Rh123

To demonstrate the multidrug resistance protein 1 (MDR1) transporter activity of single or multi-lumen cysts, polyethylene glycol (PEG) and Matrigel hydrogels were treated with 50 μM rhodamine 123 (Rh123, Sigma) in serum free medium for 1 h at 37°C. After a washing step (3× in serum free medium) the hydrogels were incubated for 2 h at 37°C, 5% CO₂ in fresh complete medium. To inhibit the MDR1 transporter activity the cysts containing hydrogels were incubated with 50 μM verapamil (Sigma-Aldrich) at 37°C for 30 min before adding Rh123. The rhodamine assay was then repeated as described above. For the quantification of the Rh123 assay the number of cysts containing Rh 123 in the luminal space and the number of cysts showing Rh 123 signal only at the level of the epithelium (blocked cysts) were counted with ImageJ.

H9c2 cells were planted in a 12-well plate to be incubated for 24 hours and the 2 μmol/L Rh123 (#R8004, Merck & Co Inc, USA) was then added into each well to be incubated for 1.5 hours in the dark, followed by 3 washes with PBS buffer. Lastly, the inverted microscope (Bato Instrument, Shanghai, China) was used to take the images and the Image-Pro software was used to analyze the images [19 (link)].

HgTx1 N-terminally labeled with Atto488 (A-HgTx) was produced by chemical synthesis by Smartox Biotechnology (France), purity 98%. Concentration of A-HgTx was measured using molar extinction coefficient 90,000 M⁻¹cm⁻¹ at 500 nm. Oligonucleotide primers listed in Table 2 were synthesized by Evrogen (Russia). LTG, ERTG, TR488, and NCer were purchased from ThermoFisher Scientific (Waltham, MA, USA). Rh123, tetraethylammonium chloride (TEA) and bovine serum albumin (BSA) were from Merck (Darmstadt, Germany). GenJector-U Transfection Reagent was from Molecta (Moscow, Russia).

Rh123, TRQ, Dulbecco's Modified Eagle's Medium (DMEM, high glucose), and fetal bovine serum were purchased from Sigma‐Aldrich. HEPES was obtained from Roth.

Ultrapure water was obtained using the Milli-Q water system (Millipore, Bedford, MA, USA). Analytical grade acetone and ethyl acetate were obtained from Merck (Darmstadt, Germany) and alcohol was obtained from Panreac AppliChem (Barcelona, Spain). HPLC-grade solvents were obtained from J.T. Baker. Dulbecco’s modified Eagle’s medium (DMEM), antibiotic-antimycotic solution and foetal bovine serum (FBS) were obtained from Gibco by Life Technologies Inc. MTT was purchased from Molecular Probes (Eugene, USA). Dimethyl sulfoxide (DMSO) was obtained from Synth (Labsynth, São Paulo, Brazil). Aβ_25–35, the genes A2M, ACHE, ADAM10, APOE, APP, GSK3β, LRP1, MAPT, PSEN1, PSEN2, HPRT1 and GAPDH, β-CD, M-β-CD, HP-β-CD, DPPP, H₂DCFDA and Rh123 were obtained from Sigma-Aldrich (St. Louis, USA). Analytical grade standards epicatechin, caffeine, catechin, ellagic acid, gallic acid (Sigma-Aldrich), pyrogallol (Fluka Analytical), gallocatechin (MP Biomedicals), epiafzelechin-(4β→8)-epicatechin, epigallocatechin, procyanidin B1 (PB1), procyanidin B2 (PB2), epigallocatechin-3-O-gallate, samarangenin A and samarangenin B, isolated by our research group [24 (link),29 ,31 ], were used for peak identification. All other chemicals used were of the highest commercially available grade.