Petrolatum

Rosa multiflora (stem), Lespedeza bicolor (aerial part), Platycladus orientalis (leaves), Castanea crenata (leaves) and Cornus officinalis (fruit) were purchased from the Gyeongbuk Forest Resource Development Institute, the Republic of Korea. The identity of the plants was confirmed by a taxonomist (Dr. Zi-Eum Im) and voucher specimens were deposited (LVPPM 2001–2005) in our laboratory. The dried and crushed parts of each plant were boiled in 30% ethanol (100 g/Liter). The extracts were filtered with Whatman filter paper Number 1 (GE Healthcare, UK Limited, UK), evaporated to dryness, and freeze-dried. A 5% (w/w) and 10% (w/w) ointments of C2RLP were made using petrolatum (Sigma-Aldrich) as a vehicle. For in vitro experiments on various cell lines, the extract was dissolved in the respective media used to grow the cells and then filtered using a 0.22-μm syringe driven filter (Merck Millipore Ltd., Carrigtwohil, Ireland).

Reagents utilized include: Silicone oil (Fisher S159-500), Borosilicate glass (Harvard Apparatus, type GC150T-10, 30-0062), Petrolatum (Sigma-Aldrich, 16415), Puromycin (BioShop, PUR333.100), Geneticin (Gibco, #11811023). Plasmids used for transfections: px459V2 (Addgene # 62988), pLVX-dTomato-C1 (Takara Bio), pEGFP-puro (Addgene #45561), or pBS/KSTau/LacZ (courtesy of Dr. Friedhelm Bladt, SLRI, Mt. Sinai Hospital, Toronto), modified from TauLacZ LTNL (Dr. Peter Mombaerts, Max Planck Institute, Frankfurt). Plasmids were prepared from bacterial stocks grown overnight using EndoFree Plasmid Maxi Kit (Qiagen, #12362), with plasmid integrity verified using gel electrophoresis and DNA transfection quality independently determined using electroporation and polyethylenimine (Sigma Aldrich, 25 kDa MW, # 408727) mediated transfection of HEK 293T cells (ATCC CRL-3216) according to standard methods together with culture lines and cell media constituents as indicated below. Analyses for beta-galactosidase activity in transfected cells were performed as previously described^{48 (link)}. Cell viability was assessed using standard trypan blue exclusion assay (Gibco 15250061)^{49 (link)}, DAPI (Sigma D9542), propidium iodide (Sigma P4170)^{50 (link)} or Calcein-AM/ethidium homodimer live/dead assay⁵¹ (Molecular Probes L-3224) methods as described previously.

BIT (CAS no. 2634-33-35, purity 97.8%; Figure 1), dimethyl sulfoxide, formic acid, lanolin (CAS no. 8006-54-0), petrolatum (CAS no. 8009-03-8), stearic acid (CAS no. 57-11-4, purity 98.5%), propylparaben (CAS no. 94-13-3), methylparaben (CAS no. 99-76-3), disodium edetate (CAS no. 6381-92-6), propylene glycol (CAS no. 57-55-6), triethanolamine (CAS no. 102-71-6, purity 99.0%), and phenacetin (CAS no. 62-44-2, IS, purity ≥ 98%; Figure 1) were purchased from Sigma-Aldrich (St. Louis, MO, USA), while homosalate (CAS no. 118-56-9) was purchased from the Tokyo Chemical Industry (Tokyo, Japan). HPLC-grade methanol, ethyl alcohol, and ethyl acetate were obtained from Burdick and Jackson Co. (Morristown, NJ, USA). Deionized water was obtained using a Milli-Q Plus Ultrapure Water System (Millipore, Bedford, MA, USA).