Lullaby ll70500

EC from human aorta (TeloHAEC, CRL-4052, ATCC, Manassas, VA, USA) and primary human umbilical vein (HUVEC, PCS-100-010, ATCC, Manassas, VA, USA) were grown as previously reported [50 (link)]. To mimic septic inflammatory condition, EC were treated up to 24 h with increasing concentrations (0.5–10 µg/mL) of lipopolysaccharides (LPS, L5543, Sigma-Aldrich, St. Louis, MO, USA). To selectively inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9), EC were incubated with 100 µg/mL of evolocumab (i-PCSK9, Repatha, Amgen Europe B.V) along with LPS 10 µg/mL for 24 h. TeloHAEC were transfected with 2 µg antagomiR hsa-miR-15b-5p (i-miR-15b, customized by Aurogene, Rome, Italy) or with corresponding antagomir Negative Control (NC, Aurogene, Rome, Italy) using the vehicle Lullaby (LL70500, OZ Biosciences, Marseille, France) in medium without serum and antibiotic. After 6 h, fetal bovine serum was added to the culture medium and incubated for further 12 h before treatments with LPS and/or i-PCSK9. The miR-15b-5p inhibition efficiency after transfection was assessed by quantitative real-time PCR (qRT–PCR). Control cells (Ctr) were grown as previously reported [11 (link)].

Human colon epithelial CCD 841 CoN (CRL-1790, ATCC, Manassas, VA, USA) and colorectal adenocarcinoma SW480 (CCL-228, ATCC, Manassas, VA, USA) and SW620 (CCL-227, ATCC, Manassas, VA, USA) cells were grown as previously reported [39 (link)]. Cells were transfected for 6 h in serum- and antibiotic-free medium with 40 nM miRNA mimic Negative Control (miR-NC, MCH00000), hsa-miR-148a miRNA mimic (miR-148a⁺, MCH01336), hsa-miR-148a miRNA agomir (a-miR-148a, MAH01336), or miRNA agomir Negative Control (a-miR-NC, MAH00000), all from Applied Biological Materials, Inc., Richmond, BC, Canada, before the addition of fetal bovine serum (FBS). Lullaby (LL70500, OZ Biosciences, Marseille, France) was used for transfection following the manufacturer’s instructions. The cells were incubated up to 72 h after transfection.