human pulmonary artery endothelial cells  (Cell Applications Inc)

Journal: PLOS One

Article Title: In vitro and in vivo characterization of wild type BMP9 and a non-osteogenic variant in models of pulmonary arterial hypertension

doi: 10.1371/journal.pone.0329089

Figure Lengend Snippet: (A) WT BMP9 and A347E variant in cell-based human MSC assay measuring SP7 expression as a marker for osteogenic differentiation in a dose-response experiment (0.0195nM-10nM). (B) pSMAD1 activation in human endothelial cells in a dose-response experiment (0.000169nM-10nM). (C-E) Human primary pulmonary artery endothelial cells were treated with WT BMP9 and A347E variant at 10nM for 24h and gene expression was measured using Taqman PCR (C) ID2, (D) TGFBI, (E) PAI1. (F) pSMAD1 activation in primary rat endothelial cells in a dose-response experiment (0.000169nM-10nM). (G) WT BMP9 and A347E variant (30 µg/kg) were dosed SC to naïve rats (n = 4-5). Lung tissue was harvested 6h post dose and SMAD7 expression was evaluated in lung tissue as a PD marker by Taqman PCR. (H) WT BMP9 (30 µg/kg; IV) and A347E variant (1, 10, 30, 100 µg/kg; SC) was dosed to cynos (n = 3). Lung tissue was harvested 6h post dose and SMAD7 expression was evaluated in lung tissue as a PD marker by Taqman PCR.

Article Snippet: Human pulmonary artery endothelial cells, hPAEC (Cell Applications) were treated with WT BMP9 (R&D Systems) or BMP9-A347E at a single dose of 10nM for 24h.

Techniques: Variant Assay, Expressing, Marker, Activation Assay, Gene Expression

Journal: Journal of respiratory biology and translational medicine

Article Title: USP11 Promotes Endothelial Apoptosis-Resistance in Pulmonary Arterial Hypertension by Deubiquitinating HINT3

doi: 10.70322/jrbtm.2025.10002

Figure Lengend Snippet: HINT3 is degraded by ubiquitination and USP11 deubiquitinates HINT3 in HPAECs in vitro . ( A ) Human pulmonary artery endothelial cells (HPAECs) were cultured for 48 h. Cell lysates were harvested and subjected to immunoprecipitation (IP) with either anti-IgG antibody or anti-HINT3 antibody and then immunoblotted with anti-USP11, anti-HINT3, or anti-β-actin (ACTB) antibody. ( B ) HPAECs were transfected for 24 h with HA-ubiquitin (1 μg, HA-Ubi) plasmid or the control plasmid (empty vector), then collected and assayed by immunoblotting for anti-HA, anti-HINT3, and ACTB (loading control) antibody. ( C ) HPAECs were pretreated with MG132 for 2 h, and then were treated with cycloheximide (CHX, 20 μg/mL) for 0, 2, 4, and 8 h, to inhibit protein synthesis, and harvested for immunoblotting. The level of HINT3 was set as 100% at time 0, and the percent HINT3 protein remaining after CHX treatment at each time point was calculated accordingly. All bars represent mean HINT3 protein levels ± SEM relative to ACTB expressed as fold-change vs. CON * p < 0.05 vs. CON, n = 3. ( D ) HPAECs were transfected with USP11 (1 μg, oxHUWE1) plasmid or the control plasmid (empty vector) for 6 h. After media replacement, HPAECs were incubated for additional 72 h. Cell lysates were collected and immunoblotted with anti-HINT3 or ACTB antibody. ( E ) HPAECs were treated with scrambled (SCR) or HINT3 (20 nM) siRNAs for 6 h, media were replaced with endothelial growth medium (EGM) containing 5% FBS. And then incubated for an additional 72 h. Western blotting was performed for HINT3 or GAPDH protein. ( F ) HPAECs were treated with dimethyl sulfoxide (DMSO) or USP11 inhibitor (mitoxantrone) for 0–4 h. Western blotting was performed for HINT3 or GAPDH protein.

Article Snippet: Human pulmonary artery endothelial cells (HPAECs) were obtained from Cell Applications, Inc. (Cell Applications, San Diego, CA, USA).

Techniques: Ubiquitin Proteomics, In Vitro, Cell Culture, Immunoprecipitation, Transfection, Plasmid Preparation, Control, Western Blot, Incubation

Journal: Journal of respiratory biology and translational medicine

Article Title: USP11 Promotes Endothelial Apoptosis-Resistance in Pulmonary Arterial Hypertension by Deubiquitinating HINT3

doi: 10.70322/jrbtm.2025.10002

Figure Lengend Snippet: HINT3 regulates BCL2 expression in HPAECs in vitro . ( A ) Human pulmonary artery endothelial cells (HPAECs) were cultured for 48 h. Cell lysates were harvested and subjected to immunoprecipitation (IP) with either anti-IgG antibody or anti-HINT3 antibody, and then immunoblotted with anti-HINT3, anti-BCL2, or anti-β-actin (ACTB) antibody. ( B ) HPAECs were treated with scrambled (SCR) or HINT3 (20 nM) siRNAs for 6 h, and the media was replaced with an endothelial growth medium (EGM) containing 5% FBS. And then incubated for an additional 72 h. Western blotting was performed for HINT3, BCL2, USP11, or GAPDH protein. ( C ) HPAECs were transfected with either the control plasmid (Mock) or USP11 (1 μg, oxUSP11) plasmid for 6 h, media were replaced with endothelial growth medium (EGM) containing 5% FBS. And then incubated for an additional 72 h. Cell lysates were collected and immunoblotted with anti-BCL2 or ACTB antibody. ( D ) Hypothetical schema defining the role of USP11/HINT3/BCL2 signaling in PAH pathogenesis. Hypoxia induces USP11, which stabilizes HINT3 levels through HINT3 deubiquitination. Increases in HINT3 stimulate anti-apoptosis marker, BCL2 expression promoting PAH pathogenesis

Article Snippet: Human pulmonary artery endothelial cells (HPAECs) were obtained from Cell Applications, Inc. (Cell Applications, San Diego, CA, USA).

Techniques: Expressing, In Vitro, Cell Culture, Immunoprecipitation, Incubation, Western Blot, Transfection, Control, Plasmid Preparation, Marker

Journal: Journal of Cell Science

Article Title: Protein kinase Cα regulates the nucleocytoplasmic shuttling of KRIT1

doi: 10.1242/jcs.250217

Figure Lengend Snippet: The PKC-dependent nucleocytoplasmic translocation of KRIT1 occurs also in endothelial cells. (A) Representative mCherry–KRIT1 fluorescence, nuclear staining (Hoechst) and merged images in adenovirally-transduced HPAECs. Subconfluent cells were treated with DMSO vehicle (CTRL; panels a–c), 1 µM BIM for 30 min (panels d–f), 20 ng/ml PMA for 2 h (panels g–i), or pre-treated for 30 min with the PKC-specific inhibitor BIM before PMA administration (BIM+PMA; panels j–l). Subcellular localization of mCherry–KRIT1 was analyzed by epifluorescence microscopy. Consistent with observations in HeLa cells, PMA promoted KRIT1 translocation from the nucleus to cytoplasm, while BIM treatment promoted nuclear accumulation. Scale bars: 20 μm. (B) Quantification of nuclear:cytoplasmic fluorescence intensity ratio. n =36 cells from five biological replicates. (C) Representative mCherry–KRIT1 fluorescence, nuclear staining (Hoechst) and merged images in adenovirally-transduced HPAECs. Confluent cells were treated with DMSO vehicle (CTRL; panels a–c), 1 μM BIM for 30 min (panels d–f), 20 ng/ml PMA for 2 h (panels g–i), or pre-treated for 30 min with BIM prior to PMA treatment (BIM+PMA; panels j–l). Subcellular localization of mCherry–KRIT1 was analyzed by epifluorescence microscopy. PMA treatment strongly promoted nuclear-to-cytoplasmic shuttling of KRIT1 in confluent endothelial cells, while BIM treatment with or without PMA promoted KRIT1 nuclear accumulation, similar to effects seen in subconfluent cells. Scale bars: 50 μm. (D) Quantification of nuclear:cytoplasmic fluorescence intensity ratio. n =37 cells from three biological replicates. Data in B and D are mean±s.e.m. ratios normalized to CTRL, * P <0.05; ** P <0.01 versus vehicle (one-way ANOVA with Tukey post hoc testing).

Article Snippet: Primary human pulmonary artery endothelial cells (HPAEC; Cell Applications Inc., San Diego, CA, USA) were cultured in DME/F-12 medium (HyClone GE Healthcare, Piscataway, NJ, USA) containing 5% FBS, 1× endothelial cell growth supplement (ECGS, ScienCell, Carlsbad, CA, USA), 15 U/ml heparin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Gibco).

Techniques: Translocation Assay, Fluorescence, Staining, Epifluorescence Microscopy

Journal: Journal of Cell Science

Article Title: Protein kinase Cα regulates the nucleocytoplasmic shuttling of KRIT1

doi: 10.1242/jcs.250217

Figure Lengend Snippet: NAC treatment promotes nuclear accumulation of KRIT1 in PMA-treated endothelial cells. (A) Representative mCherry–KRIT1 fluorescence, nuclear staining (Hoechst), and merged images in adenovirally-transduced HPAECs. Subconfluent and confluent endothelial cells were treated with DMSO vehicle (CTRL; panels a–f), 20 ng/ml PMA for 2 h (panels g–l), 10 mM NAC for 2 h (panels m–r), or co-treated with 10 mM NAC and 20 ng/ml PMA for 2 h (NAC+PMA; panels s–x). Subcellular localization of mCherry–KRIT1 was analyzed by epifluorescence microscopy. While PMA promoted KRIT1 translocation out of the nucleus, NAC treatment alone or in conjunction with PMA treatment promoted nuclear localization of KRIT1, indicating a role for redox-mediated control of PKC activation in KRIT1 nucleocytoplasmic shuttling. Scale bars: 20 µm (subconfluent), 50 μm (confluent). (B) Quantification of nuclear:cytoplasmic fluorescence intensity ratio. Data shown are mean±s.e.m. ratios normalized to CTRL. Subconfluent, n =18 cells from four biological replicates. Confluent, n =28 cells from three biological replicates. * P <0.05; ** P <0.01 versus control (one-way ANOVA with Tukey post hoc testing).

Article Snippet: Primary human pulmonary artery endothelial cells (HPAEC; Cell Applications Inc., San Diego, CA, USA) were cultured in DME/F-12 medium (HyClone GE Healthcare, Piscataway, NJ, USA) containing 5% FBS, 1× endothelial cell growth supplement (ECGS, ScienCell, Carlsbad, CA, USA), 15 U/ml heparin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Gibco).

Techniques: Fluorescence, Staining, Epifluorescence Microscopy, Translocation Assay, Control, Activation Assay