Mission short hairpin rnas shrnas

Mission short-hairpin RNAs (shRNAs) targeting human FABP5 and shRNA control vector targeting firefly luciferase were purchased from Sigma. Lentiviral shRNA transfer vectors and four expression vectors encoding viral packaging proteins (provided by Dr. Richard Mulligan, Boston Children’s Hospital) were co-transfected into HEK293 cells, as described previously [9 (link), 10 (link)]. Supernatants of HEK293T cells were collected and used to transduce HUVECs. Puromycin (2 ng/ml; Sigma) was added to the medium for 24 h for enrichment of transduced cells the day after transduction. Cells transduced with the control shRNA reached 70–80 % confluence 48–72 h after transduction.

Antibodies and fluorescent molecules were obtained from the following suppliers: TubAc, Sigma-Aldrich (T7451, St. Louis, MO); Alexa-conjugated secondary antibodies, Life Technologies; ARL13B, Abcam (ab136648, Cambridge, UK); BODIPY-CYA, BioVision (Milpitas, CA); N3-BODIPY FL, Tocris; b-actin, Abcam (ab8227); DAPI, Thermo Fisher Scientific; GFP, Abcam (ab290); GM130, BD Biosciences (610822, San Jose, CA); Hoechst 3342, Life Technologies; HRP-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories (West Grove, PA) and Cell Signaling (Grand Island, NY); HSD11β2, Abcam (ab37800 and ab115696). Small molecules were obtained from the following suppliers: 20(S)-yne Tocris Biosciences (Bristol, UK); CNX, Torcris Biosciences; CYA, Tocris Biosciences; vismodegib, Genentech (South San Francisco, CA); SAG, Merck Millipore (Billercia, MA); SAG1.5, Cellagen Technology (San Diego, CA); SHH, R&D Systems (Minneapolis, MN).
Sterols were obtained from Avanti Polar Lipids (Alabaster, AL). Mission short hairpin RNAs (shRNAs), which are described in Table S1, were obtained from Sigma-Aldrich. SHH N terminus (SHHN) was produced from HEK293-SHHN stable cells as described previously and diluted 10-fold for cell treatments (Myers et al., 2013).