Journal: EXCLI Journal

Article Title: Hypoxia-inducible factor (HIF)-3a2 serves as an endothelial cell fate executor during chronic hypoxia

doi: 10.17179/excli2021-4622

Figure Lengend Snippet: Prolonged hypoxia results in the accumulation of HIF-3α2 in human HUVECs and other endothelial cell types. HUVEC, UtMVEC, HPAEC, HCAEC, HIAEC, HPASMC, HMVEC-D and HMVEC-L, cells were exposed to hypoxia for the time periods specified and total RNA and protein lysates were collected. (A) The changes in HIF-3α2 protein levels were evaluated by western blot normalized to β-actin and total protein levels and related to the normoxic control. In all of the endothelial cell types, the HIF-3α2 isoform is expressed. The HIF-3α2 protein level analysis was performed in parallel with HIF-1α and HIF-2α analyses that were presented in Bartoszewski et al. (2019). The raw data are presented in Supplementary data (PDF). (B) Spearman's correlation of the hypoxia induced HIF-3α2 protein levels change in different human ECs. The mathematic representation of HIF-3α2 changes in human ECs exposed to hypoxia. (C) The changes in HIF-3α levels (from all ECs tested) during hypoxia time course were analyzed using the sigmoid 3-parameter function (using 200 iterations, P < 0.005). The error bars represent SE.

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Cellworks (Caltag Medsystems Ltd, UK) and cultured in EGM-2 Bulletkit Medium (Lonza).

Techniques: Western Blot, Control

Journal: EXCLI Journal

Article Title: Hypoxia-inducible factor (HIF)-3a2 serves as an endothelial cell fate executor during chronic hypoxia

doi: 10.17179/excli2021-4622

Figure Lengend Snippet: Hypoxic accumulation of HIF-3α results from both increased expression and prolyl hydroxylase inactivation in HUVECs. (A) The Spearman's correlation of the hypoxia induced- HIF3A mRNA level changes in different human ECs. (B) The mathematic representation of HIF3A mRNA levels during hypoxia in human ECs. The changes in mRNA levels obtained from all ECs tested during the hypoxia time course were analyzed The changes in HIF3A levels in HUVECs during hypoxia time course were analyzed using the sigmoid 3 parameter function (using 200 iterations, P < 0.005). The error bars represent SE. (C) Schematic presentation of firefly luciferase (FLuc) and renilla luciferase reporter cloning vector pEZX-FR02 (GeneCopoeia) containing the HIF3A human (NM_152794) gene region comprising ODD (amino acids 450-576, including the Proline 492 (a PHD substrate) was fused with firefly luciferase (without the ATG start codon) in-frame downstream of the HIF3A . (D) Hypoxia-related stabilization of the HIF-3α ODD reporter. HUVECs following the transfection with the HIF-3α-ODD reporter vector were exposed to hypoxia for indicated time points and luciferase activity measured with Dual-Luciferase Reporter Assay System. The data were normalized to the control vector (without HIF-3α-ODD) and expressed as a fold change over the normoxic signal. R.L.U. - relative light units, Data represent the mean ± SD of three independent experiments (6 replicates each). * P < 0.05 was considered significant. (E) PHD inhibition-related stabilization of HIF-3α ODD reporter. HUVECs following the transfection with the HIF-3α-ODD reporter vector were treated with 1 mM DMOG for 16 hours and luciferase activity was measured with Dual-Luciferase Reporter Assay System. The data were normalized to control vector (without HIF-3α-ODD) and expressed as a fold change over the normoxic signal. R.L.U. - relative light units, Data represent the mean ± SD of two independent experiments (4 replicates each). * P < 0.05 was considered significant. The raw data are presented in Supplementary data (XLSX).

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Cellworks (Caltag Medsystems Ltd, UK) and cultured in EGM-2 Bulletkit Medium (Lonza).

Techniques: Expressing, Luciferase, Cloning, Plasmid Preparation, Transfection, Activity Assay, Reporter Assay, Control, Inhibition

Journal: EXCLI Journal

Article Title: Hypoxia-inducible factor (HIF)-3a2 serves as an endothelial cell fate executor during chronic hypoxia

doi: 10.17179/excli2021-4622

Figure Lengend Snippet: HIF-3α contributes to hypoxia-related elevated caspase 3 levels in HUVECs. (A) The silencing efficiency of HIF3A in hypoxia exposed HUVECs. Cells were transfected with siRNA against the HIF3A or control siRNA and exposed to hypoxia for the time periods specified and total RNA lysates were collected. HIF3A mRNA levels were quantified by qRT-PCR and normalized to TBP and 18S rRNA levels and expressed as a percentage of control siRNA transfected samples. Data represent the mean ± SD of two independent experiments (3 replicates each). * P < 0.05 was considered significant. (B) HIF3A knockdown does not affect hypoxia-induced expression of GLUT1 and (C) VEGFA , but does reduce (D) DDIT3 mRNA levels in hypoxia exposed HUVECs. (E) The caspase 3/7 activity was monitored by luminescence and expressed in Relative Light Units (R.L.U.). HUVECs with HIF3A knockdown or transfected with control siRNA for each time point of exposure to hypoxia were seeded in six technical replicates, and the experiments were repeated three times. Error bars represent standard deviations, *P < 0.05 was considered significant. The raw data are presented in Supplementary data (XLSX).

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Cellworks (Caltag Medsystems Ltd, UK) and cultured in EGM-2 Bulletkit Medium (Lonza).

Techniques: Transfection, Control, Quantitative RT-PCR, Knockdown, Expressing, Activity Assay