human pulmonary microvascular endothelial cells hpmvec  (PromoCell)

Journal: Redox Biology

Article Title: MOTS-c attenuates lung ischemia-reperfusion injury via MYH9-Dependent nuclear translocation and transcriptional activation of antioxidant genes

doi: 10.1016/j.redox.2025.103681

Figure Lengend Snippet: MOTS-c attenuates oxidative stress and preserves endothelial barrier integrity in lung ischemia-reperfusion injury (A) Schematic diagram of the rat LIRI model via unilateral pulmonary hilar clamping. (B) Western blot analysis of MOTS-c expression levels in sorted endothelial cells, epithelial cells, neutrophils, and macrophages from LIRI and sham rat lungs (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (C) Immunofluorescence staining of lung tissue sections from LIRI rats, colocalizing MOTS-c with various cell markers. Red, MOTS-c; cyan, CD31; green, EpCAM; orange, CD11b; pink, CD68; blue, DAPI (nuclei). (D) Time-dependent increase in extracellular MOTS-c release from HPMVECs during HR (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (E) Intracellular MOTS-c expression dynamics in HPMVECs during HR (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (F) Representative immunofluorescence images of VE-cadherin (scale bars: 25 μm), rhodamine-phalloidin (scale bars: 25 μm), FITC-dextran permeability (scale bars: 50 μm), and DCFH-DA ROS staining (scale bars: 100 μm) in HPMVECs under normoxia, hypoxia-reoxygenation (HR), HR with actinonin (150 μM) pretreatment (48 h), and HR with MOTS-c overexpression conditions. (G) TEER measurements reflecting barrier function in HPMVECs treated with actinonin, MOTS-c overexpression, or vehicle during HR (n = 6, one-way ANOVA post hoc Student-Newman-Keuls test). (H–K) Quantitative analyses of FITC-dextran permeability (H), ROS fluorescence intensity (I), flow cytometry of ROS levels (J), MDA content (K), and GSH/GSSG ratio (L) in HPMVECs under indicated conditions (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (L – M) HR-induced inflammatory activation assessed by IL-6 secretion in cell culture medium (L) and ICAM-1 expression via Western blot (M) (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). Data are presented as the mean ± SEM. Abbreviations: LIRI, lung ischemia-reperfusion injury; H&E, hematoxylin & eosin; BALF, bronchoalveolar lavage fluid; H4R6, hypoxia for 4 h and reoxygenation for 6 h; H4R12, hypoxia for 4 h and reoxygenation for 12 h; H4R24, hypoxia for 4 h and reoxygenation for 24 h; HR, hypoxia reoxygenation.

Article Snippet: 6.Cell culture and hypoxia-reoxygenation (HR) model Human primary pulmonary microvascular endothelial cells (HPMVECs) were purchased from the ScienCell Research Laboratories (#3200, USA) and cultured in Endothelial Cell Medium (ScienCell, #1001, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Permeability, Over Expression, Fluorescence, Flow Cytometry, Activation Assay, Cell Culture

Journal: Redox Biology

Article Title: MOTS-c attenuates lung ischemia-reperfusion injury via MYH9-Dependent nuclear translocation and transcriptional activation of antioxidant genes

doi: 10.1016/j.redox.2025.103681

Figure Lengend Snippet: MOTS-c undergoes MYH9-dependent nuclear translocation in response to ROS production (A) Representative fluorescence images of HPMVECs stably expressing MOTS-c-GFP under normoxia, hypoxia-reoxygenation (HR), normoxia with tBHP (100 μM), and HR with NAC (10 mM) and quantification of the proportion of MOTS-c-GFP in the nucleus. Green, MOTS-c-GFP; blue, DAPI (nuclei). Scale bars: 20 μm. (B) The relative fluorescence intensity of DCFH-DA staining under the same conditions as in (A) and its linear correlation analysis with the proportion of MOTS-c-GFP in the nucleus. (C) Western blot analysis of GFP expression in cytoplasmic and nuclear fractions of MOTS-c-GFP HPMVECs under the same conditions as in (A) (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (D) Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of MOTS-c-GFP immunoprecipitates from normoxic and HR-treated HPMVECs, identifying MYH9 and γ-Actin as the top binding partners. (E) Volcano plot of IP-MS results labeled with MYH9 and γ-Actin, demonstrating no significant difference between normoxic and HR conditions. (F) Western blot validation of MOTS-c-GFP interactions with MYH9 and γ-Actin in immunoprecipitates from normoxic and HR-treated HPMVECs (n = 3). (G) Molecular docking simulation using GRAMM, illustrating the interaction between MOTS-c and the MYH9-γ-Actin complex. (H) Representative fluorescence images of MOTS-c-GFP HPMVECs transfected with siRNA-MYH9 or siRNA-NC under HR conditions and quantification of the proportion of MOTS-c-GFP in the nucleus, demonstrating reduced nuclear GFP fluorescence in MYH9-depleted cells. Green, MOTS-c-GFP; blue, DAPI (nuclei). Scale bars: 20 μm. (I) Western blot analysis of GFP expression in cytoplasmic and nuclear fractions of MOTS-c-GFP HPMVECs transfected with siRNA-MYH9 or siRNA-NC under HR conditions (n = 3, two-tailed t -test). (J) Western blot analysis of γ-Actin levels in GFP immunoprecipitates from MOTS-c-GFP HPMVECs transfected with siRNA-MYH9 or siRNA-NC, demonstrating reduced γ-Actin binding upon MYH9 depletion (n = 3, two-tailed t -test). Data are presented as the mean ± SEM. Abbreviations: HR, hypoxia reoxygenation; tBHP, tert -butyl hydroperoxide; NAC, N-acetylcysteine.

Article Snippet: 6.Cell culture and hypoxia-reoxygenation (HR) model Human primary pulmonary microvascular endothelial cells (HPMVECs) were purchased from the ScienCell Research Laboratories (#3200, USA) and cultured in Endothelial Cell Medium (ScienCell, #1001, USA).

Techniques: Translocation Assay, Fluorescence, Stable Transfection, Expressing, Staining, Western Blot, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Protein-Protein interactions, Labeling, Biomarker Discovery, Transfection, Two Tailed Test

Journal: Redox Biology

Article Title: MOTS-c attenuates lung ischemia-reperfusion injury via MYH9-Dependent nuclear translocation and transcriptional activation of antioxidant genes

doi: 10.1016/j.redox.2025.103681

Figure Lengend Snippet: CK2A-Mediated MYH9 Ser1943 Phosphorylation Governs MOTS-c Nuclear Translocation and Endothelial Protection (A) Left : Higher-energy collision-induced dissociation (HCD) MS/MS spectrum of the human MYH9 phosphopeptide KGAGDGSDEEVDGK ([M+2H] 2+ ion at m / z 722.2874). Predicted b- and y-ions are annotated. Right : MYH9 Ser1943 phosphorylation intensity in normoxia versus HR groups (n = 3, two-tailed t -test). (B) Immunoprecipitation of MOTS-c-GFP with MYH9 in HPMVECs under normoxia or HR, followed by Western blot analysis of p-MYH9/t-MYH9 ratios (n = 3, two-tailed t -test). (C) Western blot analysis of MYH9 Ser1943 phosphorylation in HPMVECs treated with tBHP (100 μM) under normoxia or NAC (10 mM) during HR (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (D) Immunoprecipitation of MYH9 with CK2A and PKCα in MOTS-c-GFP HPMVECs under normoxia or HR (n = 3, two-tailed t -test). (E) Western blot analysis of MYH9 phosphorylation in HPMVECs pretreated with CK2A inhibitor CX4945 (10 μM, 2 h) or PKCα inhibitor GO6983 (100 nM, 2 h) prior to HR (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (F) Representative fluorescence imaging of MOTS-c-GFP nuclear translocation in HPMVECs pretreated with CX4945 (10 μM, 2 h) or GO6983 (100 nM, 2 h) prior to HR and quantification of the proportion of MOTS-c-GFP in the nucleus. Green, MOTS-c-GFP; blue, DAPI (nuclei). Scale bars: 25 μm. (G) Subcellular fractionation and Western blot analysis of GFP localization in cytoplasmic and nuclear compartments (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (H) Western blot analysis of MYH9 phosphorylation in HPMVECs pretreated with NAC (10 mM), CX4945 (10 μM), or GO6983 (100 nM) for 2 h, followed by tBHP (100 μM) stimulation for 16 h (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (I) TEER measurements in MOTS-c-overexpressing HPMVECs pretreated with CX4945 (10 μM, 2 h) during HR (n = 6, two-tailed t -test). (J) Representative immunofluorescence images of FITC-dextran permeability (scale bars: 50 μm) and DCFH-DA ROS staining (scale bars: 100 μm) in HPMVECs pretreated with CX4945 (10 μM, 2 h) during HR (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). Data are presented as the mean ± SEM. Abbreviations: HR, hypoxia reoxygenation; IP, immunoprecipitation; tBHP, tert -butyl hydroperoxide; NAC, N-acetylcysteine.

Article Snippet: 6.Cell culture and hypoxia-reoxygenation (HR) model Human primary pulmonary microvascular endothelial cells (HPMVECs) were purchased from the ScienCell Research Laboratories (#3200, USA) and cultured in Endothelial Cell Medium (ScienCell, #1001, USA).

Techniques: Phospho-proteomics, Translocation Assay, Tandem Mass Spectroscopy, Two Tailed Test, Immunoprecipitation, Western Blot, Fluorescence, Imaging, Fractionation, Immunofluorescence, Permeability, Staining

Journal: Redox Biology

Article Title: MOTS-c attenuates lung ischemia-reperfusion injury via MYH9-Dependent nuclear translocation and transcriptional activation of antioxidant genes

doi: 10.1016/j.redox.2025.103681

Figure Lengend Snippet: Nuclear MOTS-c Exhibits Transcriptional Factor-like Activity by Directly Binding to Antioxidant Gene Promoters (A) Volcano plot of RNA-seq data from MOTS-c-overexpressing HPMVECs subjected to hypoxia-reoxygenation (HR). Differentially expressed genes were filtered (|fold change| >2, Q-value <0.05), with key antioxidant enzymes and pro-inflammatory factors highlighted in red. (B) Gene Ontology (GO) enrichment analysis of differentially expressed genes, categorized into biological processes (BP), cellular components (CC), and molecular functions (MF). (C) Left : Venn diagram of genes identified in three independent ChIP-seq experiments. Right : Peaks corresponding to overlapping genes. (D) Left : Intersection of ChIP-seq-derived genes with promoters containing AREs. Right : Genomic coordinates of overlapping genes. (E) Intersection of RNA-seq and ChIP-seq datasets and core analysis results. (F – G) RT-qPCR and Western blot validation of HMOX1, NQO1, GPX2, and PRDX6 expression in HPMVECs with MOTS-c modulation under HR (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (H) Luciferase reporter gene assays confirming transcription factor-like effects of MOTS-c on the AREs of antioxidant genes and the promoter regions of IL6 (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). (I) ChIP-qPCR confirming MOTS-c binding to promoter regions of HMOX1, NQO1, GPX2, and PRDX6 in MOTS-c-overexpressing HPMVECs post-HR (n = 3, two-tailed t -test). (J – K) RT-qPCR and Western blot analysis of HMOX1, NQO1, GPX2, and PRDX6 expression in MOTS-c-overexpressing HPMVECs pretreated with MYH9 siRNA or CK2A inhibitor CX4945 (10 μM, 2 h) under HR (n = 3, one-way ANOVA post hoc Student-Newman-Keuls test). Data are presented as the mean ± SEM. Abbreviations: HR, hypoxia reoxygenation; IP, immunoprecipitation; ChIP-seq, chromatin immunoprecipitation sequencing; ARE, antioxidant response element; RNA-seq, RNA sequencing; RLU, relative light unit.

Article Snippet: 6.Cell culture and hypoxia-reoxygenation (HR) model Human primary pulmonary microvascular endothelial cells (HPMVECs) were purchased from the ScienCell Research Laboratories (#3200, USA) and cultured in Endothelial Cell Medium (ScienCell, #1001, USA).

Techniques: Activity Assay, Binding Assay, RNA Sequencing, ChIP-sequencing, Derivative Assay, Quantitative RT-PCR, Western Blot, Biomarker Discovery, Expressing, Luciferase, ChIP-qPCR, Two Tailed Test, Immunoprecipitation