human pulmonary microvascular endothelial cells pmvecs  (Procell Inc)

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: A) Cartoon of BMP9 signaling in HHT. B) Western blot verification of BMP9 in PMVECs. C-E) siRNA-mediated knockdown verification of ALK1, ENG, and SMAD4 by qPCR. F and G) Validation of BMP9 signaling in ALK1, ENG, and SMAD4 knockdown conditions in PMVECs by qPCR (read out ID1).

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Western Blot, Knockdown, Biomarker Discovery

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: Functional consequence of ALK1, ENG, and SMAD4 knockdown in PMVECs (proliferation, apoptosis and tube formation).

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Functional Assay, Knockdown

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: A and B) Venn diagram of common upregulated or downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated with BMP9 (20ng/ml) for 2 or 24hrs (RNAseq). C) Heatmap of the common upregulated and downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated with BMP9 for 2 and 24hrs. D) SinyGo biological processes analysis of the common upregulated and downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated with BMP9 for 2 and 24hrs. E) Panther pathway analysis of the common upregulated and downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated BMP9 for 2 and 24hrs.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Knockdown

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: STRING network analysis of the common upregulated and downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated with 2h and 24h of BMP9.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Knockdown

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: Validation of common upregulated or downregulated genes signatures with BMP9 stimulation at 2 (A and B) or 24 hrs (C and D) and functional consequences of the knockdown of LYVE1, GPNMB and MC5R in PMVECs. After 72 hrs knockdown of LYVE1, MC5R, and GPNMB PMVECs proliferation (E), apoptosis (F), tube formation (G) were assessed.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Biomarker Discovery, Functional Assay, Knockdown

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: Drug prediction based on the common upregulated downstream targets after ALK1, ENG and SMAD4 knockdown and experimental validation in PMVECs. A) Experimental strategy: the common upregulated genes 117 (2h) and 112 (24h) after knockdown of the three HHT genes and stimulation with BMP9 for 2 and 24hrs (HHT disease signature) were uploaded separately on the Clue query app. The criteria of the drugs rank include samples >=3, normalized connectivity score, tas value >=0.20, and FDR value. B) top scoring 5 HHT (positive values) and anti-HHT drugs (negative values, indicated by brown color) are represented. (C-K) Effect of Brivanib on the expression of the common persistent downstream targets (LYVE1, GPNMB, RRAGD, ANKRD33, HS3ST2, MC5R, SLC25A47, FGF19, SHISA9, FRG2C, HIST1H2BE, and CPA4) after ALK1, ENG and SMAD4 knockdown was assessed by qRT-PCR in PMVECs. MOA, mode of action; cs, connectivity score; tas, transcriptional activity score. Data are represented as mean ± standard error mean (n=3). Two-way repeated measures ANOVA with a Bonferroni post-hoc test, * P= <0.5, ** P= <0.01, *** P= <0.001, **** P= <0.0001.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Knockdown, Biomarker Discovery, Expressing, Quantitative RT-PCR, Activity Assay

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: Effect of Brivanib on the expression of the common persistent downstream targets after ALK1, ENG and SMAD4 knockdown were assessed by qRT-PCR in PMVECs (A-C).

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Expressing, Knockdown, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: A) PMVECs were treated with 10uM Brivanib or DMSO for 24 hrs followed by 20ng/mL VEGF or PBS for 10 mins. Protein was harvested and the effect of Brivanib on VEGF-induced phosphorylation of ERK1/2 was assessed in PMVECs by western blotting. B) The effect of Brivanib on VEGF-induced cell proliferation was assessed by MTT assay in PMVECs. C) Angiogenesis was assessed in a Matrigel tube formation assay using PMVECs treated with either Brivanib 1, 10, 50 uM or DMSO. D) Proposed model for the mechanism by which Brivanib might influence AVM and HHT pathogenesis.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Phospho-proteomics, Western Blot, MTT Assay, Tube Formation Assay