human carotid artery ecs (hcaecs)  (Lonza)

Journal: Angiogenesis

Article Title: Oxidized phospholipids stimulate production of stem cell factor via NRF2-dependent mechanisms

doi: 10.1007/s10456-017-9590-5

Figure Lengend Snippet: SCF mRNA is induced in endothelial and monocytic cells by OxPLs and PGA2 in a time- and concentration-dependent manner a Endothelial cells were stimulated with OxPAPC (100 µg/ml, 6 h). Total RNA was extracted using Trizol reagent and analyzed by microarray hybridization (HCAEC; three independent experiments and hybridizations) or qRT-PCR (HAEC, HUVEC; quadruplicate samples from one representative experiment out of three). Levels of SCF mRNA were normalized to β2-microglobulin mRNA. b HUVECs were stimulated with OxPAPC (100 µg/ml) for indicated time periods, followed by analysis of SCF mRNA. c HUVECs were treated with indicated concentrations of OxPAPC for 6 h. qRT-PCR was used for SCF mRNA quantification. d , e Levels of SCF mRNA were analyzed in aortas of aged (12 months old) wild type and ApoE − / − mice ( d ) or in aortas of wild type and ApoE − / − young animals fed for 8 weeks either with chow (6.5% fat) or high-fat diet (15% fat) ( e ). Total RNA was prepared from homogenized aortas using Trizol reagent. SCF mRNA expression was analyzed by qRT-PCR and normalized to β2-microglobulin mRNA levels

Article Snippet: Human umbilical vein ECs (HUVECs), human carotid artery ECs (HCAECs) and human aortic ECS (HAECs) (all from Lonza) were grown at 37 °C in 5% CO 2 in medium M199 containing 20% FCS, 1 U/ml heparin, ECGS growth supplement (Promocell), 2 mmol/l glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and used up to passage 5.

Techniques: Concentration Assay, Microarray, Hybridization, Quantitative RT-PCR, Expressing

Journal: Angiogenesis

Article Title: Oxidized phospholipids stimulate production of stem cell factor via NRF2-dependent mechanisms

doi: 10.1007/s10456-017-9590-5

Figure Lengend Snippet: OxPAPC induces expression of cell-associated and secreted SCF protein and stimulates c-Kit phosphorylation. a Two forms of SCF mRNA were quantified in OxPAPC-stimulated HUVECs (100 µg/ml, 6 h) by qRT-PCR using primers selective for individual splice variants. b SCF protein was analyzed by ELISA in extracts of HUVECs or in conditioned media after 12 h of stimulation with 100 µg/ml OxPAPC. c Conditioned media produced by cells stimulated with indicated concentrations of OxPAPC stimulate phosphorylation of c-Kit receptor in HUVECs. d Panel presents ELISA data on the levels of SCF protein in conditioned media. e Pretreatment of cells with c-Kit tyrosine kinase inhibitor imatinib (30 min, 20 µM) attenuated c-Kit phosphorylation in HUVECs incubated with conditioned media from OxPAPC-stimulated cells

Article Snippet: Human umbilical vein ECs (HUVECs), human carotid artery ECs (HCAECs) and human aortic ECS (HAECs) (all from Lonza) were grown at 37 °C in 5% CO 2 in medium M199 containing 20% FCS, 1 U/ml heparin, ECGS growth supplement (Promocell), 2 mmol/l glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and used up to passage 5.

Techniques: Expressing, Phospho-proteomics, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Produced, Incubation

Journal: Angiogenesis

Article Title: Oxidized phospholipids stimulate production of stem cell factor via NRF2-dependent mechanisms

doi: 10.1007/s10456-017-9590-5

Figure Lengend Snippet: Induction of SCF by OxPAPC depends on the transcription factor NRF2. a , b , c and d HUVECs were transfected with siRNAs targeting ATF4 ( a ), PERK ( b ), NRF2 ( c ), or KEAP ( d ). Twenty-four hours after transfection cells were stimulated with OxPAPC (100 µg/ml, 6 h). Levels of SCF mRNA were analyzed by qRT-PCR in total RNA prepared using Trizol reagent and normalized to β2-microglobulin mRNA. e Protein kinase CK2 inhibitor TBB attenuates induction of SCF by OxPAPC. Cells were pretreated with TBB (20 µM, 30 min) and thereafter stimulated with OxPAPC (100 µg/ml, 6 h); SCF mRNA was quantified as described above. f miR-155 potentiates induction of SCF by OxPAPC. HUVECs were transfected with the RNA oligonucleotide mimicking miR-155 for 24 h and stimulated by OxPAPC (100 µg/ml, 6 h). Levels of SCF mRNA were quantified by qRT-PCR. g Steady-state levels of SCF mRNA are decreased in aortas of NRF2 − / − mice. Total RNA was prepared from aortas of 6 months old NRF2 − / − or wild-type mice and analyzed by qRT-PCR method. The levels of NRF2 mRNA were normalized to β2-microglobulin mRNA

Article Snippet: Human umbilical vein ECs (HUVECs), human carotid artery ECs (HCAECs) and human aortic ECS (HAECs) (all from Lonza) were grown at 37 °C in 5% CO 2 in medium M199 containing 20% FCS, 1 U/ml heparin, ECGS growth supplement (Promocell), 2 mmol/l glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and used up to passage 5.

Techniques: Transfection, Quantitative RT-PCR