced the amount of TRAP ‐positive osteoclasts attached to the bone (b) in the area of tumor (arrow) and thus increased the osteolytic phenotype. (C) Different ES cell lines with constitutive CHM 1 knockdown and respective controls were analyzed by qRT ‐ PCR for expression of osteolytic genes such as HIF 1A , IL 6 , JAG 1 , and VEGF . Data are mean ± SEM of two independent experiments; t‐ test. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: Tube Formation Assay, Transfection, Control, Staining, Knockdown, Quantitative RT-PCR, Expressing

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 delayed proliferation in ES in vitro . (A) Analysis of contact‐dependent growth of constitutively sh. CHM 1‐ and sh.control‐infected ES cell lines with xCELL igence. Left panel: Cellular impedance was measured every four hours (relative cell index). Data are mean ± SEM (hexaplicate/group); t ‐test. Right panel: doubling time of constitutive A673, SK ‐N‐ MC , and TC ‐71 CHM 1 sh RNA infectants. Data are mean ± SEM of two independent experiments/cell line (hexaplicate/group); t ‐test. B. Effect of CHM 1 knockdown on anchorage‐independent growth in A673, SK ‐N‐ MC , and TC ‐71 cells using methylcellulose matrices. Left panel: A representative experiment with SK ‐N‐ MC cells was shown as macrograph. Right panel: The average number of colonies of at least two different experiments with three different ES cell lines was shown after stable CHM 1 suppression. (C) Left panel: evaluation of tumorigenicity of constitutive A673 and TC ‐71 CHM 1 sh RNA infectants in immunodeficient Rag2 −/− γc −/− mice (3–5 mice per group). Right panel: post ex vivo CHM 1 expression using qRT ‐ PCR . Data are mean ± SEM , t ‐test. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: In Vitro, Control, Infection, Knockdown, Ex Vivo, Expressing, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 enhances metastasis in ES in vivo . (A) Analysis of invasiveness of ES cell lines through Matrigel after transfection with specific CHM 1 sh RNA constructs. Data are mean ± SEM of two independent experiments; t ‐test. (B) Upper panel: qRT ‐ PCR of MMP 9 expression after stable CHM 1 knockdown. Data are mean ± SEM of two independent experiments; t ‐test. Lower panel: analysis of the invasive potential of A673 and SK ‐N‐ MC cells after transient transfection with two specific MMP 9 si RNA s 48 h before seeding. Data are mean ± SEM ; t ‐test. (C) Analysis of metastasis using A673 and TC ‐71 cells with stable CHM 1 suppression and respective controls (four to five mice per group). Left panel: Representative lungs with corresponding H&E staining of A673‐injected mice are shown (scale bar 5 or 2 mm). Right panel: Average number of apparent metastases per mouse in lung and liver tissues is illustrated; t ‐test. (D) DotBlot of relative CHM 1 expression in ES osseous tumor samples compared to ES lung metastases samples using microarray analysis of 14 patient tumor samples. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: In Vivo, Transfection, Construct, Quantitative RT-PCR, Expressing, Knockdown, Staining, Injection, Microarray

Journal: Nucleic Acids Research

Article Title: Validation of human microRNA target pathways enables evaluation of target prediction tools

doi: 10.1093/nar/gkaa1161

Figure Lengend Snippet: HiTmIR overview and representative selection of miR-34a. ( A ) Combined experimental and computational workflow of HiTmIR. Three computational steps are carried out consecutively before target gene sets are validated by an automated reporter assay. ( B ) Immunocytochemistry of D2R expression in differentiated LUHMES cells. ( C ) Immunocytochemistry of TH expression in differentiated LUHMES cells. (B, C) Expression of dopaminergic markers in differentiated LUHMES cells were analyzed by immunocytochemistry with antibodies against TH and D2R. The nuclei were visualized by DAPI staining. Scale bars are 25 μm. ( D ) Heatmap of the 50 most down-regulated miRNAs in LUHMES cells that were differentiated toward dopaminergic neurons and treated with MPP+ to induce a PD-like phenotype. ( E ) Heatmap of the 50 most up-regulated miRNAs. (D, E) Shown are z-scores of quantile-normalized expression values. ( F ) Validation of microarray results by qRT-PCR of up-regulated and down-regulated miRNAs. Bars present the log 2 fold change between PD-like and controls together with the respective standard deviation. ( G ) Increased expression of miR-34a-5p in the blood of patients, spanning an age range from 20 to 80 years. The orange line shows a smoothed spline with 8 degrees of freedom and the shaded area represents the 95% confidence interval.

Article Snippet: Lund human mesencephalic (LUHMES) cells were purchased from the American Type Culture Collection (ATCC) and transfected for GFP-expression.

Techniques: Selection, Reporter Assay, Immunocytochemistry, Expressing, Staining, Biomarker Discovery, Microarray, Quantitative RT-PCR, Standard Deviation

Journal: Molecular Cancer

Article Title: SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex

doi: 10.1186/s12943-018-0824-y

Figure Lengend Snippet: SALL1 expression is down-regulated in human breast cancer. a and b Gene expression levels of SALL1 in different cancer cell lines (in a ) and in tumor tissues (in b ) using Real-time PCR analyses. Tumor cell lines include breast cancer (human MDA-MB-231, MCF7, BC80, 31, 30, 29, 16, 12, and 10), melanoma (human Mel1938, Mel1586, Mel1860, Mel1363, Mel1526 and Mel1628), prostate cancer (PC3 and DU145), colon cancer (SW480), and lymphoma (L428 and L504). Normal breast cell lines (BN6, BN16, MCF10A and MCF12A), Fibroblasts (F163, F160, F158 and F112) and 293 T cells were included as controls. mRNA levels in each cell line and tissue were normalized to the relative quantity of GAPDH expression and then adjusted to SALL1 levels in 293 T cells (set as 1). Results shown in the histogram are mean ± SD from three independent experiments. c and d Association analyses of SALL1 expression with specific breast cancer subtypes. The data sets were accessed from the TCGA breast cancer Argilent microarray expression database downloaded from the cBioPortal ( http://www.cbioportal.org /). The box plot indicated the log 2 transformed mRNA median expression level of SALL1 in the tissues. N indicated the number of sample size of each tissue type. Mann-Whitney analysis was used to compare the SALL1 expression across the different breast cancer subtypes and normal tissues, and ** p < 0.01 within the comparison groups. e SALL1 expression in tumor cells in breast cancer tissues was determined using the immunohistochemical staining. f and g SALL1 expression levels in breast cancer tissues with different ER and HER2 status. SALL1 + cell population in ER + patients was significantly higher than that in ER − patients. Furthermore, SALL1 + cell numbers in HER2 + patients were much higher than that in HER2 − patients. Tissue immunohistochemical staining and cell number counting were identical as in ( e ). Significance was determined by unpaired T test

Article Snippet: Breast tumor cell lines (human MDA-MB-231, MCF7, BC80, 31, 30, 29, 16, 12, 10, and murine 4 T1 and E0771), Melanoma cell lines (Mel1938, Mel1586, Mel1860, Mel1363, Mel1526 and Mel1628, and murine B16F0), prostate cell line PC3 and DU145, colon cancer cell line SW480 and lymphoma L428 and L504, as well as normal breast cells and fibroblast cells, were either obtained from the American Tissue Culture Collection (ATCC) or established by our group, and maintained in RPMI 1640 medium containing 10% fetal calf serum (FCS) and penicillin-streptomycin (Invitrogen, Inc. San Diego, CA).

Techniques: Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Microarray, Transformation Assay, MANN-WHITNEY, Comparison, Immunohistochemical staining, Staining

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Effects of hypoxia on cell markers, morphology, and migration (A) Expression in breast cancer MCF7 and MDA-MB-231 cells of E-cadherin (epithelial marker), fibronectin (epithelial marker), HIF-1α (hypoxia marker), β-catenin, and GLUT1. Cells were cultured at 37°C in 5% CO 2 atmosphere for normoxic treatment, and in 1% O 2 / 5% CO 2 / 94% N 2 atmosphere for hypoxic treatment. Cells were harvested, lysed in T-PER Reagent, and protein content was determined by BCA assay. Western blotting was performed as described in M&M. (B) Morphological changes under normoxic and hypoxic conditions. Cells (2 × 10 5 per well) were grown in 6-well plates for 24 h under the two conditions. Photos were taken by phase-contrast microscopy at 200× magnification. (C) Cell migration assessed by wound assay. Cell monolayers under the two conditions were scratched with pipette tip. Cells were washed with ice-cold 1× PBS and cultured in serum-free medium. Pictures of wounds were taken at 0 and 24 h by phase-contrast microscopy (100× magnification).

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, Expressing, Marker, Cell Culture, BIA-KA, Western Blot, Microscopy, Transferring

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MALDI-TOF-MS spectra of N-glycans from MCF7 cells MCF7 cells were cultured in 10-cm dishes under normoxic and hypoxic conditions, and N-glycans were separated and desalted as described in M&M. Lyophilized N-glycans were dissolved in MW, and an aliquot of mixture with DHB solution was spotted on MTP AnchorChip sample target and air-dried. MALTI-TOF-MS was performed in positive-ion mode. Experiments were performed in biological triplicate, and representative N-glycan spectra are shown. Peaks (signal-to-noise ratio > 5) were selected for relative proportion analysis. Detailed structures were analyzed using the GlycoWorkbench program. Proposed structures are indicated by m/z value.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Cell Culture, Glycoproteomics

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Relative proportions of various types of N-glycans in MCF7 and MDA-MB-231 cells under normoxia and hypoxia.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques:

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Variation of fine glycan structures detected by lectin microarray analysis (A) Variation of levels of glycans from MCF7 (upper) and MDA-MB-231 (lower) cells, detected by 37 lectins, is presented as a heatmap. Lectin microarray analysis was performed as described as M&M. Red: fluorescence signal activation. Green: signal inhibition. Black: missing data. (B) Altered glycan levels evaluated by lectin histochemistry. Four lectins (Con A, MAL-I, LCA, PHA-E) were applied, and lectin histochemistry was performed as described in M&M. Signals are shown from merge images of Cy3-conjugated lectins and DAPI staining of nuclei in MCF7 (left) and MDA-MB-231 (right) under normoxic and hypoxic conditions (60× magnification). (C) Expression in MCF7 and MDA-MB-231 cells of HIF-1α, MGAT3, and tubulin.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Glycoproteomics, Microarray, Fluorescence, Activation Assay, Inhibition, Staining, Expressing

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Differential glycopatterns in normoxia- vs. hypoxia-treated MCF7 cells revealed by lectin microarray analysis.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Microarray

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MGAT3 overexpression suppresses hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-transfected MCF7 cells. Cells were stably transduced with a GFP-marked lentivirus carrying mock gene or MGAT3 gene, harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M using anti-MGAT3 and anti-GFP antibody. (B) Levels of bisecting GlcNAc structures in mock- and MGAT3-transfectants. Whole cell lysates of the two transfectants were subjected to PHA-E lectin blotting as described in M&M. (C) Proliferation of transfectant cells. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (E) Colony formation ability. The two transfectants (2500 cells each) were cultured in 6-cm dishes for 1–2 week, fixed, stained with crystal violet solution, and photographed. Acetic acid was added to dissolve crystal violet, and OD 595 was determined (D) . * p < 0.05; *** p < 0.001. (F) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (H) . * p < 0.05. (G) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure , harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Expressing, Transfection, Stable Transfection, Transduction, Western Blot, Cell Culture, MTS Assay, Staining, Migration, BIA-KA

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MGAT3 knockdown promotes hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-shRNA-transfected MCF7 cells. Cells were stably transduced with lentivirus carrying anti-MGAT3 shRNAs (MCF7/shMGAT3-1/2) or shNC (MCF7/mock), harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M. (B) Cell proliferation. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (C) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (E) . * p < 0.05; ** p < 0.01. (D) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, β-catenin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure , harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Knockdown, Expressing, shRNA, Transfection, Stable Transfection, Transduction, Western Blot, Cell Culture, MTS Assay, Migration, BIA-KA

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Upregulation of the transcription factors POU3F2 and NTF3 during neuronal differentiation of NT2D1. a The protocol for neuronal induction of NT2D1 cells is schematized. b β3-tubulin staining for neuronal cells in NT2D1 cells untreated (non) and treated with neuronal induction medium at the indicated time points. c Quantification of β3-tubulin-positive cells. d Immunoblotting analysis for POU3F2, POU3F3, β3-tubulin, and NTF3 in NT2D1 cells untreated or treated with neuronal induction medium at the indicated time points. The values show the expression relative to that of untreated cells (to which a value of 1 was assigned). e Microarray analysis showed that neuronal induction for 6 h increased the expression of NTF3 and GADD45 in NT2D1 cells. f NTF3 mRNA expression after neuronal induction was analyzed by real-time PCR. The levels of mRNA were calculated as the relative expression compared with that of non-induced NT2D1 cells. GAPDH mRNA was used as a control. * p < 0.05; *** p < 0.001. g Phospho-TrkC (Tyr820) staining in treated and untreated NT2D1 cells. Values are presented as mean ± SEM of three independent experiments for c and f

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Staining, Western Blot, Expressing, Microarray, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Identification of the POU3F2 binding site on the NTF3 promoter. a Transcription factor response elements predicted by the Transcription Element Search System for the nucleotide sequence of the NTF3 promoter region (− 1823 to + 243). The transcription start site is indicated as + 1. b Comparison of NTF3 promoter sequence conservation between different species. c Biotin-labeled oligonucleotides containing the intact or mutated POU3F2 binding site were hybridized with total lysates prepared from NT2D1 cells. The POU3F2-DNA complexes were precipitated by streptavidin agarose beads. POU3F2 was analyzed by Western blot analyses. The input of nuclear extracts was used as loading control. Three independent experiments were performed. d Chromatin was prepared from NT2D1 cells treated with induction medium for 0, 2, and 6 h. Cell lysates were mixed with antibodies against POU3F2 or IgG and then precipitated. The precipitates were analyzed by PCR for the presence of the NTF3 promoter sequence. The DNA purified from the sonicated chromatin was directly analyzed by PCR using the ChIP primer, which was used as an input control (Input). e The values of the ChIP DNA were normalized to that of the NT2D1 cells at 0 h (as a control). Values of fold-change over the control are presented as mean ± SEM of three independent experiments for d . * p < 0.05 compared with the control

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Binding Assay, Sequencing, Comparison, Labeling, Western Blot, Control, Purification, Sonication

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 on NTF3 promoter activity. a Schematic representation of NTF3-luciferase chimeric constructs. The negative numbers refer to the numbers of bases upstream of the transcription start (+ 1) site of the NTF3 gene. b NT2D1 cells were transiently transfected with the pGL3 basic vector or NTF3 promoter constructs of different lengths. The luciferase activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. c NT2D1 cells were transfected with the pGL3 basic vector, pNTF3-1902, and pNTF3-1902 POU3F2 mut. Approximately 24 h later, cells were treated with neuronal induction medium. The transcriptional activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. Values are presented as mean ± SEM of three independent experiments for b and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 silencing on neuronal differentiation and NTF3 expression in NT2D1 cells. a POU3F2 expression in POU3F2-knockdown (shPOU3F2) and control (shLuc) NT2D1 cells was determined by Western blot analyses after neuronal induction for 6 h. GAPDH was used as a loading control. The values represent the relative expression compared with that of the non-induced shLuc cells (to which a value of 1 was assigned). b NTF3 mRNA expression of the cells described in a was analyzed by real-time PCR. mRNA levels were calculated relative to that of the non-induced shLuc cells. * p < 0.05; *** p < 0.001. c Neuronal morphology of shLuc and shPOU3F2 cells that were treated with neuronal induction medium for 24 h or left untreated (non). d Quantification of cell numbers of shLuc and shPOU3F2 described in c . All the percentages of the shLuc and shNTF3 cells were compared to that of the non-induction shLuc cells (to which a value of 100% was assigned). e β3-tubulin staining was performed on shLuc and shPOU3F2 cells, which were treated with neuronal induction medium for 0, 6, or 24 h or left untreated, after which neuronal cells were detected. Values represent the mean ± SEM of three independent experiments for b and d

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Expressing, Knockdown, Control, Western Blot, Real-time Polymerase Chain Reaction, Staining

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of NTF3 silencing and NTF3 recombinant protein treatment on the viability and neuronal differentiation of NT2D1 cells. a NTF3 mRNA levels in NTF3-knockdown (shNTF3) and control (shLuc) NT2D1 cells, which were treated with neuronal induction medium for 0, 24, or 48 h or left untreated (Non), were determined by real-time PCR. mRNA levels were calculated as the relative expression compared with the untreated shLuc cells. *** p < 0.001. b Phase contrast microscopy images of untreated shLuc and shNTF3 cells and those cells 24 h after neuronal induction with concomitant treatment of rNTF3 (5, 20 ng/ml) or vehicle. c Quantification of neuron number of shLuc and shNTF3 cells as described in b . All the percentages of neurons differentiated from shLuc and shNTF3 cells were compared to that of neurons differentiated from the vehicle-treated shLuc cells (to which a value of 100% was assigned). * p < 0.05; ** p < 0.01. d A suggested model of the POU3F2/NTF3 pathway that mediates the process of neuron differentiation. Values are presented as mean ± SEM of at least three independent experiments for a and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Recombinant, Knockdown, Control, Real-time Polymerase Chain Reaction, Expressing, Microscopy