Rhodamine 123 r123

The compounds lucifer yellow (LY; Mw = 457.25 g mol⁻¹), rhodamine 123 (R123; Mw = 380.82 g mol⁻¹) and elacridar (GF; Mw = 563.65 g mol⁻¹) and other materials like bovine serum albumin and dimethyl sulfoxide, were purchased from Sigma-Aldrich (St. Quentin Fallavier, France).
All powdered compounds were dissolved in dimethyl sulfoxide or Krebs-Ringer HEPES (RH) buffer (NaCl 150 mM, KCl 5.2 mM, CaCl₂ 2.2 mM, MgCl₂ 0.2 mM, NaHCO₃ 6 mM, glucose 2.8 mM, HEPES 5 mM, sterile water for injection—pH: 7,4). The source and origin of all other materials used in this study are detailed throughout the methodology.

P-gp activity is assessed by the accumulation of the fluorescent substrates Rhodamine 123 (R123) (Sigma) and FLUO-3-AM (Thermo) [23 (link), 57 (link)]. The specific P-gp inhibitor cyclosporine A (CsA) is used as a control to monitor P-gp activity [23 (link)]. For other inhibitor experiments PSC-833 (Sigma), MK571 (Sigma), or Ko143 (Sigma) were used to inhibit P-gp, MRPs, and BCRP respectively. Uninfected BECs are used as a control for comparison. After infection, cells are washed with Hank’s Balanced Salt Solution (HBSS) (Thermo) and pre-incubated with or without inhibitor (10 μM CsA, 10 μM PSC-833, 10 μM MK571, or 1 μM Ko143) for 1 h at 37 °C + 5% CO₂. Following pre-incubation, cells were incubated with 10 μM R123 or FLUO-3-AM with or without inhibitors mentioned for 2 h at 37 °C + 5% CO₂. After incubation, cells are washed twice in PBS and 200 μl of RIPA buffer (Thermo) is added and placed on a shaker for 10 min at room temperature protected from light. Fluorescence was measured on a plate reader (Tecan). Next, a BCA protein assay (Thermo) was conducted on each well and fluorescence values are normalized to BCA to account for relative cell number as previously described [23 (link)].

The following curcumin formulations were used for experiments: A native curcuma extract (150 mmol/L in DMSO; Jupiter Ley, Okkal, India), liposomal curcumin (75 mmol/L in H₂Odd; Longvida^®; Verdure Sciences, Noblesville, IN, USA), a curcuma extract with turmeric oils (150 mmol/L in DMSO; BCM-95^®; Arjuna Natural Extracts, Kerala, India), a curcuma extract with adjuvants (75 mmol/L in DMSO; TISSO Naturprodukte GmbH, Wenden, Germany), submicron-particle curcumin (150 mmol/L in H₂Odd; Theracurmin CR-043P; Theravalues Corp., Tokyo, Japan), phytosomal curcumin (50 mmol/L in H₂Odd; Meriva^®; Indena S.p.A., Milan, Italy) a curcumin-γ-cyclodextrin complex (75 mmol/L in H₂Odd; Cavacurmin^®; Wacker Chemie, Munich, Germany) and curcumin incorporated into PS80 micelles consisting of 93% PS80 and 7% curcuminoid powder (30 mmol/L in H₂Odd; NovaSOL^® Curcumin; AQUANOVA AG, Darmstadt, Germany). For experiments, rhodamine 123 (R123; stock 1 mg/mL in ethanol), verapamil hydrochloride (stock 50 mmol/L in H₂Odd), elacridar (stock 2 mg/mL in DMSO) and PS80 were purchased from Sigma-Aldrich (Steinheim, Germany). Empty PS80 micelles were from AQUANOVA AG (Darmstadt, Germany). β-Glucuronidase type H-1 from Helix pomatia (EC 3.2.1.31) was purchased from Sigma-Aldrich (Taufkirchen, Germany). Methanol, ethylacetate, ethanol and acetonitrile were HPLC grade.

All chemicals used in this study were of analytical grade. Unless stated otherwise, they were purchased from Merck (Darmstadt, Germany) and Roth (Karlsruhe, Germany). Fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA), Pisum sativum agglutinin (FITC-PSA) and rhodamine 123 (R123) were obtained from Sigma-Aldrich (Steinheim, Germany), whereas propidium iodide (PI) was purchased from Thermo Fisher Scientific Inc. (Darmstadt, Germany).

5-HD with the purity of 99%, 4-aminopyridine (4-AP), and rhodamine-123 (R-123) were purchased from Sigma (USA). Polyclonal mouse anti-rat α-actin antibody (α-SM actin), polyclonal goat anti-rat Kv1.5 antibody, goat anti-rat MCP-1 antibody, and rabbit anti-rat TGF-β1 antibody were purchased from Santa Cruz (USA). Cell counting kit-8 (CCK) was purchased from Tongue Chemical Institutes (Japan).

As₂O₃, α-TOC, L-AA, Primers [Nrf2, Bcl2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)], Triton X 100, dichlorofluorescein diacetate, and Rhodamine-123 (R-123) were purchased from Sigma-Aldrich, Bangalore, India. RapiGest SF was purchased from Waters India Pvt Ltd, Bangalore, India. Fetal bovine serum (FBS), antibiotic–antimycotic solution, Roswell Park Memorial Institute-1640 medium, ethidium bromide, and all other chemicals were purchased from Himedia Pvt. Ltd (Mumbai, India). As₂O₃ (1 mM) and L-AA (10 mM) were prepared in aqueous solutions and kept as stock solutions. Stock solution of α-TOC (10 mM) was prepared in 0.2% ethanol. These solutions were diluted to working solutions of As₂O₃ (10 μM), L-AA (100 μM), and α-TOC (50 μM) on the experiment day before use.