Has 2

Total RNA was isolated from lesional skin tissue using TRIzol reagent (Life Technologies) and purified with an RNeasy Mini kit (Qiagen). Quantitative RT-PCR was performed using validated gene expression assays for ADORA1, ADORA2A, ADORA2B, ADORA3, COL1A1, interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), HAS-2, and FN (Sigma-Aldrich). Cyclophilin A (PPIA) and β-actin were used as endogenous controls to normalize transcript levels of total RNA of each sample.
The following primers were used for this study: ADORA1 forward 5′-TGTGCCCGGAAATGTACTGG-3′, reverse 5′-TCTGTGGCCCAATGTTGATAAG-3′; ADORA2A forward 5′-TTCCACTCCGGTACAATGGC-3′, reverse 5′-CGATGGCGAATGACAGCAC-3′; ADORA2B forward 5′-GCGTCCCGCTCAGGTATAAAG-3′, reverse 5′-CGGAGTCAATCCAATGCCAAAG-3′; ADORA3 forward 5′-ACGGACTGGCTGAACATCAC-3′, reverse 5′-AGACAATGAAATAGACGGTGGTG-3′; COL1A1 forward 5′-GCTCCTCTTAGGGGCCACT-3′, reverse 5′-CCACG TCTCACCATTGGGG-3′; IL-6 forward 5′-ACCGCTATGAAGTTCCTCTC-3′, reverse 5′-CTCCGACTTGTGAAGTGGTA-3′; MCP-1 forward 5′-AGCATCCACGTGTTGGCTC-3′, reverse 5′-TGGGATCATCTTGCTGGTG-3′; HAS-2 forward 5′-TGTGAGAGGTTTCTATGTGTCCT-3′, reverse 5′-ACCGTACAGTCCAAATGAGAAGT-3′; PPIA forward 5′-GAGCTGTTTGCAGACAAAGTTC-3′, reverse 5′-CCCTGGCACATGAATCCTGG-3′; β-actin forward 5′-GGCTGTATTCCCCTCCATCG-3′, reverse 5′-CCAGTTGGTAACAATGCCATGT-3′. Data were analyzed with GraphPad Prism software using the 2^–ΔΔCt method.

Human umbilical vein endothelial cells (HUVEC) were grown and maintained in basal media supplemented with VascuLife EnGS LifeFactors Kit (Lifeline Cell Technology, Oceanside, CA). Cells were plated on 0.2% (w/v) gelatin-coated cell culture plates (Thermo Fisher Scientific, Waltham, MA) and utilized within the first five passages. Antibodies for Western Blotting (WB) were purchased from the following sources and utilized at the designated dilutions: GAPDH (14C10, 1:10,000 dilution; Cell Signaling, Danvers, MA), LC3B (L7543, 1:1,000 dilution; Sigma), and HAS2 (sc-514737 and sc-365263 at 1:250 and 1:150, respectively; Santa Cruz Biotechnology, Dallas, TX). The following siRNA was purchased from Santa Cruz Biotechnology and used at the designated amounts: ATG5 siRNA (sc-41445, 100 pM) and scramble control siRNA-A (sc-37007, 100 pM).