Passive lysis buffer (plb)

The effects of endoplasmic reticulum stress on puromycinylated protein levels were determined as previously described (Halliday et al., 2015 (link)). In brief, 10⁶ HEK293 cells were plated in 6-well plates. Two days later, culture media was changed to fresh media, and cells were treated with vehicle (DMSO) or thapsigargin in the presence or absence of the indicated concentration of inhibitors for 2.5 h. For puromycin labelling, 10 μg/ml puromycin was added during the last 10 min before harvest. Cells were lysed with passive lysis buffer (Sigma) supplemented with protease inhibitor cocktail (Roche). After centrifugation at 13 000 rpm for 20 min, supernatants were mixed with SDS-PAGE sample buffer. To detect puromycinylated protein 20 μg of total protein, respectively, was subjected to 12% SDS-PAGE and transferred onto PVDF membrane. Immunoblot detection was conducted using primary antibodies for puromycinylated protein (1:5000; Proteintech). Scanned images were quantified using ImageJ software.

To ascertain whether miR-646 inhibited VEGF-A expression, TargetScan was used, which is an online publicly available algorithm, to analyze miR-646 sequence 3'-UTR (NM_001025369) of VEGF-A. Sequence-combined index comprehensive ranking was performed to confirm that the combined prediction score for the miR-646 binding site in the 3'UTR was the highest scoring site. Once it was established that miR-646 could target VEGF-A, subsequent experiments were performed. EPCs were transfected with either wild type or mutant VEGF-A constructs without the miR-646 binding site. This part of the experiment allowed EPC cells to grow to 70-80% confluence in 24-well plates and Lipofectamine^TM 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used with a luciferase reporter vector (200 ng) (Promega Corporation), and appropriate miRNA (50 nM, mimics and inhibitors) were co-transfected. Following the experimental protocol, after 72 h, the cells were washed and lysed with passive lysis buffer (Sigma-Aldrich; Merck KGaA). The relative activity of luciferase was then assessed using a dual luciferase vector assay (Thermo Fisher Scientific, Inc.). Finally, relative reporter gene activity was determined by normalizing to Renilla luciferase activity.