Endothelial cell growth kit

HEK-293T, B16-F10, A549, H460, and HUVEC cells were originally purchased from ATCC; HUASMC were purchased from PromoCell (C-12500); H82 cells were kindly provided by Julien Sage (Stanford School of Medicine); KP (238N1) and KPT (2985T2) lung adenocarcinoma cells were generated previously from tumors in Kras^LSL-G12D; p53^f/f and Kras^LSL-G12D; p53^f/f; R26^LSL-Tom mice. HEK-293T, 238N1, 2985T2 and B16-F10 cells were cultured in DMEM containing 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. A549, H460 and H82 cells were cultured in RPMI1640 media containing 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. HUVECs were cultured in Vascular Cell Basal Medium (ATCC, PCS-100–030) with Endothelial Cell Growth Kit (ATCC, PCS-100–041); HUASMC were cultured in Smooth Muscle Cell Growth Medium 2 (PromoCell, C-22062). All cell lines were confirmed to be mycoplasma negative (MycoAlert Detection Kit, Lonza).
Pitstop (ab120687) and Dyngo 4a (ab120689) were purchased from Abcam.
All plasmids used in this study are listed in Supplementary file 1 and key plasmids for multiple G-baToN systems will be available on Addgene.

Primary human aortic endothelial cells (HAEC) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Vascular Cell Basal Complete Medium (ATCC, Manassas, VA, USA) containing 2% of heat-inactivated fetal bovine serum (FBS, ATCC, Manassas, VA, USA), 0.5% of streptomycin-penicillin (ATTC, Manassas, VA, USA), 0.5% of Gentamicin/Amphoterin-B (ATCC, Manassas, VA, USA), 0.5% of Phenol Red (ATCC, Manassas, VA, USA) and an Endothelial Cell Growth Kit (ATCC, Manassas, VA, USA) was used for cell maintenance. Medium was replaced every 2–3 days, and cells were trypsinized before reaching confluence with Trypsin-EDTA (0.05%) Phenol Red (Gibco, Gaithersburg, MD, USA).

Human umbilical vein/vascular endothelial (HUVEC, ATCCPCS-100-013) was purchased from ATCC and cultured in with vascular cell basal medium (ATCC, PCS-100-030) supplemented with endothelial Cell Growth Kit (ATCC, PCS-10-040).

Primary HUVECs (ATCC, United States) were cultured in Vascular Cell Basal Medium supplemented with Endothelial Cell Growth Kit (ATCC, United States) at 37°C in a humidified atmosphere of 5% CO₂. HUVECs from 2 to 8 passages were used for the indicated experiments. For the indicated transfections, HUVECs were seeded in 6-well plates and cultured for 24 h to reach 50–60% confluency. Then HUVECs were transfected with 40 nM agomir-505, 40 nM antagomir-505, 40 nM agomir-NC or 40 nM antagomir-NC using Lipofectamine RNAi MAX (Invitrogen, United States) following the manufacturer’s instructions. For LPS or TNF-α treatment, LPS (10 ng/mL) or TNF-α (2, 10 or 20 ng/mL) was added 24 h after the indicated transfections. THP-1 cells (ATCC, United States) were cultured in RPMI-1640 medium (Gibco, United States) supplemented with 10% fetal bovine serum (FBS) (Gibco, United States). For TNF-α stimulation, THP-1 cells were plated in 6-well plates and incubated with 10 ng/mL TNF-α for 24 h. Peripheral blood mononuclear cells (PBMCs) were isolated from C57/BL6 mice using Ficoll-Paque PREMIUM reagent (GE Healthcare, United States). PBMCs were cultured in RPMI-1640 medium supplemented with 10% FBS, followed by 10 ng/mL TNF-α stimulation for 24 h or 5 ng/mL LPS stimulation for 6 h.

Human umbilical vein endothelial cells (HUVEC; CRL-4053) were purchased from American Type Culture Collection (ATCC) and cultured in Vascular Cell Basal Medium (ATCC PCS-100-030) supplemented with Endothelial Cell Growth Kit (ATCC PCS-110-041), penicillin (100 U/mL), and streptomycin (100 U/mL). Cells were maintained at 37 °C and 5% CO₂ in a humidified incubator. To evaluate effects of C2CD4B, cells were cultured for 1 h with the recombinant protein (100 ng/mL). To characterize intracellular signaling, HUVECs were pretreated with the following pharmacological inhibitors: phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor (wortmannin, 10 μM, 1 h); the AKT inhibitor X (10 μM, 1 h); and Go6976 (500 nM, 30 min).

Human HCC cell lines SNU-398 and SK-HEP-1, and Human umbilical vein endothelial cell (HUVEC) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Human HCC cell line Huh7 was purchased from National Collection of Authenticated Cell Cultures of Chinese Academy of Sciences (Shanghai, China). SNU-398 was maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA USA) added with 10% fetal bovine serum (FBS, Invitrogen). SK-HEP-1 was maintained in Eagle’s Minimum Essential Medium (Invitrogen) added with 10% FBS. HUVEC was maintained in Vascular Cell Basal Media (ATCC) supplemented with Endothelial Cell Growth Kit (ATCC). Huh7 was maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) added with 10% FBS. All cells were cultured at 37 °C containing 5% CO₂ under normoxia. Under hypoxia, cells were cultured at 37 °C containing 1% O₂, 94% N₂, and 5% CO₂. The cells were authenticated using STR profiles. All cells were routinely tested as mycoplasma-free.

Primary human aortic ECs (HAECs) and the human Jurkat cell line were purchased from ATCC (Manassas, VA, USA). HAECs were cultured with Vascular Cell Basal Medium (ATCC, PCS-100-030) and Endothelial Cell Growth Kit (ATCC, PCS-100-041). Jurkat cells were cultured in RPMI-1640 medium (ATCC, 30-2001) with 10% Fetal Bovine Serum (FBS, ATCC 30-2020).