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: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are localized and secreted by human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A Human lung tissue from control subjects, idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF was immune-stained with IL-11, IL-11Rα and αSMA and with secondary fluorescence antibodies. Representative images are showed. White colour represents co-localization of both antibodies. Yellow arrows indicate endothelial cells. B HPAECs and C HPASMCs were isolated from pulmonary arteries of control subjects, IPF and PH associated to IPF patients and cultured until passage 1. Cell culture supernatants were collected to measure IL-11 by ELISA. Data are presented as scatter dot blot with median and interquartile range values of n = 6 patients in each group. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Staining, Fluorescence, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are increased in whole lung homogenates, isolated pulmonary arteries and serum of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF. The protein expression of IL-11 and IL-11Rα in A , B isolated pulmonary arteries (70–500 µm of internal diameter), C , D serum, and E , F lung tissue homogenates. Protein expression was measured using ELISA kits. H CD31 protein expression was measured in isolated pulmonary arteries as endothelial cells marker by ELISA. H , I Human lung tissue from control subjects, IPF and PH associated to IPF was immune-stained with IL-11, IL-11Rα and alpha smooth muscle actin (αSMA) antibodies. Representative images are showed from non-fibrotic lung areas and fibrotic areas. J Vascular wall thickening was quantified in a total of 20–30 pulmonary arteries per patient. K , L Immunohistochemical score quantification of IL-11 and IL-11Rα in a total of 20–30 pulmonary arteries per patient. Scale bar: 100 µm. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison. M Spearman ρ correlation of IL-11 expression in isolated pulmonary arteries from PH + IPF and mean pulmonary artery pressure (mPAP). N indicates the number of patients in each graph

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Marker, Staining, Immunohistochemical staining, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: SiRNA-IL-11 transiently transfection attenuates bleomycin-induced lung fibrosis and pulmonary hypertension in transgenic Tie2-GFP mice. Wild-type (WT) siRNA(−) Tie2-GFP mice and IL-11-KO siRNA-IL-11 Tie2-GFP mice received a single intratracheal dose of bleomycin (1.5 U/kg) on day 1 ( n = 11) during 14 days. siRNA-IL-11 was administered intravenously and intranasally three times a week from day 1 to day 14. At day 14 the following parameters were measured. A Masson’s trichrome histological images are showed. Scale bar: 100 µm. B Ashcroft score lung fibrotic index, C hydroxyproline amount in lung tissue D right ventricular systolic pressure (RVSP) mmHg, E right ventricular (RV) hypertrophy measured by the ratio of RV/left ventricular (LV) + septo in mg/mg, F pulmonary artery remodeling and G inflammatory cells in bronchoalveolar lavage fluid (BALF) were measured. H Immunohistochemical analysis of αSMA, IL-11 and IL-11Rα. Scale bar: 50 µm. Black arrows show pulmonary arteries. I Co-immunofluorescence of αSMA/Tie2-GFP. Scale bar: 25 µm. White arrows indicates co-localizations. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Transfection, Transgenic Assay, Immunohistochemical staining, Immunofluorescence, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and soluble IL-11Rα induce human pulmonary artery endothelial cell (HPAEC) to mesenchymal transition (EnMT) and human pulmonary artery smooth muscle cell (HPASMC) to myofibroblast-like transition. A HPAEC and B HPASMC were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or their combination during 48 h replacing culture medium and stimulus each 24 h. Experiments were done between passages 2–3. Gene mRNA transcripts of different genes measured by quantitative PCR (qPCR) as 2 −ΔCt . Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin for protein. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 4 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα activates intracellular signal. A Human pulmonary artery endothelial cells (HPAEC) and B human pulmonary artery smooth muscle cells (HPASMC) were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination during 30 min. Experiments were done between passages 2–3. Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin or non-phosphorylated protein as indicate. Representative blots are sowed. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα promotes time-dependent proliferation and senescence in human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A HPAECs or B HPASMCs were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination at indicated times. Experiments were done between passages 2–3. Cell proliferation was measured by the BrDU kit at 24 h, 48 h, 72 h and 96 h. Cell senescence was measured after 72 h of cell stimulation using β-galactosidase histology and P21 expression. Results were expressed as % senescence (β-galactosidase blue positive cells) relative to the total number of cells in each field. P21 expression was measured by quantitative PCR (qPCR) as 2 −ΔCt and western blot. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3–4 control subjects performed in triplicate). P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Cell Stimulation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Dot Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Isolation, characterization, and functional study of extracellular vesicles derived from Leishmania tarentolae

doi: 10.3389/fcimb.2022.921410

Figure Lengend Snippet: Dot blot using anti-GP63 (upper row) confirmed the presence of this GPI-anchored extracellular vesicle (EV) marker in EVs of both species and dot blot using anti-GFP (lower row) confirmed the presence of this cytosolic EV marker within EVs of both species. (A, F) L. tarentolae GFP+ extract as a positive control for tEV (35 µg protein in total). (B, G) L. tarentolae GFP+ EVs or tEV (1 µg in total). (C, H) L. major GFP+ extract as a positive control for mEV (35 µg in total). (D, I) L. major GFP+ EVs or mEV (1 µg in total). (E, J) Negative control (phosphate-buffered saline). The middle row depicts a separate dot blot using normal mouse sera as the primary antibody, which serves as a negative control. (a) L. tarentolae GFP+ extract (same as A, F ). (b) L. tarentolae GFP+ EVs or tEV (similar to B, G ). (c) L. major GFP+ extract (same as C, H ). (d) L. major GFP+ EVs or mEV (similar to D, I ). PC, positive control; tEV, L. tarentolae EV; mEV, L. major EV; NC, negative control.

Article Snippet: The GP63 antibody was provided by the Pasteur Institute of Iran, the anti-GFP-HRP goat polyclonal antibody was obtained from Acris antibodies GmbH (Germany), and the beta actin monoclonal antibody was purchased from ProteinTech Group Inc. (Chicago, IL, USA).

Techniques: Dot Blot, Marker, Positive Control, Negative Control

Journal: RNA biology

Article Title: Enterotoxigenic Escherichia coli infection promotes enteric defensin expression via FOXO6-METTL3-m 6 A-GPR161 signalling axis.

doi: 10.1080/15476286.2020.1820193

Figure Lengend Snippet: Figure 1. E. coli K88 simultaneously promotes the expression of β-defensin with m6A methylation. IPEC-J2 cells infected with E. coli K88 (MOI = 10:1) were analysed at various times, and the cells without infection constituted the control group. (A) The mRNA levels of DEFb1, DEFb2 and β-actin were measured by q-PCR, and the results are presented relative to those of Gapdh. (B) Immunoblotting was used to analyse the protein levels of DEFb1 and DEFb2 after transfection of the Flag fusion expression vector. The right panel shows the relative protein levels quantified by densitometry and normalized to the level of GFP. (C) m6A Dot blot was to measure the m6A levels with purified mRNA. Methylene blue staining was used as a loading control. The right panel shows the relative levels quantified by densitometry and normalized to the level of the control. The data are expressed as the mean ± SEM; statistically significant difference relative to the control: *P < 0.05, **P < 0.05, n = 3 biological replicates.

Article Snippet: The followin antibodies were used for immunoprecipitation (IP) and immunoblot analysis (IB): GPR161 (13398-1-AP, rabbit, 1:1000), FOXO6 (19122-1-AP, rabbit, 1:1000), GFP (50430-2-AP, rabbit, 1:1000), FLAG (20543-1-AP, rabbit, 1:1000), METTL3 (15073-1-AP, rabbit, 1:1000) and β-actin (60008-1-Ig, mouse, 1:1000) were purchased from Proteintech (Wuhan, China); m6A antibody (202111, mouse, 1:1000) was obtained from Synaptic Systems (Germany, Go ̈ttingen, Germany).

Techniques: Expressing, Methylation, Infection, Control, Western Blot, Transfection, Plasmid Preparation, Dot Blot, Purification, Staining

Journal: RNA biology

Article Title: Enterotoxigenic Escherichia coli infection promotes enteric defensin expression via FOXO6-METTL3-m 6 A-GPR161 signalling axis.

doi: 10.1080/15476286.2020.1820193

Figure Lengend Snippet: Figure 2. METTL3 depletion prevents β-defensin induction after E. coli K88 infection. IPEC-J2 cells with or without METTL3 knocking down were infected with E. coli K88 (MOI = 10:1) and analysed at various times. (A-B) The mRNA levels of DEFb1 (A) and DEFb2 (B) were measured by q-PCR, and the results are presented relative to the level of sh-Scramble 0 h group, and normalization to Gapdh. (C) Immunoblotting was used to analyse the protein levels of DEFb1 and DEFb2 after the transfection of the Flag fusion expression vector. The right panel shows the relative protein levels quantified by densitometry and normalized to the level of GFP. (D– F) METTL3-depleted IPEC-J2 cells were transfected with the Mettl3 plasmid or vector, followed by E. coli K88 infection and analysed at indicated times. The results from the q-PCR analysis of the mRNA levels of DEFb1 (D) and DEFb2 (E) are presented relative to the level of sh-Scramble 0 h group, and normalization to Gapdh. Immunoblotting was used to analyse the protein levels of Flag-labelled DEFb1 and DEFb2 (F). The right panel shows the relative protein levels quantified by densitometry and normalized to the level of GFP. The data are expressed as the mean ± SEM; *P < 0.05, **P < 0.05, n = 3 biological replicates.

Article Snippet: The followin antibodies were used for immunoprecipitation (IP) and immunoblot analysis (IB): GPR161 (13398-1-AP, rabbit, 1:1000), FOXO6 (19122-1-AP, rabbit, 1:1000), GFP (50430-2-AP, rabbit, 1:1000), FLAG (20543-1-AP, rabbit, 1:1000), METTL3 (15073-1-AP, rabbit, 1:1000) and β-actin (60008-1-Ig, mouse, 1:1000) were purchased from Proteintech (Wuhan, China); m6A antibody (202111, mouse, 1:1000) was obtained from Synaptic Systems (Germany, Go ̈ttingen, Germany).

Techniques: Infection, Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress

doi: 10.1038/s41467-025-56666-4

Figure Lengend Snippet: a Schematic of the construct and strategy for detection of MERCs. b Schematic of the MERBiT system. c Representative images of V5-TOMM20-SmBiT and LgBiT−3×HA-Sec61β localization in HeLa cells stably expressing V5-TOMM20-SmBiT and LgBiT−3×HA-Sec61β (MERBiT cells). Cells were stained with V5, HA, HSP60 and calnexin antibodies. HSP60 is used as a mitochondrial marker and calnexin is used as an ER marker. d , Representative immunoblots for each component of MERBiT cells. The lysates of MERBiT cells were analyzed by immunoblotting for V5 (V5-TOMM20-SmBiT), HA (LgBiT-3×HA-Sec61β), TOMM20, HSP60, calnexin, and α-tubulin. Black and white arrowheads indicate tagged and endogenous TOMM20, respectively. e Luminescence of MERBiT cells. Quantification of the luminescence of HeLa cells, MERBiT cells, and stably expressing V5-TOMM20-SmBiT HeLa cells. Data are mean ± s.e.m. ( n = 9). f Quantification of MERCs reduction during recovery from starvation in MERBiT cells. Cells were starved in HBSS for 1 h and then recovered in 10% FBS DMEM for the indicated times before luminescence was measured. Data are mean ± s.e.m. ( n = 3, triplicate). g, h Effects of knockdown of different MERCs tethering factors on MERBiT luminescence in MERBiT cells. Cells were transfected with the indicated siRNAs and then luminescence was measured or WB was performed with the indicated antibodies to confirm protein expression levels. Data are mean ± s.e.m. ( n = 3, triplicate). i MERCs linker increases luminescence. MERBiT cells were transfected with MERCs linker (pCAG-AKAP1(1-30 aa)-mTagBFP-V5-SACM1L (521-587 aa)) and luminescence was detected. Data are mean ± s.e.m. ( n = 3, triplicate). Statistical significance was analyzed by one-way analysis of variance (ANOVA) (e, f, g) or Student’s t -test, Two-tailed (i). P values are indicated as; ** p < 0.01; **** p < 0.0001.

Article Snippet: For immunoblotting: Mouse VAPB antibody (66191-1-Ig, 1:1000), rabbit PDZD8 antibody (25512-1-AP, 1:1000), rabbit polyclonal anti-PTPIP51 (RMDN3) antibody (20641-1-AP, 1:1000) and rabbit monoclonal anti-TOMM20 antibody (11802-1-AP, 1:2000) were purchased from Proteintech.

Techniques: Construct, Stable Transfection, Expressing, Staining, Marker, Western Blot, Knockdown, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress

doi: 10.1038/s41467-025-56666-4

Figure Lengend Snippet: a RMDN3 and VAPB are critical tethering factors for MERCs formation induced by antimycin A and rotenone stimulation. MERBiT cells were transfected with the indicated siRNAs for 3 days and treated with or without rotenone (50 nM) and antimycin A (50 nM) for 1 h before luminescence measurements. Data are mean ± s.e.m. ( n = 3, triplicate). b Interaction between RMDN3 and VAPB increase in rotenone and antimycin A treatment. HeLa cells were transfected with the indicated vectors and treated with rotenone (50 nM) or antimycin A (50 nM) for 1 h. Cell lysates were subjected to IP assay (left). Ratio of RMDN3-VAPB interaction (right). Data are mean ± s.e.m. ( n = 3). c Schematic model of the RMDN3 domain. d The expression levels of RNAi-resistant RMDN3 vectors. The HeLa cells were transfected with RMDN3 siRNA for 2 days and then transfected with the indicated vectors such as RMDN3 RNAi-resistant vectors for 1 day. e FFAT but not TPR domain is important for mitochondrial ROS-induced MERCs formation. The MERBiT cells were transfected with the indicated siRNAs for 2 days. Then transfected with empty vectors or indicated RNAi-resistant vectors for 1 day. Before measuring luminescence, cells were treated with or without rotenone (50 nM) and antimycin A (50 nM) for 1 h. Data are mean ± s.e.m. ( n = 3, triplicate). f Threonine 160 mutant of RMDN3 decrease phosphorylation by antimycin A stimulation. HeLa cells were transfected with the indicated vectors and then treated with or without antimycin A (50 nM). Cell lysates were subjected to IP assay and then beads were incubated with or without lambda phosphatase (λPP). Pull-down lysates were subjected to Phos-tag-PAGE or SDS-PAGE. g Phosphorylation of RMDN3 T160 was important for interaction with VAPB by antimycin A treatment. HeLa cells were transfected with the indicated vectors and treated with antimycin A (50 nM) for 1 h. Cell lysates were subjected to IP assay and IB assay (left). Ratio of RMDN3-VAPB interaction (right). Data are mean ± s.e.m. ( n = 3). h Phosphorylation of RMDN3 T160 is important for MERCs formation induced by antimycin A stimulation. The MERBiT cells were transfected with the indicated siRNAs for 2 days and then transfected with vectors for 1 day before treatment with or without antimycin A (50 nM) for 1 h, and then the luminescence was measured. Data are mean ± s.e.m. ( n = 3, triplicate). Statistical significance was analyzed by one-way analysis of variance (ANOVA) ( a , b , e , g , h ). P values are indicated as * p < 0.05; ** p < 0.01; **** p < 0.0001; n.s., not significant.

Article Snippet: For immunoblotting: Mouse VAPB antibody (66191-1-Ig, 1:1000), rabbit PDZD8 antibody (25512-1-AP, 1:1000), rabbit polyclonal anti-PTPIP51 (RMDN3) antibody (20641-1-AP, 1:1000) and rabbit monoclonal anti-TOMM20 antibody (11802-1-AP, 1:2000) were purchased from Proteintech.

Techniques: Transfection, Expressing, Mutagenesis, Phospho-proteomics, Incubation, SDS Page

Journal: Nature Communications

Article Title: ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress

doi: 10.1038/s41467-025-56666-4

Figure Lengend Snippet: a Rotenone or antimycin A treatment of RMDN3 and VAPB knockdown cells reduced cell viability. The HeLa cells were transfected with the indicated siRNAs for 5 days with or without rotenone (50 nM) and antimycin A (50 nM) for 2 days before cell viability was measured. Cell viability was determined and expressed as a fold change of si-NT. Viable cells were detected by cell viability assay using Cell Counting Kit-8. b The HeLa cells were transfected with the indicated siRNAs for 5 days with or without rotenone (50 nM) and antimycin A (50 nM) for 2 days before measuring cell viability. oxNAC (50 µM), NACS2 (50 µM) and mito-TEMPO (100 nM) were treated for 3 days before measuring cell viability. Cell viability was determined and expressed as a fold change of si-NT. Viable cells were detected by cell viability assay using Cell Counting Kit-8. c Lack of TPR domain does not rescue cell viability of RMDN3 knockdown with rotenone or antimycin A treatment. HeLa cells were transfected with RMDN3 siRNA for 5 days and with the indicated vectors for 3 days before measuring cell viability. Rotenone (50 nM) and antimycin A (50 nM) were treated for 2 days before cell viability was measured. Cell viability was determined and expressed as a fold change of si-NT. Viable cells were detected by cell viability assay using Cell Counting Kit-8. FLAG-resi-RMDN3 WT, FLAG-resi-RMDN3ΔFFAT, and FLAG-resi-RMDN3ΔTPR are RMDN3 RNAi-resistant vectors. d HeLa cells were transfected with RMDN3 siRNA for 5 days with or without rotenone (50 nM) and antimycin A (50 nM) for 2 days before measuring cell viability. The indicated inhibitors were treated for 2 days before measuring cell viability. Cell viability was determined and expressed as a fold change of si-NT. Viable cells were detected by cell viability assay using Cell Counting Kit-8. z-VAD-FMK (20 µM), necrostatin-1 (20 µM), ferrostatin-1 (Fer-1) (5 µM), and deferoxamine (DFO) (100 µM). Data are mean ± s.e.m. (n = 3, triplicate), and statistical significance was analyzed by one-way analysis of variance (ANOVA) (a-d). P values are indicated as **** p < 0.0001; n.s., not significant.

Article Snippet: For immunoblotting: Mouse VAPB antibody (66191-1-Ig, 1:1000), rabbit PDZD8 antibody (25512-1-AP, 1:1000), rabbit polyclonal anti-PTPIP51 (RMDN3) antibody (20641-1-AP, 1:1000) and rabbit monoclonal anti-TOMM20 antibody (11802-1-AP, 1:2000) were purchased from Proteintech.

Techniques: Knockdown, Transfection, Viability Assay, Cell Counting

Journal: Nature Communications

Article Title: ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress

doi: 10.1038/s41467-025-56666-4

Figure Lengend Snippet: a, b Suppression of RMDN3 does not affect the induction of thermogenic genes. Cells were transfected with the indicated siRNAs and harvested at 0 or 4 days after differentiation. The cell lysates were analyzed by immunoblotting with the indicated antibodies ( a ). mRNA levels of differentiation markers were measured by qRT-PCR. Data were normalized to s18 mRNA and expressed relative to si-NT on day 4 ( b ). c Representative images of lipid droplets (LDs) in cells treated with the indicated siRNAs. Cells were fixed on day 4. The LDs and mitochondria were labeled with LipidTOX and anti-TOMM20 antibodies. The LDs and mitochondria were quantified for total LD area in ( d ), average LD size in ( e ), total mitochondrial area in ( f ), and the ratio of total LD area to total mitochondrial area in ( g ) from the ROI of ( c ). 2−3 cells from three independent experiments for the control and si-RMDN3#1 cells, respectively. Data are mean ± s.e.m. ( n = 3) h Lipid peroxide production increases with NE stimulation. MitoPeDPP (10 µM) was stained 30 min after stimulation with or without NE (1 µM) for 1 h and MitoPeDPP signals were detected in brown adipocytes (day 6). i, j RMDN3 and VAPB binding is increased by mitochondrial ROS generated under NE stimulation. Cell lysates were subjected to IP assay with anti-RMDN3 antibody and IB assay with the indicated antibodies ( j ). Ratio of RMDN3-VAPB interaction, plotted data for NE (1 µM) with or without Mito-TEMPO (10 µM) treatment versus control. Data are mean ± s.e.m. (n means three independent experiments). k Phosphorylation of RMDN3 by mitochondrial ROS and binding with VAPB is necessary for the suppression of lipid peroxide production. Cells were transfected with the indicated siRNAs and expressed human RMDN3 or human RMDN3 T160A before measuring MitoPeDPP fluorescence. NE was treated for 1 h. Data are mean ± s.e.m. (n means three independent experiments). Statistical significance was analyzed by one-way analysis of variance (ANOVA) ( b , j , k ) or Student’s t-test, Two-tailed ( d – h ). P values are indicated as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n.s., not significant.

Article Snippet: For immunoblotting: Mouse VAPB antibody (66191-1-Ig, 1:1000), rabbit PDZD8 antibody (25512-1-AP, 1:1000), rabbit polyclonal anti-PTPIP51 (RMDN3) antibody (20641-1-AP, 1:1000) and rabbit monoclonal anti-TOMM20 antibody (11802-1-AP, 1:2000) were purchased from Proteintech.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Labeling, Control, Staining, Binding Assay, Generated, Phospho-proteomics, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: Fibrillar form of α-synuclein-specific scFv antibody inhibits α-synuclein seeds induced aggregation and toxicity

doi: 10.1038/s41598-020-65035-8

Figure Lengend Snippet: Characterization of purified scFv-pF and scFv-pC. ( a ) Dot blot showing specific binding of scFv-pF and -pC to fibrillar and oligomeric forms of α-syn. Indicated amounts of full-length α-syn monomers (M), fibrils (F) or oligomers (O) (upper panel) and 1 μg of A-beta 42, Tau, IAPP (bottom panel) were spotted onto a nitrocellulose membrane. The membranes were probed with scFv-pF/-pC and Syn-F2 for α-syn, 82E1 for A-beta 42, 5E2 for Tau, R10/99 for IAPP antibodies, and Syn-1 for full-length α-syn. ( b ) In vitro seeding of α-syn aggregation assay showing inhibition by Syn-F2, scFv-pF and -pC. α-Syn monomers (25 µM) were seeded with 1 µM α-syn seeds, which were incubated in the presence or absence of Syn-F2 (1 µM), scFv-pF (40 µM) and scFv-pC (8 µM) for 6 hours with continuous shaking at 37 °C. The extent of fibrillation was estimated by the Th-S fluorescence assay at indicated time-points. The assay was performed in triplicate (average of triplicate measurements ± standard deviations). Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison test. (****p < 0.0001). ( c ) Electron microscopy images of negatively stained samples collected at time 0 and 6 hours from experiment in ( b ) show that mature amyloid fibrils (300–700 nm long) are formed in seeds and monomer incubated samples at 6 hours time point which is inhibited by Syn-F2, scFv-pF and scFv-pC antibodies. Arrowhead indicate presence of seeds at the time-point 0 and 6 hours samples. Magnification 28500x. Scale bar = 500 nm.

Article Snippet: Proteins were transferred to nitrocellulose membrane and developed using anti-6X-His tag mouse antibody (1:2500, Abcam) followed by incubation with HRP-conjugated IgG goat anti-mouse antibody (Thermo Scientific) at a dilution of 1:10,000 using SuperSignal West-Pico -Chemiluminescent Substrate.

Techniques: Purification, Dot Blot, Binding Assay, Membrane, In Vitro, Inhibition, Incubation, Fluorescence, Comparison, Electron Microscopy, Staining

Journal: Autophagy

Article Title: Systematic analysis of ATG13 domain requirements for autophagy induction

doi: 10.1080/15548627.2017.1387342

Figure Lengend Snippet: The amino acid sequence V348-M373 comprises the RB1CC1 binding region in ATG13. ( A ) atg13 KO MEFs retrovirally transfected with empty vector or cDNA encoding either HA-ATG13 or HA-ATG13(ΔV348-M373) were lysed and cleared cellular lysates were subjected to immunopurifications with anti-HA-agarose or protein A/G beads in combination with anti-RB1CC1 or anti-ATG101 antibodies, respectively. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for RB1CC1, ULK1, ATG13, HA, or ATG101. ( B ) atg13 KO MEFs stably expressing HA-ATG13 or the indicated mutants were seeded onto glass cover slips. The next day cells were used for proximity ligation assay as described in the material and methods section (anti-HA antibody: covance MMS-101P). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data are represented as mean ± SEM. Samples without significant difference display identical letter (Student t test, 2-sample assuming unequal variances; minimum of 24 images was analyzed). ( C ) atg13 KO MEFs stably expressing HA-APEX2 alone or fused to either wild-type ATG13 or the indicated mutants were pre-incubated with phenol-biotin for 30 min and peroxidase was activated by adding H 2 O 2 for 1 min. Cells were washed 3 times with quenching solution and lysed. Biotinylated proteins were purified using streptavidin agarose. Purified proteins were analyzed by immunoblotting for RB1CC1, HA, or ATG101. ( D ) S100 extracts of cells described in ( A ) were separated by size-exclusion chromatography on a Superose 6 increase column. Fractions were analyzed by immunoblotting for the indicated proteins. Diagrams show protein levels for each fraction at a ratio of the input and normalized to the fraction containing the highest concentration of the analyzed protein. Curves for controls (KO and ATG13) are reused in figures 5 and .

Article Snippet: Antibodies against ACTB/β-actin (clone AC-74, Sigma-Aldrich, A5316), ATG101 (Sigma-Aldrich, SAB4200175), ATG13 (Sigma-Aldrich, SAB4200100), ATG13 phospho (p)-S318 (Rockland Immunochemicals, 600-401-C49), ATG14 (MBL, PD026), ATG16L1 (MBL, PM040), HA (Covance, MMS-101R, now BioLegend, 901501), LC3 (for immunoblotting: Cell Signaling Technology, 2775 [detects endogenous levels of total LC3B protein; cross-reactivity may exist with other LC3 isoforms according to manufacturer specification]; for immunofluorescence: MBL International, PM036 [reacts with LC3A/LC3B/LC3C according to manufacturer specification]), RPS6KB1 phospho (p)-T389 (clone 1A5, Cell Signaling Technology, 9206), RB1CC1 (for immunoblotting: Bethyl Laboratories, A301-536A; for immunofluorescence: Proteintech, 17250-1-AP), SQSTM1/p62 (PROGEN Biotechnik, GP62-C), ULK1 (clone D8H5, Cell Signaling Technology, 8054), ULK1 phospho (p)-S757 (Cell Signaling Technology, 6888), VCL/Vinculin (Sigma-Aldrich, V9131), and WIPI2 (Serotec, MCA5780GA) were used.

Techniques: Sequencing, Binding Assay, Transfection, Plasmid Preparation, Purification, SDS Page, Western Blot, Stable Transfection, Expressing, Proximity Ligation Assay, Staining, Incubation, Size-exclusion Chromatography, Concentration Assay

Journal: Autophagy

Article Title: Systematic analysis of ATG13 domain requirements for autophagy induction

doi: 10.1080/15548627.2017.1387342

Figure Lengend Snippet: Composition of the ULK1 complex is influenced by the ATG13-ULK1 interaction but not by the LIR motif of ATG13. ( A ) S100 extracts of atg13 KO MEFs stably expressing the indicated HA-ATG13 variants were separated by size-exclusion chromatography on a Superose 6 increase column. Fractions were analyzed by immunoblotting for the indicated proteins. Diagrams show protein levels for each fraction at a ratio of the input and normalized to the fraction containing the highest concentration of the analyzed protein. Curves for controls (KO and ATG13) are reused in figures 2 and . ( B ) Cells described in ( A ) were seeded onto glass cover slips one day prior to stimulation with full medium (DMEM) or starvation medium (EBSS) for 1 h. Cells were fixed, permeabilized and stained for HA (covance MMS-101P) and RB1CC1. An inverse confocal laser scanning microscope was used for imaging. Puncta and colocalization per cell quantification was done using fiji software. A minimum of 127 cells per stimulation was analyzed. Data represent mean + SEM. Statistical analysis using the Student t test, 2-sample assuming unequal variances was performed comparing EBSS to DMEM for each individual cell line. * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bar: 20 µm. ( C ) atg13 KO MEFs stably expressing HA-ATG13 or the indicated mutants were seeded onto glass cover slips one day prior to stimulation with starvation medium (EBSS) for 2 h. Cells were fixed, permeabilized and stained for HA (covance MMS-101P) and LC3. An inverse confocal laser scanning microscope was used for imaging. Scale bar: 20 µm.

Article Snippet: Antibodies against ACTB/β-actin (clone AC-74, Sigma-Aldrich, A5316), ATG101 (Sigma-Aldrich, SAB4200175), ATG13 (Sigma-Aldrich, SAB4200100), ATG13 phospho (p)-S318 (Rockland Immunochemicals, 600-401-C49), ATG14 (MBL, PD026), ATG16L1 (MBL, PM040), HA (Covance, MMS-101R, now BioLegend, 901501), LC3 (for immunoblotting: Cell Signaling Technology, 2775 [detects endogenous levels of total LC3B protein; cross-reactivity may exist with other LC3 isoforms according to manufacturer specification]; for immunofluorescence: MBL International, PM036 [reacts with LC3A/LC3B/LC3C according to manufacturer specification]), RPS6KB1 phospho (p)-T389 (clone 1A5, Cell Signaling Technology, 9206), RB1CC1 (for immunoblotting: Bethyl Laboratories, A301-536A; for immunofluorescence: Proteintech, 17250-1-AP), SQSTM1/p62 (PROGEN Biotechnik, GP62-C), ULK1 (clone D8H5, Cell Signaling Technology, 8054), ULK1 phospho (p)-S757 (Cell Signaling Technology, 6888), VCL/Vinculin (Sigma-Aldrich, V9131), and WIPI2 (Serotec, MCA5780GA) were used.

Techniques: Stable Transfection, Expressing, Size-exclusion Chromatography, Western Blot, Concentration Assay, Staining, Laser-Scanning Microscopy, Imaging, Software

Journal: Autophagy

Article Title: Systematic analysis of ATG13 domain requirements for autophagy induction

doi: 10.1080/15548627.2017.1387342

Figure Lengend Snippet: Disruption of the RB1CC1-binding region in ATG13 inhibits mutual recruitment to the phagophore. atg13 KO MEFs retrovirally transfected with cDNA encoding either HA-ATG13 or HA-ATG13(ΔV348-M373) were seeded onto glass cover slips one day prior to stimulation with full medium (DMEM) or starvation medium (EBSS) for 1 h. Cells were fixed, permeabilized and stained for HA (covance MMS-101P) and RB1CC1. An inverse confocal laser scanning microscope was used for imaging. Puncta and colocalization per cell quantification was done using fiji software. Data represent mean + SEM. A minimum of 168 cells per stimulation was analyzed. Statistical analysis using the Student t test, 2-sample assuming unequal variances was performed comparing EBSS to DMEM for each individual cell line. No statistical significance with P < 0.05 was obtained. Columns for control (ATG13) are reused in figures 4 , , and supplementary figure S9. Scale bar: 20 µm.

Article Snippet: Antibodies against ACTB/β-actin (clone AC-74, Sigma-Aldrich, A5316), ATG101 (Sigma-Aldrich, SAB4200175), ATG13 (Sigma-Aldrich, SAB4200100), ATG13 phospho (p)-S318 (Rockland Immunochemicals, 600-401-C49), ATG14 (MBL, PD026), ATG16L1 (MBL, PM040), HA (Covance, MMS-101R, now BioLegend, 901501), LC3 (for immunoblotting: Cell Signaling Technology, 2775 [detects endogenous levels of total LC3B protein; cross-reactivity may exist with other LC3 isoforms according to manufacturer specification]; for immunofluorescence: MBL International, PM036 [reacts with LC3A/LC3B/LC3C according to manufacturer specification]), RPS6KB1 phospho (p)-T389 (clone 1A5, Cell Signaling Technology, 9206), RB1CC1 (for immunoblotting: Bethyl Laboratories, A301-536A; for immunofluorescence: Proteintech, 17250-1-AP), SQSTM1/p62 (PROGEN Biotechnik, GP62-C), ULK1 (clone D8H5, Cell Signaling Technology, 8054), ULK1 phospho (p)-S757 (Cell Signaling Technology, 6888), VCL/Vinculin (Sigma-Aldrich, V9131), and WIPI2 (Serotec, MCA5780GA) were used.

Techniques: Disruption, Binding Assay, Transfection, Staining, Laser-Scanning Microscopy, Imaging, Software, Control

Journal: Autophagy

Article Title: Systematic analysis of ATG13 domain requirements for autophagy induction

doi: 10.1080/15548627.2017.1387342

Figure Lengend Snippet: Mutation of residues I131, R133, V134 and Y138 in ATG13 is sufficient to inhibit its interaction with ATG101. ( A ) Computational alanine scanning of the ATG13-ATG101 interface was performed using the structure of the human ATG13-ATG101 HORMA heterodimer (PDB ID: 5C50, ) and the DrugScore PPI webserver (upper panel). ΔΔ G denotes binding free energy differences for wild-type residue-to-Ala mutations; residues yielding ΔΔ G > 1 kcal mol −1 are considered binding hot spots. In the middle panel, per-residue effective binding energies (ΔG binding ) computed by the MM-GB/SA approach ( , ) are shown. Residues considered hot spots according to both methods are colored in red. In the lower panel, the localization of these residues in the ATG13 interface is shown. ( B ) atg13 KO MEFs stably expressing VenusC-ATG101 and VenusN-ATG13 (wild-type or the indicated mutants) were trypsinized and analyzed for Venus fluorescence using a flow cytometer. The median of fluorescence intensity for each sample was normalized to control cells lacking VenusN-ATG13 expression (“none”) and was plotted in a bar diagram representing mean ± SEM. Samples without significant difference display identical letters (Student t test, 2-sample assuming unequal variances). Representative data are plotted in a histogram. Cell lysates were examined for expression of the indicated proteins by immunoblotting. ( C ) atg13 KO MEFs retrovirally transfected with empty vector or cDNA encoding either HA-ATG13 or the indicated variants were lysed and cleared cellular lysates were subjected to immunopurification with anti-ATG101 antibodies and a protein A/G-Sepharose mix. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for RB1CC1, ULK1, or HA. ( D ) atg13 KO MEFs stably expressing the indicated HA-tagged ATG13 variants were grown on glass cover slips one day prior to incubation with starvation medium (EBSS) for 2 h, fixation and permeabilization. Immunofluorescence for HA (covance MMS-101P) and RB1CC1 was performed. An inverse confocal laser scanning microscope was used for imaging. Please note that we detected a high number of HA-positive puncta varying in size and intensity, of which only a minor portion colocalized with RB1CC1 puncta. This might be due to the exogenous expression of HA-ATG13 variants. Puncta and colocalization per cell quantification was done using fiji software. A minimum of 89 cells per stimulation was analyzed. Data represent mean + SEM. Statistical analysis using the Student t test, 2-sample assuming unequal variances was performed comparing EBSS to DMEM for each individual cell line. ** P < 0.01, *** P < 0.001. Scale bar: 20 µm.

Article Snippet: Antibodies against ACTB/β-actin (clone AC-74, Sigma-Aldrich, A5316), ATG101 (Sigma-Aldrich, SAB4200175), ATG13 (Sigma-Aldrich, SAB4200100), ATG13 phospho (p)-S318 (Rockland Immunochemicals, 600-401-C49), ATG14 (MBL, PD026), ATG16L1 (MBL, PM040), HA (Covance, MMS-101R, now BioLegend, 901501), LC3 (for immunoblotting: Cell Signaling Technology, 2775 [detects endogenous levels of total LC3B protein; cross-reactivity may exist with other LC3 isoforms according to manufacturer specification]; for immunofluorescence: MBL International, PM036 [reacts with LC3A/LC3B/LC3C according to manufacturer specification]), RPS6KB1 phospho (p)-T389 (clone 1A5, Cell Signaling Technology, 9206), RB1CC1 (for immunoblotting: Bethyl Laboratories, A301-536A; for immunofluorescence: Proteintech, 17250-1-AP), SQSTM1/p62 (PROGEN Biotechnik, GP62-C), ULK1 (clone D8H5, Cell Signaling Technology, 8054), ULK1 phospho (p)-S757 (Cell Signaling Technology, 6888), VCL/Vinculin (Sigma-Aldrich, V9131), and WIPI2 (Serotec, MCA5780GA) were used.

Techniques: Mutagenesis, Binding Assay, Residue, Stable Transfection, Expressing, Fluorescence, Flow Cytometry, Control, Western Blot, Transfection, Plasmid Preparation, Immu-Puri, Purification, SDS Page, Incubation, Immunofluorescence, Laser-Scanning Microscopy, Imaging, Software

Journal: Autophagy

Article Title: Systematic analysis of ATG13 domain requirements for autophagy induction

doi: 10.1080/15548627.2017.1387342

Figure Lengend Snippet: Summary of the effects of mutations in ATG13 interaction interfaces on autophagy induction by amino acid starvation or MTOR inhibition. The heat map shows i) the percentage of LC3-II signal detected by immunoblotting for the indicated stimuli in the presence of bafilomycin A 1 (columns 1 to 3), ii) the increase of WIPI2 and ATG16L1 puncta formation after autophagy induction by the indicated stimuli (columns 4 to 6), or iii) the percentage of colocalization events of HA-ATG13 variants and RB1CC1 after treatment with the indicated stimuli (columns 7 and 8). All values were normalized to the control, which was set to 100% (ATG13, first row). The range for mapping was defined from 5.7 to 159. The value for LIR mut under EBSS stimulation (267.6%) was set to out of range, and the color code dark red was manually assigned (n.d., not determined).

Article Snippet: Antibodies against ACTB/β-actin (clone AC-74, Sigma-Aldrich, A5316), ATG101 (Sigma-Aldrich, SAB4200175), ATG13 (Sigma-Aldrich, SAB4200100), ATG13 phospho (p)-S318 (Rockland Immunochemicals, 600-401-C49), ATG14 (MBL, PD026), ATG16L1 (MBL, PM040), HA (Covance, MMS-101R, now BioLegend, 901501), LC3 (for immunoblotting: Cell Signaling Technology, 2775 [detects endogenous levels of total LC3B protein; cross-reactivity may exist with other LC3 isoforms according to manufacturer specification]; for immunofluorescence: MBL International, PM036 [reacts with LC3A/LC3B/LC3C according to manufacturer specification]), RPS6KB1 phospho (p)-T389 (clone 1A5, Cell Signaling Technology, 9206), RB1CC1 (for immunoblotting: Bethyl Laboratories, A301-536A; for immunofluorescence: Proteintech, 17250-1-AP), SQSTM1/p62 (PROGEN Biotechnik, GP62-C), ULK1 (clone D8H5, Cell Signaling Technology, 8054), ULK1 phospho (p)-S757 (Cell Signaling Technology, 6888), VCL/Vinculin (Sigma-Aldrich, V9131), and WIPI2 (Serotec, MCA5780GA) were used.

Techniques: Inhibition, Western Blot, Control

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: Density of UG nucleotide sequences 100bp upstream and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMS; A ) or cross-linking induced truncation sites (CITS; B ) in relation to random sequences (red line). Grey shading represents 95% confidence regions. ( C ) Schematic of HaloTag immunoprecipitation and dot blot procedure. ( D ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of HaloTag-labeled proteins in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. ( E ) Diagram illustrating insertion of the HaloTag open reading frame into the endogenous TARDBP locus immediately 5’ to the TDP43 start codon, resulting in a fusion of HaloTag to the N-terminus of TDP43. ( F ) Halo-TDP43 HEK293T cells labeled live with JF646 Halo dye (red), then fixed, permeabilized, and immunostained with anti-TDP43 antibody (green) prior to imaging. DAPI (blue) marks the nucleus of each cell. Scale bar = 10µm. ( G ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or exogenous HaloTag. Additional replicates shown in Sup. Fig. 1.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Modification, Isolation, Immunoaffinity Purification, Labeling, Imaging

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) HaloTag-TDP43 immunoprecipitation was followed by DART-seq to delineate m6A sites within TDP43 target RNAs. HaloTag-TDP43 HEK293T cells were transfected with APOBEC1-YTH or APOBEC1-YTHmut and crosslinked before immunoaffinity purification of HaloTag-labeled proteins. Immunoprecipitated RNAs were then sequenced and C-T transitions were identified in the context of DRACH motifs (red shaded box, D=A/G/T, R=A/G, H=A/C/T). Absolute counts ( B ) and relative frequency ( C ) of base pair transitions observed by RNA-seq in each condition. Shaded boxes represent transition types expected from APOBEC1 activity. ( D ) Example m6A sites identified by DART-seq in RPL10A . C-T transitions are highlighted in red, and DRACH motifs in pink. Green arrow, transcription start site; red hexagon, transcription stop site; thick blue bars, coding exons; thin blue bars, untranslated region. ( E ) Absolute count and relative distribution ( F ) of DART-seq reads in cells expressing APOBEC1-YTH and APOBEC1-YTHmut. UTR, untranslated region; CDS, coding sequence. ( G ) Scatter plot of TDP43 targets, determined by fold enrichment in precipitated RNA from HaloTag-TDP43 cells (expressing APOBEC1-YTH and APOBEC10YTHmut) compared to cells transfected with HaloTag. Red dots signify transcripts showing > 2-fold enrichment in both APOBEC1-YTH and APOBEC1-YTHmut expressing cells. TARDBP , yellow dot, identified as high confidence target. ( H ) Stacked bar graph showing percentage of m6A modified RNA in TDP43 targets (red) and non-targets (black). ( I ) Cumulative distribution of RNA methylation in TDP43 targets (red) and non-targets (black). p = 1.87×10 −55 by Kolmogorov Smirnov test. ( J ) Euler diagram depicting overlap between TDP43 targets identified in this study, and those identified by TDP43 cross linking and immunoprecipitation followed by RNA-sequencing (CLIP-seq) in HEK293T cells (Hallegger et al ., 2021) . **p=1.5×10 −117 , hypergeometric test. ( K ) Pie charts demonstrating the percentage of methylated RNA among TDP43 targets (pink) and non-targets (grey). **p<1×10 −5 chi-square test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunoprecipitation, Transfection, Immunoaffinity Purification, Labeling, RNA Sequencing, Activity Assay, Expressing, Sequencing, Modification, Methylation

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) TARDBP gene map, illustrating TDP43 binding region (TBR), the location of the DRACH motif (pink square), and the C-T transition (red box) identified by DART-seq within this domain, representing an m6A site. ( B ) Schematic of the TARDBP minigene reporter, consisting of the mCherry ORF upstream of TARDBP exon 6 and 3.4 Kb of the TARDBP 3’ UTR. The A residue adjacent to the detected C-T transition via DART-seq in the WT reporter (mCherry-TBR) was mutated to a G, precluding methylation the mutant reporter (mCherry-mTBR). Red, methylated residue; blue line, DRACH motif; dagger, C-T transition from DART-seq. ( C ) HaloTag-TDP43 was isolated by immunoaffinity purification from HaloTag-TDP43 HEK293T cells expressing mCherry-TBR or mCherry-mTBR, and reporter RNA detected in elution fractions by qRT-PCR. ( D ) Outline of TDP43 autoregulation assay. Excess TDP43 binds to the reporter, triggering reporter splicing, destabilization, and reduced mCherry fluorescence. ( E ) Primary rodent neurons were transfected with WT (mCherry-TBR) or mutant (mCherry-mTBR) reporters, together with EGFP or TDP43-EGFP. After 7d, mCherry expression was assessed by fluorescence microscopy. Scale bar= 20 µm. Normalized RFP (mCherry) intensity in primary neurons expressing WT mCherry-TBR reporter ( F ) or mutant mCherry-mTBR ( G ) reporter together with EGFP or TDP43(WT)-EGFP. Cherry-TBR+GFP n= 160, Cherry-TBR+TDP43(WT)-GFP n= 58, Cherry-mTBR+GFP n= 105, Cherry-mTBR+TDP43(WT)-GFP n= 44. Data in C plotted as mean ± SD, collected from 3 biological replicates. ns= not significant, *p< 0.05, **p< 0.01; one-way ANOVA with Tukey’s test. Data in F and G plotted as mean ± SD, color coded by biological replicate. ns = not significant, *p < 0.05; Welch’s t-test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Binding Assay, Residue, Methylation, Mutagenesis, Isolation, Immunoaffinity Purification, Expressing, Quantitative RT-PCR, Fluorescence, Transfection, Microscopy

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Genome-wide analysis of RNA methylation via epitranscriptomic array. RNA was extracted from control (n= 3) and sporadic ALS (sALS) patient (n= 4) spinal cord samples, prior to m6A RNA immunoprecipitation. The resulting samples were separated into methylated and non-methylated RNA, then labeled with distinct fluorescent dyes (red and green stars) prior to hybridization, allowing relative quantification of methylation at each annotated locus. ( B ) Principal component analysis (PCA) plot comparing methylation levels of control (grey) and ALS (red) patient samples. ( C ) Hierarchical clustering of mRNA methylation profiles from control and ALS mRNA samples. ( D ) Volcano plot depicting fold change in mRNA methylation levels in ALS compared to control spinal cord. ( E ) Hierarchical clustering of lncRNA methylation profiles from control ALS lncRNA samples. ( F ) Volcano plot showing fold change in lncRNA methylation levels in ALS compared to control spinal cord. In D and F , grey horizontal vertical lines represent p= 0.05 and fold change (FC)= 2. ( G ) Euler diagram demonstrating overlap (n= 322, p= 5.09×10 −119 , hypergeometric test) among TDP43 substrates and methylated transcripts identified in HEK293T cells, in additional to hypermethylated transcripts determined via m6A array in sALS spinal cord. Comparisons were limited to the subset of transcripts expressed in both HEK293T cells and human spinal cord (nTPM>2). ( H ) Based on comparisons with the GEO transcription factor loss-of-function database via Enrichr , there was strong enrichment for TDP43-regulated genes not only among the set of 2034 transcripts hypermethylated in sALS spinal cord, but also among the 322 TDP43 targets that were also hypermethylated in sALS (A1 in G ). Combined score = (log 10 p * Z-score). ( I ) Immunohistochemical staining for m6A in control and sALS spinal cord sections. Scale bars= 50 µm. ( J ) Quantification of m6A antibody reactivity in spinal cord neurons from control (n= 110 neurons) and sALS (n= 277 neurons) sections. Plot shows mean +/- SD, color coded by patient. ****p< 0.0001 via Mann-Whitney test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Genome Wide, Methylation, Control, RNA Immunoprecipitation, Labeling, Hybridization, Quantitative Proteomics, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Representative images of rodent primary neurons transfected with plasmids expressing Cas9-2A-EGFP and sgRNA targeting the neuronal protein NeuN or negative control (LacZ). 5d after transfection, neurons were fixed and immunostained for NeuN (red). White dashed circles indicate nucleus stained with Hoechst (blue). ( B ) NeuN antibody reactivity measured in EGFP-positive neurons expressing sgLacZ (n= 565) or sgNeuN (n= 654), ****p < 0.0001 by Mann-Whitney. ( C ) Schematic depicting m6A writers (green), erasers (red), and readers (orange) targeted by CRISPR/Cas9. ( D ) Primary neurons expressing EGFP and TDP43-mApple were assessed at regular 24h intervals by fluorescence microscopy, and their survival assessed by automated image analysis. Individual neurons are assigned unique identifiers (yellow number) and tracked until their time of death (red), indicated by cellular dissolution, blebbing, or neurite retraction. Scale bar= 20µm. ( E ) Cumulative hazard plot depicting risk of death for neurons expressing TDP43(WT) + non-targeting (NT) (red line), mApple + NT (grey line), or TDP43(WT) + Atxn2 sgRNA (purple line). †p<2.0 ×10 −16 , Hazard ratio (HR)= 3.45; ***p= 5.81 ×10 −4 , HR= 0.80). ( F ) Forest plot showing HR for TDP43-overexpressing neurons upon knockdown of m6A writers (green), erasers (dark red), and readers (orange), in comparison to nontargeting (NT) control. Dashed line indicates HR= 1, representing the survival of the reference condition, neurons expressing TDP43-mApple and NT sgRNA. Values >1 indicate increased toxicity, whereas values <1 denote relative protection. Error bars represent 95% CI. ( G ) Alkbh5 knockout significantly increases TDP43 associated toxicity. †p=3.11 ×10 −5 , HR= 1.59; ***p= 2.65×10 −11 , HR= 2.03. ( H ) Ythdf2 knockout significantly extends survival in TDP43-expressing neurons. ***p <2.0 ×10 −16 , HR= 1.69; †p= 6.2 ×10 −6 , HR= 0.71. ( I ) YTHDF2 overexpression is toxic to neurons. ***p= 3.07×10 −5 , HR= 1.30. ( J ) METTL3/14 overexpression enhances TDP43-dependent toxicity in neurons. †p = 5.53 ×10 −4 , HR= 1.32; ***p =4.16 ×10 −6 , HR= 1.31. p values in E, G-J determined via Cox proportional hazards analysis, with a minimum 3 of biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Transfection, Expressing, Negative Control, Staining, MANN-WHITNEY, CRISPR, Fluorescence, Microscopy, Dissolution, Knockdown, Comparison, Control, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Immunostaining of YTHDF2 in control and sALS patient spinal cord samples. Scale bar= 50 µm. ( B ) Quantification of YTHDF2 immunoreactivity in spinal cord neurons from control (n= 117 neurons) and sALS (n= 193 neurons) samples. Plot shows mean +/- SD, color coded by sample. ****p< 0.0001 via Mann-Whitney test. ( C ) Strategy used to create isogenic iPSCs expressing native TDP43(WT)-Dendra2 or TDP43(M337V)-Dendra2. ( D ) Representative images of untransduced (grey) and transduced (green) iNeurons expressing shRNA against YTHDF2 (shYTHDF2) and a GFP reporter. Time of death (red circles) for each cell is used to determine cumulative risk of death, plotted in ( E ) and ( F ). Scale bar= 20µm. shRNA-mediated knockdown of YTHDF2 significantly extended the survival of TDP43(M337V)-Dendra2 iNeurons ( E ; †p= 8.42×10 −12 , HR= 6.25; ***p= 4.82×10 −9 , HR=0.32; #p= 0.08, HR= 1.84) as well as mutant C9ORF72 iNeurons ( F , †p= 1.42×10 −11 , HR= 2.85; ***p= 1.42×10 −16 , HR= 0.32). ns= not significant. Values in ( E , F ) calculated by Cox proportional hazards analysis, with a minimum 3 biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunostaining, Control, MANN-WHITNEY, Expressing, shRNA, Knockdown, Mutagenesis