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: 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: Frontiers in Cell and Developmental Biology

Article Title: VE-PTP controls a fluid shear stress set point that governs cell morphological responses through Tie-2

doi: 10.3389/fcell.2025.1603517

Figure Lengend Snippet: VE-PTP inhibition increases shear stress induced cellular morphological responses in a Tie-2-FOXO1 dependent way. (A–C) Effect of Tie-2 siRNA on AKB-9778-mediated promotion of cell alignment and elongation in 5 dyn/cm 2 of shear stress. HUVECs transfected with control or Tie-2 siRNA were exposed to shear stress for 24 h with or without AKB-9778 in EBM-2 flow medium. The resulting cells were stained for VE-cadherin (green) and VE-PTP (red) (A) . Percentage of parallel cell alignment (B) and elongation (C) were quantified with Fiji/ImageJ. (D) siRNA-mediated Tie-2 silencing efficiency. HUVECs transfected with control or Tie-2 siRNA were treated with AKB-9778 for 4 hours. The resulting cells were lysed and immunoblotted with antibodies against Tie-2, VE-PTP and α-Tubulin. (E–G) Effect of FoxO1 siRNA on VE-PTP siRNA-mediated promotion of cell alignment and elongation in 5 dyn/cm 2 of shear stress. HUVECs transfected with control, VE-PTP or FoxO1 siRNA were exposed to shear stress for 24 h in EBM-2 flow medium. The resulting cells were stained for VE-cadherin (green), FoxO1 (red) and Hoechst (blue) (E) . Percentage of parallel cell alignment (F) and elongation (G) were quantified with Fiji/ImageJ. (H) siRNA-mediated FoxO1 and VE-PTP silencing efficiency. HUVECs were transfected with control, VE-PTP or FoxO1 siRNA. The resulting cells were lysed and immunoblotted with antibodies against FoxO1, VE-PTP and α-Tubulin. Mean ± SEM; n = 7 (A–C) or 4 (E–G) ; P values are calculated with two-way ANOVA followed by Dunnett’s multiple tests ( (C) ; vs. control siRNA + AKB-9778 and (F) ; vs. VE-PTP siRNA). Scale bars: 50 μm (A,E) .

Article Snippet: The following antibodies were used for immunofluorescence and immunoblotting: Rabbit monoclonal anti-human FoxO1 (clone C29H4 #2880, Cell Signaling, 1:1,000 for immunoblotting and 1:200 for immunofluorescence staining); rabbit monoclonal anti-human LC3B (clone D11 #3868, Cell Signaling, 1:1,000 for immunoblotting); mouse monoclonal anti-human Tie-2 (clone Ab33, #05-584, Sigma, 1 μg/mL for immunoblotting or 5 μg/mL for immunoprecipitation); mouse monoclonal anti-human α-tubulin (clone B-5-1-2 #T6074, Sigma, 0.5 μg/mL for immunoblotting); mouse monoclonal anti-phosphotyrosine (clone 4G10, #05-321, Sigma, 0.5 μg/mL for immunoblotting); mouse monoclonal anti-human VE-cadherin (clone F-8 #sc-9989, Santa Cruz Biotechnology, 1:100 for immunofluorescence staining); rabbit polyclonal anti-human VEGFR2-pY1054/59 (#44-1047G, invitrogen, 1:250 for immunofluorescence staining) rabbit polyclonal anti-human VE-PTP (VE-PTPh1-8, homemade , 10 μg/mL for immunofluorescence staining); rabbit polyclonal anti-human VE-PTP (VE-PTP-C, homemade , 1 μg/mL for immunoblotting).

Techniques: Inhibition, Shear, Transfection, Control, Staining

Journal: Journal of Translational Medicine

Article Title: AIMP2 accumulation in brain leads to cognitive deficits and blood secretion in Parkinson’s disease

doi: 10.1186/s12967-024-05666-x

Figure Lengend Snippet: Intracellular AIMP2 accumulation leads to AIMP2 secretion and intercellular transmission. ( A ) Representative immunofluorescence images of endothelial marker CD31 and AIMP2 in the cortical brain subregions from two- and six-month-old AIMP2 transgenic mice and age-matched littermate controls. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar = 10 μm. ( B ) Quantification of AIMP2 signal intensities in CD31-positive brain endothelium in the indicated mouse groups ( n = 5 in two-month-age group, n = 5 in control six-month-age group, and n = 8 in AIMP2 Tg six-month-age group). ( C ) Anti-AIMP2 dot blot assessment of GFP-AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with either GFP or GFP-AIMP2 constructs. Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( D ) Quantification of secreted AIMP2 in the media from SH-SY5Y cells transfected with GFP or GFP-AIMP2 based on the dot blot result in the panel C ( n = 3 separate experiments per group). ( E ) Anti-GFP dot blot assessment of GFP-AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with either GFP or GFP-AIMP2 constructs. Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( F ) Quantification of relative anti-GFP dot blot optical densities for experimental groups in the panel E ( n = 3 separate experiments per group). ( G ) Dot blot assessment of AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with GFP-AIMP2 construct (0, 1, 2 μg). Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( H ) Quantification of secreted AIMP2 in the media from SH-SY5Y cells transfected with the indicated combination of GFP and GFP-AIMP2 ( n = 3 separate experiments per group). ( I ) Representative immunofluorescence images showing GFP-AIMP2 uptake into HUVECs. HUVECs were treated with conditioned media (48 h) from GFP or GFP-AIMP2 transfected SH-SY5Y cells. Scale bar = 50 μm. ( J ) Percentage GFP-positive HUVECs in the indicated experimental groups ( n = 3 separate experiments per group). ( K ) Quantification of GFP-AIMP2 immunofluorescence signals in HUVECs in the indicated experimental groups ( n = 3 separate experiments per group). Quantitative data are expressed as the mean ± SEM, and statistical significance was determined by ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001. ns, non-significant

Article Snippet: The following primary antibodies were used: rabbit antibody against AIMP2 (Proteintech; #10424-1-AP, 1:5,000), mouse antibody against MAP2 (Sigma-Aldrich; #M4403, 1:5,000), rabbit antibody against MAP2 (Abcam; #ab32454, 1:5,000), mouse antibody against CD31 (BD Biosciences; #550274, 1:1,000), and rabbit antibody against GFP (Cell Signaling Technology; #2555S, 1:5,000).

Techniques: Transmission Assay, Immunofluorescence, Marker, Transgenic Assay, Control, Dot Blot, Transfection, Construct, Staining

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: 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 cell biology

Article Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity.

doi: 10.1038/ncb2332

Figure Lengend Snippet: Figure 6 Autophagy controls intracellular MBd levels. (a) Single-plane confocal microscopy images of MBds within LC3-positive autophagosomes in MEFs expressing GFP–LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MBd markers: Cep55, MKLP1 or MgcRacGAP. Autophagosomes: GFP–LC3 or LC3. Note that MKLP1 (blue) and MgcRacGAP (red) are co-localized (magenta) in the autophagosome (green), indicating that MBds are sorted into autophagosomes. Scale bars, 2 µm. (b) Decreasing autophagy levels by deletion of the Atg5 gene (left) or depletion of ATG7 by siRNA (right) significantly increases the percentage of MBd+ cells (P = 0.0019 and P = 0.021, respectively, n = 3). Immunoblots confirm loss of the Atg5–Atg12 conjugation in mutant cells and depletion of ATG7 (asterisk). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (c) Rapamycin (Rapa) and LiCl co-treatment induces autophagy and decreases the percentage of MBd+ cells (left, HeLa; P = 0.0056, n = 3). Immunoblots showing

Article Snippet: Lee); Centriolin (1:200, ref. 9); Flag (1:200, Sigma, F7425); GAPDH (1:8,000; Santa Cruz, SC-32233); GFP (1:1,000; Abcam, ab6556, and Santa Cruz, sc-9996); GT335 (1:100; a gift from P. Denoulet); β1-integrin (1:50; BD Phramingen); K15 (1:100; Lab Vision, MS-1068-P); LC3 (1:10 for immunofluorescence, Nano Tools, LC3-5F10; 1:300 for immunoblotting, Novus Bio NB100-2331); LAMP2 (1:50, H4B4 from DSHB); MgcRacGAP (1:500, Abcam, ab2270); MKLP1 (1:1,000 for immunofluorescence, 1:200 for immunohistochemistry, 1:10 for immuno-electron microscopy, Santa Cruz, sc-867); NBR1 (1:500, Abnova, H00004077-B01P); p62, human samples (1:500, BD Trans Lab, 610833); p62, mouse samples (1:1,000, Progen, GP62-C); RFP (1:200, Clontech, 632496); Na/K-ATPase (1:15, α6F, DSHB); α-tubulin (1:100 for immunofluorescence, 1:400 for immunoblotting, Sigma, T9026a; 1:100 for immunofluorescence, Millipore, CBL270); α-tubulin– FITC (fluorescein isothiocyanate) (1:300, Sigma, F2168); TRA-1-60–biotin (1:200, eBioscience, 13-8863); ubiquitin (1:2,000, BDBioSci, no 550944);WGA-Alexa Fluor 555 (1:200, Molecular Probes, W32464); ZO-1–FITC (1:50, Zymed, 33-9111).

Techniques: Confocal Microscopy, Expressing, Staining, Western Blot, Conjugation Assay, Mutagenesis

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. MEFs harboring TetO-OKSM and M2rtTA cassettes were transfected with siRNA targeting different canonical NuRD components (indicated in the illustration), 2 and 4 days after reprogramming initiation following DOX administration. Reprogramming was then evaluated by AP staining at day 8. B. Reprograming efficiency following siRNA treatments was evaluated using AP staining, after 8 days of reprogramming (n=3, two-sided Student’s t-test p values are indicated). C. Cell growth curves of MEFs treated with siRNA for the indicated NuRD components (two-sided Student’s t-test p values are indicated). Knockdown (KD) of Gatad2a, unlike KD of Chd4, Mbd3 and Hdac2, does not severely inhibit cell proliferation. D. Representative images of cells treated with siRNA targeting Mbd3 or Gatad2a and exposed to BrdU in order to evaluate proliferation. E. Quantitative evaluation of BrdU incorporation test, which shows normal proliferation in siScramble and siGatad2a, unlike in cells treated with siMbd3 (n=8, two-sided Student’s t-test p values are indicated Student’s t-test). F . Knockdown for different canonical NuRD components does not show a significant elevation in cell death or apoptosis. Viability and apoptosis induction were measured using FACS following Annexin-PI staining. G. Reprogramming efficiency following siRNA treatments targeting different NuRD components, at different time points. KD was performed at two distinct cycles: the early one (Regimen 1, marked in black) started one day prior to DOX induction, and the second one (Regimen 2, marked in grey) started one day post-DOX induction. H. iPSC reprogramming efficiency following different siRNA treatments. was evaluated at day 8. (n=3 per each condition, two-sided Student’s t-test p values are indicated). Gatad2a siRNA improves reprogramming whether administrated prior or post DOX administration, unlike siRNA targeting Mbd3 or Chd4, in which only in Regimen #2 iPSC colony formation efficiency was increased.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Transfection, Staining, Knockdown, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A . Scheme demonstrating strategy for generating secondary isogenic Gatad2a WT and KO lines, and comparing their reprogramming efficiency side by side. 2i/LIF-KSR conditions were introduced at day 3.5 during the 8-day course. More detailed information is provided in Supplementary regarding different systems used herein. B . Targeting scheme of Gatad2a locus to generate knockouts by CRISPR/Cas9. C . Western blot validation of Gatad2a knockout in iPSC harboring M2rtTA and TetO-OKSM cassettes in comparison to its parental isogenic line. D . Representative images of Gatad2a -/- iPSC derived E13.5 chimera. Red arrow highlights mCherry+ chimera which originates from mCherry labeled iPSCs that were microinjected. E. Bulk iPSC reprogramming as in F , but experiment was terminated after 6 days and iPSC colony formation was evaluated by Alkaline Phosphatase staining (AP+). F . Representative flow cytometry measurements of ΔPEOct4-GFP reactivation dynamics in polyclonal/bulk Gatad2a-WT and Gatad2a-KO isogenic cell lines. Throughout the course of the reprogramming experiment the cells were not passaged to avoid any biases. Reprogramming of secondary MEFs seeded as single cells. G. Representative summaries of single-cell iPSC reprogramming efficiency experiment. Secondary isogenic Gatad2a WT and KO reprogrammable MEFs carrying constitutively expressed mCherry-NLS and naïve pluripotency specific ΔPE-Oct4-GFP reporter were sorted and seeded as single-cell per well. Reprogramming was initiated by DOX administration according to panel A . Reprogramming efficiency was assessed after 8 days based on the number of wells in which mCherry+ cells formed an ΔPE-Oct4-GFP positive colony. Throughout the course of the reprogramming experiment the cells were not passaged to avoid any biases. H. The indicated secondary Gatad2a +/+ and Gatad2a -/- somatic cell types were isolated and subjected to single cell reprogramming and evaluation of iPSC efficiency following 8 days of DOX. Reprogramming efficiency was assessed after 8 days based on the number of wells in which mCherry+ cells formed an ΔPE-Oct4-GFP positive colony. In summary, these results indicate that complete inhibition of Gatad2a (also known as P66a), a NuRD specific subunit, does not compromise somatic cell proliferation as previously seen upon complete Mbd3 protein elimination, and yet disrupts Mbd3/NuRD repressive activity on the pluripotent circuitry and yields 90-100% highly-efficient reprogramming within 8 days as similarly observed previously in Mbd3 hypomorphic Mbd3 flox/- donor somatic cells ( ; ).

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: CRISPR, Western Blot, Biomarker Discovery, Knock-Out, Comparison, Derivative Assay, Labeling, Staining, Flow Cytometry, Isolation, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Three different clonal sets of isogenic secondary OSKM reprogrammable cells/sets were generated: (i) MEFs were reprogrammed to iPSC following viral infection of FUW-M2rtTA and FUW-OKSM. mCherry constitutive marker and ΔPE-Oct4-GFP markers were then introduced to the iPSC. (ii) R26-M2rtTA +/- m. Col1a-OKSM +/- ES were derived from E3.5 embryos following mating of mice. (iii) ES Kh2 m.Col1a-TetO-2XFlag-Mbd3 were injected to blastocysts, and MEF were harvested at E12.5. MEF were then reprogrammed following viral infection of FUW-OKSM. All described cell lines were then subjected to Gatad2a-KO using CRISPR/Cas9, followed by an injection of both the KO and its isogenic WT to blastocysts. Chimeric fibroblasts were separated from the donor cells by Puromycin selection. B . Summary of CRISPR/Cas9 strategy to generate Gatad2a null cells. sgRNA targeted sequence is indicated. C . Correct targeting efficiency by the strategy described in B to generate mouse Gatad2a KO PSCs as determined both by Western blot analysis and PCR sequencing. D . Representative Western blot analysis for sub cloned lines following targeting Gatad2a with sgRNA. Blue arrows indicate examples of KO clonal lines. E. Transcriptome landscape (RNA-seq), alongside ATAC-seq and H3K27Ac ChIP-seq, of fibroblasts (FSP-1 and Thy1) and pluripotent (Nanog and Sall4) related genes. IGV Data range is indicated at the top right corner of the signal.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Generated, Infection, Marker, Derivative Assay, Injection, CRISPR, Selection, Sequencing, Western Blot, Clone Assay, RNA Sequencing, ChIP-sequencing

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Mass spectrometry analysis of NuRD complex components binding efficiency to Mbd3 in Gatad2a-KO and its isogenic Gatad2a-WT line, in MEF cells during reprogramming by OSKM. Only putative NuRD components are presented and can be seen in an equal strength at both platforms, except for Chd4, which does not bind Mbd3 in Gatad2aKO. Flag-Tagged Mbd3 was used to establish a platform for studying Mbd3-binding proteins, by correct targeting of TetO-Mbd3-Flag into the M. Col1a locus. Isogenic Gatad2a-KO were generated from this line with CRISPR/Cas9 and used for the IP and MS analysis indicated above (See ). B. Flag-Mbd3 CoIP in Gatad2a-WT and Gatad2a-KO cells. Experiments were conducted both in MEF cells and ESs. C. Cells during reprogramming were treated with siRNA targeting Gatad2a, and pellets collected after four days. CoIP of Chd4 shows that siGatad2a prevents Mbd3 binding to Gatad2a but also to Chd4. D. Gatad2a-KO MEF with overexpression of Gatad2a (transgenic recovery; abbreviated as Gatad2a-Tg) or Mock were subjected to Chd4-CoIP. Gatad2aoverexpression recovers the binding of Chd4 to Mbd3 and does not affect its binding to Mta2. E. The coiled coil region of Mbd3 is highly conserved between different organisms, and different proteins in the MBD family. The highlighted amino acids are crucial for Gatad2a binding to Mbd2 or Mbd3. F. A scheme of Flag tagged Mbd3, and mutant forms of Mbd3: lacking the Coiled coil region (#CCR-Mbd3) or the methyl-binding domain (#MBD-Mbd3). G. WT-Mbd3 and both mutants were over-expressed in 293T cells and were subjected to Flag-Mbd3 CoIP to examine their protein interactions. While the deletion of MBD prevents the binding of Klf4, only the deletion of the coiled coil region abolished the binding to Chd4 and Gatad2a. H-I. Mbd3 fl/- secondary MEF, harboring #PE-Oct4-GFP reporter, were transfected with two different forms of Flag tagged WTMbd3 and ΔCCR-Mbd3. The cells were then subjected to reprogramming. While WT-Mbd3 significantly reduced reprogramming efficiency ( p Value<0.0001, two-sided Student’s t-test, n=3), ΔCCR-Mbd3 expression was not able to inhibit deterministic reprogramming in the cells, consistent with its inability to interact and recruit Chd4 to the assembled complex.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Mass Spectrometry, Binding Assay, Generated, CRISPR, Over Expression, Transgenic Assay, Mutagenesis, Transfection, Expressing

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. A scheme of Gatad2a constructs and mutants used. Gatad2a-WT, Gatad2a-Truncated (lacking the CCR2 of Gatad2a) and HA-tagged-CCR1-Gatad2a construct. B. Flag-Mbd3 and different Gatad2a constructs were cotransfected in 293T cells. Flag-Mbd3 binds both forms of Gatad2a (WT and truncated), as shown by Flag-Mbd3 CoIP. C. Dot blot analysis to validate protein expression following HA-Gatad2aCCR1 peptide overexpression. D. Overexpression of Flag-Mbd3 with HA-Gatad2a-CCR1 or control (HA-GFP) in 293T cells, followed by Co-IP for anti-Flag-Mbd3. Over-expression of Gatad2a-CCR1 reduces the binding of Mbd3 to endogenous Gatad2a and Chd4, comparing to the control specimen. Further, CCR can cause a reduction in Mbd3 binding to endogenous Gatad2a and Chd4, without changing other NuRD components (such as Hdac2) binding. E. Overexpression of STEMCCA-OKSM vector, HA-MBD (methyl-binding domain of Mbd3) or HA-GFP in 293T cells, was followed by Co-IP with anti-HA. The results demonstrate that MBD domain can bind to pluripotency factors such as Oct4 and to the NuRD component Hdac2 but cannot bind Gatad2a or Chd4. F. Summarizing scheme for three different approaches for neutralizing Mbd3-Gatad2a-Chd4 axis: (i) by deleting the CCR domain of Mbd3 (ii) KO or KD of Gatad2a protein and (iii) overexpression of an exogenous Gatad2a-CCR competitive peptide that interferes with Gatad2aCCR interaction with Mbd3.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Construct, Dot Blot, Expressing, Over Expression, Control, Co-Immunoprecipitation Assay, Binding Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Rosa26-M2rtTA Col1a:TetO-2XFlag-Mbd3 ES cells were subjected to different differentiation protocols, and cells from 5 distinct states (naïve ESC, EpiLC, EBs, MEF, 4-day OSKM Reprogramming) were subsequently subjected to CoIP with anti- Flag-Mbd3. Lysates were then analyzed by western blot, and reacted with different antibodies against different NuRD components, Pluripotency factors, and other epigenetic proteins. While some of the proteins show constitutive binding to Mbd3 throughout all differentiation states (Mta2, Prmt5)- other proteins show differential binding (Oct4, Klf4, Cdk2ap1) and the latter is not correlated with the proteins level in the cell. B. Rosa26-M2rtTA Col1a:TetO-2XFlag-Mbd3 ES cells were either maintained in ground state naïve conditions or in priming conditions (Fgf2/Activin A). Lysates were subjected to Co-IP with anti-Flag-Mbd3 and examined by Western blot. Oct4 protein expression is significantly reduced after priming, but its binding to Mbd3 can be detected only in the primed pluripotent state. C. Mbd3 expression in ES cells treated with growth media containing different small molecules, after 72 hours of treatment. Unlike other treatments, PKCi Go6983 (5 μM) treatment resulted in a radical decrease in Mbd3 protein expression. D. PKCi Go6983 effect on Mbd3 level is seen after approximately 48 hours, in different concentration ranging from 0.5 to 10 μM. E. WT EpiSCs reversion efficiency to naïve ESCs in different conditions. Anova test P values are indicated. (one representative experiment out of 3 performed is shown). F. Isogenic WT and Gatad2a KO ESCs were expanded on feeder free gelatin coated plates in N2B27 LIF only or LIF/PKCi conditions. Phase images and Oct4-GFP signal maintenance are shown after 8 passages (P8). Oct4-GFP. G. ES cells treated with naïve ground state condition were treated with shRNA targeting Ubc9 or Scramble. Cells were lysed and subsequent CoIP of Chd4 shows a decrease in Gatad2a binding to the protein. H. Cells induced in naïve ground state 2i/LIF conditions and subsequent shRNA targeting either for Ubc9 or scramble negative control. The cells were lysed and fractioned – Cytoplasm, Nucleoplasm and Chromatin fractions, proteins were analyzed by western blot. The NuRD components Mbd3 and Gatad2a, but not Mta2, can be seen mainly in the chromatin fraction, and their expression is significantly reduced following shUbc9 treatment.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Western Blot, Binding Assay, Co-Immunoprecipitation Assay, Expressing, Concentration Assay, shRNA, Negative Control

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Western blot showing Mbd3 protein levels in ESCs expanded in the indicated growth conditions. Mbd3 levels were decreased following PKCi (Go6983) treatment in ES cells maintained in different conditions (FBS and KSR based growth media, supplemented with 2i). B. Reduction in Mbd3 protein expression is maintained following long term PKCi (Go6983) treatment, as examined in different concentrations (0.5-5 μM). Media was exchanged ever 48h. C. Western blot analysis for different NuRD components in ESC cells with and without Go6983. Analysis shows that other NuRD components expression level is not affected from PKCi (Go6983) treatment. D. as in A, but a different PKCi was used, termed GFX which does not inhibit atypical PKC pathway. E. Mbd3 protein levels following shRNA treatment of mouse V6.5 ESCs for atypical PKCzeta isoform. F. Mbd3 levels in MEFs following treatment with Go6983 are not affected. G. RT-PCR analysis for Mbd3 transcript abundance 24 and 48h following PKCi Go6983 treatment, do not show significant changes and reduction in Mbd3 transcript level. This suggests that the depletion in Mbd3 protein levels shown in A-C is post-translational. H. Go6983 causes mild depletion in Mbd3 in MEFs following 3 days of OSKM expression. I . Gatad2a +/+ WT MEFs carrying ΔPE -Oct4-GFP reporter and constitutive mCherry markers (secondary system i) were subjected to iPSC reprogramming protocols in in and iPSC efficiency was quantified at day 8. In the last two conditions included in the panel, the MEFs were pre-treated with control and Ubc9 shRNA following with Neomycin selection , and then subjected to DOX mediated iPSC reprogramming. Anova P values are indicated. J. A Schematic representation of Gatad2a known domains and confirmed SUMOylation sites . Importantly, harnessing prediction tools for identification of possible SUMO consensus sites results in 2 additional sites inside the coiled coil regions (Chi-squared test p value<0.05). K. 2-d08 specific small molecule inhibitor for SUMOylation was applied during co-IP experiments for Chd4 at the indicated increasing concentrations known to deplete global SUMOylation levels. Cells were lysed the CoIP of Mbd3 shows a decrease in Gatad2a binding to Chd4, as similarly seen with shRNA depletion of Ubc9 . L. As in with immunoblot for SUMO2/3 on the same exact gel series.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Control, Selection, Co-Immunoprecipitation Assay, Binding Assay

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