Escin

Legs or imaginal discs were fixed in 4% formaldehyde (PFA) PBS solution for 20 min followed by rinsing in PBS with 0.2% Triton X-100 (PBT) three times. To label muscle, legs were incubated in 1:50 phalloidin in a PBS solution with the following reagents to improve tissue penetrance: 1% triton X-100, 0.5% DMSO, 0.05 mg/ml Escin (Sigma-Aldrich, E1378), and 3% normal goat serum. Legs were allowed to incubate for one week at 4 °C with occasional rocking. After staining, legs were rinsed 3 x with PBS-Tx, 1 x with PBS, and mounted onto slides in Vectashield with or without DAPI.
Each slide was labeled according to experimental condition. Prior to analysis, we taped-over the labels. Categorizations in Table 1 were performed with the experimenter blinded to experimental condition.

Nitrotetrazolium blue chloride (NBT), xanthine, xanthine oxidase, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), potassium persulfate, 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (ferrozine), iron (II) chloride tetrahydrate (FeCl₂ × 4H₂O, 1,3,5-Tri(2-pyridyl)-2,4,6-triazine (TPTZ), iron (III) chloride (FeCl₃), aluminum chloride (AlCl₃), potassium acetate, Folin−Ciocalteu reagent, sodium nitrite, sodium molybdate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, 2(3)-t-Butylhydroquinone monomethyl ether (BHA), 2(3)-t-Butyl-4-hydroxyanisole, hyaluronic acid (IV), escin, hyaluronidase from bovine testes, hexadecyltrimethylammonium bromide (CTAB), L-tyrosine, koji acid, and tyrosine from mushrooms were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The standards of eleutheroside B ≥98.0% (HPLC), eleutheroside E ≥98.0% (HPLC), eleutheroside E1 ≥98.0% (HPLC), protocatechuic acid ≥97%, p-hydroxybenzoic acid 99%, vanillic acid ≥97%, caffeic acid ≥98%, and ferulic acid ≥99% were also purchased from Sigma-Aldrich. Solvents used for extraction were purchased from Avantor Performance Materials (Gliwice, Poland).

Stocks of aminoglycoside antibiotics (All purchased from Sigma-Aldrich, St. Louis, MO, USA) were dissolved in sterile H₂O at a 100 mg/mL concentration, and stored at 4 °C. PTC124 (3-[5-(2-Fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid, MedChemExpress, Cat. #HY-14832), escin (Sigma-Aldrich, Cat. #E1378), VX-661 (MedChemExpress, Monmouth Junction, NJ, USA Cat. #HY-15448), NMDI-14 (Ethyl 2-(((6,7-dimethyl-3-oxo-1,2,3,4-tetrahydro-2-quinoxalinyl)acetyl)amino)-4,5-dimethyl-3-thiophenecarboxylate, EMD Millipore, Burlington, MA, USA, Cat. #530838), and amlexanox (MedChemExpress Cat. #HY-B0713) were dissolved in dimethylsulphoxide (DMSO). SMG1i (2-chloro-N,N-diethyl-5-((4-(2-(4-(3-methylureido)phenyl)pyridin-4-yl)pyrimidin-2-yl)amino)benzenesulfonamide) was received from Dr. Robert Bridges from Rosalind Franklin University of Medicine and Science through the CFFT compound distribution program, and was dissolved in DMSO. Working solutions were all ≤0.1% DMSO, with the exception of NMDI-14, which was 1% DMSO. The increased DMSO did not prevent G418-facilitated FIS (Supplementary Figure S9). Stocks of forskolin (Sigma-Aldrich, Cat. #F6886) were dissolved in 100% EtOH and stored at −20 °C.

The roots of Eurycoma longifolia (SK 3317/18) were harvested from Bentong, Pahang, Malaysia. The samples were then dried and shredded into chip form, about 1 cm in size. Human breast cancer cell line (MCF-7) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium. Dimethyl sulfoxide (DMSO, 99.9%), vanillin (≥97%), oleanolic acid (≥97%), and escin (≥95%) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Ethanol, acetic acid, perchloric acid, sulfuric acid, formic acid, ethyl acetate, butanol, chloroform, acetonitrile, and acetone were sourced from Merck, Darmstadt, Germany.

KCl, NaCl, HEPES, EDTA, nonactin, pentane, ethanol, methanol, calcein, triton X-100, sephadex G-50, digitonin, tribulosin, dioscin, diosgenin, solasodine, escin, uvaol, lupeol, betulin, nystatin (NyS), and gramicidin A (GrA) were purchased from Sigma-Aldrich (St. Louis, USA). Syringomycin E (SyrE) was isolated and purified as described previously [37 (link)], and kindly offered by Dr. J.Y. Takemoto (Utah State University, USA). The chemical structures of tested agents are presented in Figure 6. The purity of saponins and related compounds was ≥95% (except for betulin (≥98%), lupeol (≥94%), diosgenin (≥93%), and digitonin (~50%)). All experiments were performed at room temperature (25 °C).
Lipids, 1,2-diphytanoil-sn-glycero-3-phosphocholine (DPhPC), 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine (HOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DPPG), and cholesterol (CHOL) were obtained from Avanti Polar Lipids (Pelham, NY, USA).

Escin and MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant Wnt3a and secreted frizzled related proteins (sFRPs) were purchased from R&D systems (Minneapolis, MN, USA).

Sephadex G-200 (discontinued product) was originally obtained from Pharmacia Biotech (now GE Healthcare). The Microsource Spectrum Collection and small-molecule library containing 2560 compounds included in the US and International Drug Collections, together with Natural Product and Discover libraries, was purchased from Microsource Discovery Systems, Inc. DMSO and H₂O₂ (30%) were from Thermo Fisher Scientific. DTPA and all other chemicals were from Sigma–Aldrich and were from the highest available purity grade. Specialty chemicals (>95% purity guarantee by manufacturer) present in the Microsource library that were purchased in powder form included the following: gambogic acid (drug G) from Shanghai Tauto Biotech Co, closantel (drug C), hematoporphyrin (drug H), hexachlorophene (drug X), and docusate sodium (drug DS) from MCE (MedChemExpress), dihydrogambogic acid (drug D), chaulmoogric acid (drug M) and hematein (drug I) from MicroSource, Inc, bixin (drug B), sennoside A (drug S), tetradecyl sulfate sodium (drug T), agaric acid (drug A) and escin (drug E) from Sigma, avocadene (drug V) and all other drugs were taken directly from the Microsource microtiter plate library as 10 mM solutions in DMSO.