Cell Culture
Human dermal microvascular endothelial cells (HDMECs) were purchased from Promocell (Heidelberg, Germany) and were cultured in endothelial cell basal media (EBM) MV2 growth media (C-22221; Promocell, Heidelberg, Germany), supplemented with 5% (v/v) fetal calf serum (FCS) and EGF (5 ng/ml), VEGF (0.5 ng/ml), FGF-2 (10 ng/ml), long R3 insulin growth factor-1 (20 ng/ml), hydrocortisone (0.2 μg/ml) and ascorbic acid (1 μg/ml) (supplement pack C-39221; Promocell, Heidelberg, Germany).
siRNA transfection
siRNA duplexes were obtained from Qiagen (Crawley, UK). HDMECs were transfected with siRNA duplexes at concentrations of 1-10 nM using 0.1% (v/v) Lipofectamine RNAiMAX (Invitrogen, Paisley, UK), according to the manufacturer's instructions. Transfection reactions were performed in serum-free OptiMEM (Invitrogen, Paisley, UK). Cell media was changed to serum containing media 4 hours after transfection.
RT-qPCR
Total RNA was extracted from HDMECs using the RNeasy mini kit (Qiagen, Crawley, UK), 24 and 72 hours post-transfection. DNase treatment was performed using the on-column DNase digestion (Qiagen, Crawley, UK). One μg total RNA was used for cDNA synthesis with M-MLV reverse transcriptase and either random hexamers or oligo(dT) (18T) primers. RT-qPCR was performed using Power SYBR Green Mastermix (Applied Biosystems, Warrington, UK). Primers were designed using the Invitrogen oligoperfect designer web tool, and were designed to give an amplicon of approximately 150 base pairs (figure 1A). Primer sequences were screened using a BLAST search to confirm specificity, and the PCR products run on an agarose gel to confirm that products of the expected size were detected. The efficiency of each primer set for RT-qPCR was determined to be between 95 and 100%. Alternatively pre-designed primers from Qiagen (Crawley, UK) were purchased. Reactions were analyzed upon an ABI 7000 real-time PCR machine using the following cycle conditions: 50°C for 10 minutes, 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Results were normalized against β-actin expression.
Immunoblotting
Protein lysates from HDMECs were prepared in LDS sample buffer containing 2.5% (v/v) β-mercaptoethanol. Proteins were resolved by SDS-PAGE on 4-12% NuPage Gels, and transferred onto nitrocellulose membranes (GE Healthcare, Bucks, UK). Membranes were blocked with 5% BSA. Antibodies to the C-terminus of PKCε and to actin were obtained from Millipore (Watford, UK) and Santa Cruz (Heidelberg, Germany) respectively. Membranes were washed 6 times with TBS-T, and incubated with peroxidase-conjugated secondary antibodies (GE Healthcare, Bucks, UK/Sigma, Dorset, UK). Blots were detected using an enhanced chemiluminescence (ECL) detection kit (GE Healthcare, Bucks, UK).