primary human aortic ecs (haecs)  (ScienCell)

Journal: Redox Biology

Article Title: Big data analytics for MerTK genomics reveals its double-edged sword functions in human diseases

doi: 10.1016/j.redox.2024.103061

Figure Lengend Snippet: Proteomics in HAECs with MerTK gene knockout or control. ( A ) MerTK expression in HAECs incubated with apoptotic Jurkat cells for 1 h. ( B ) Immunochemical staining for MerTK expression in the aortic arch from WT mice. ( C ) Efferocytosis of apoptotic Jurkat cells by HAECs after 1 h of co-incubation. P < Apoptotic Jurkat cells were labeled with green PKH67 (Sigma) and HAECs were labeled with red PKH26 (Sigma). Green cells are apoptotic Jurkat cells that were not engulfed by HAECs. Green/red small round cells are apoptotic Jurkat cells that were engulfed by HAECs. Large red cells are HAECs. (D) Volcano plot illustration in MerTK KO vs. control. Relative protein abundance (log2) plotted against significance level (-log10 P-value), showing significantly (p < 0.05) downregulated (blue), upregulated (red) or non-differentially expressed proteins (grey). (E) Graphic summarization for pathways in MerTK KO vs. control. (F) MerTK KO activates apoptosis signaling. (G) Canonical pathway analysis in MerTK KO vs. control. Color depends on z-score. Blue signifies negative value; orange signifies positive value; and grey signifies no activity pattern. Size is proportional to the number of genes that overlap the pathway. (H) Machine learning analysis for activated or inhibited disease pathways. (I–K) IPA prediction shows that MerTK KO activates premature aging, kidney failure and heart failure. Proteomics data were analyzed by IPA. Data were analyzed with GraphPad Prism 9.4.1 and shown as the mean ± SD (n = 3–5). P < 0.05 was considered statistically significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary human aortic ECs (HAECs) and the human Jurkat cell line were purchased from ATCC (Manassas, VA, USA).

Techniques: Gene Knockout, Control, Expressing, Incubation, Staining, Labeling, Quantitative Proteomics, Activity Assay

Journal: bioRxiv

Article Title: PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1

doi: 10.1101/2021.05.26.445830

Figure Lengend Snippet: A-B) PGC1α protein expression (A) or mRNA expression (B) from HUVECs were measured by either western blots or RT-qPCR after cells were subjected to either static or laminar flow shear stress for 48 hours. C) PGC1α mRNA expression measured by RT-qPCR from HUVECs after exposure of 48 hours of laminar or oscillatory FSS. D) Bright-field image of Mouse lung endothelial cells (MLECs) isolated from either wild-type (WT) or PGC1α ECKO mice after exposure to laminar FSS. E) MLECs were isolated from WT and PGC1α-ECTG mice, and RT-qPCR were performed for different genes related to endothelial function after exposure to laminar FSS. F) HAECs were either treated with scrambled or PGC1α siRNA, and RT-qPCR was performed for different genes related to endothelial function after exposure to laminar FSS for 48 hours. G-H) Aortae were isolated from either WT (G) or PGC1α-ECTG (H) mice and mRNA was isolated, and RT-qPCR was done for the arch and thoracic region for disturbed and laminar flow. (I) Sample sites of disturbed (oscillatory) vs. laminar fluid shear stress in mouse aorta. (J) En face staining with β-Catenin and DAPI in WT and PGC1α -ECKO aorta. Scale bar, 20Lμm. (K) Composite data of length/width ratio of the endothelium in the thoracic region of mouse aortae. n □=□3 – 6 in each group. All the experiments were repeated 3 – 5 times. Statistically significant differences between groups are indicated (* P □<□0.05 by Student’s t-test ). The data are mean□±□SEM. Scale bar, 5□μm.

Article Snippet: Human Aortic ECs (HAECs) (#PCS-100-011) and Human Umbilical Vein ECs (HUVECs) (#PCS100010) cells were purchased from ATCC and cultured in EBMTM-2 Endothelial Cell Growth Basal Medium-2 containing bullet kit growth factor supplements (Lonza), 5% fetal bovine serum, 100 units/mL Penicillin, 100 μg/mL Streptomycin, and 2 mM L-glutamine (Invitrogen).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Shear, Isolation, Staining

Journal: bioRxiv

Article Title: PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1

doi: 10.1101/2021.05.26.445830

Figure Lengend Snippet: A-B) HAECs were either treated with scrambled or ERRα siRNA (A) or scrambled or KLF4 siRNA (B), and RT-qPCR was performed for different shear stress-related genes after exposure of cells with laminar FSS for 48 hours. C) HUVECs were lysed and immunoprecipitation (IP) was performed either with control IgG or ERRα antibody and immunoblotting was done with antibodies against PGC1α and KLF4. Lysates were examined by probing with GAPDH antibody. D-E) MLECs were isolated from control and PGC1α -ECTG mice, and either RT-qPCR (D) or immunoblot analysis (E) were performed with the probes and antibodies as indicated. F) Lysates prepared from HAECs treated with control shRNA or shRNA against PGC1α (48□hrs) were examined by immunoblot analysis using antibodies for TERT, PGC1α and GAPDH. G) MLECs were isolated from WT and PGC1α ECKO mice aortae and mRNA expression was measured by RT-qPCR for TERT gene before and after exercise. H) Aortae were isolated, and mRNA expression was measured for TERT by RT-qPCR in the arch and thoracic region of WT mice. I-J) ChIP-qPCR analysis of PGC1α recruitment to the TERT promoter region was performed in HUVECs. N □=□3 - 4 in each group. All the experiments were repeated 3 – 6 times. Statistically significant differences between groups are indicated (* P □<□0.05 by Student’s t-test ). The data are mean□±□SEM.

Article Snippet: Human Aortic ECs (HAECs) (#PCS-100-011) and Human Umbilical Vein ECs (HUVECs) (#PCS100010) cells were purchased from ATCC and cultured in EBMTM-2 Endothelial Cell Growth Basal Medium-2 containing bullet kit growth factor supplements (Lonza), 5% fetal bovine serum, 100 units/mL Penicillin, 100 μg/mL Streptomycin, and 2 mM L-glutamine (Invitrogen).

Techniques: Quantitative RT-PCR, Shear, Immunoprecipitation, Control, Western Blot, Isolation, shRNA, Expressing, ChIP-qPCR

Journal: bioRxiv

Article Title: PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1

doi: 10.1101/2021.05.26.445830

Figure Lengend Snippet: A) HMOX1 and KLF4 mRNA expression in HAECs after LSS. B) MLECs were isolated from control and PGC1α-ECTG mice and immunoblot analysis was performed with the antibodies as indicated. C) Lysates prepared from HAECs after treatment with either control or ERR siRNA (48□hrs) were examined by immunoblot analysis using HMOX1 and GAPDH antibodies. D) MLECs were isolated from WT, and PGC1α ECKO mice aortae and mRNA expression was measured by RT-qPCR for the HMOX1 gene before and after exercise. E) HUVECs were exposed with laminar or oscillatory FSS with or without TERT inhibitor and immunoblot analysis was performed with the antibodies as indicated. F) HAECs were either treated with scrambled or HMOX1 siRNA and RT-qPCR was performed for different genes related to endothelial function after the exposure of laminar FSS for 48 hours. G) Bright-field image of HAECs after exposure of laminar FSS in the presence of either control (CuPP 1mM) or two different HMOX1 inhibitors (ZnPP 1mM and OB 24 hydrochloride 1mM). H) Cell and mitochondrial morphology (MitoRed fluorescence) were imaged in the presence of Laminar flow with and without HMOX1 inhibition. I) D) Schematic diagram of LSS-induced PGC1α-TERT-HMOX1 pathway. Scale bar, 5□μm. n □=□3 - 6 in each group. All the experiments were repeated 3 – 5 times. Statistically significant differences between groups are indicated (* P □<□0.05 by Student’s t-test ). The data are mean□±□SEM.

Article Snippet: Human Aortic ECs (HAECs) (#PCS-100-011) and Human Umbilical Vein ECs (HUVECs) (#PCS100010) cells were purchased from ATCC and cultured in EBMTM-2 Endothelial Cell Growth Basal Medium-2 containing bullet kit growth factor supplements (Lonza), 5% fetal bovine serum, 100 units/mL Penicillin, 100 μg/mL Streptomycin, and 2 mM L-glutamine (Invitrogen).

Techniques: Expressing, Isolation, Control, Western Blot, Quantitative RT-PCR, Fluorescence, Inhibition