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Supporting Information to: Induction of CYP1A by Green Tea Extract in Human Intestinal Cell Lines M. I. Netsch1,2 H. Gutmann1 C. B. Schmidlin2 C. Aydogan2 J. Drewe1 Affiliation 1
Department of Research and Clinical Pharmacology, University Hospital, Basel,
Switzerland 2
Frutarom Switzerland Ltd., R&D Department Phytopharmaceuticals, Waedenswil,
Switzerland Correspondence Juergen Drewe, MD, MSc Department of Clinical Pharmacology and Toxicology University Clinic Basel Petersgraben 4 4031 Basel Switzerland Phone: +41-61-265-3848 Fax: +41-61-265-8581 E-mail:
[email protected]
© Georg Thieme Verlag KG · DOI 10.1055/s-2006-931537 (10.1055/s-2006-931611) · Planta Med 2006; 72: e149–e152 · Netsch MI et al.
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Materials and Methods
Protein extraction Proteins were extracted by lysing a frozen ( 20 °C) cell pellet in 1 mL protein extraction buffer (pH 7.4), containing 20 mM Tris-HCl, 0.5 mM Na3VO4 and 1% (v/v) Igepal CA-630. Proteases were inhibited with 1 mM PMSF, 8 µM leupeptin, 5 µM bestatin, 2 µg/mL aprotinin, 6 µM E-64 and 1 µM pepstatin. The suspension was centrifuged at 15000 rpm for 10 min at 4 °C and supernatant was frozen at 70 °C. Protein quantification was performed with the BCA Protein Assay (Pierce, Rockford, Il, USA). Samples were transferred into a 96-well microtiter plate and UV-detection was performed with a Spectra Max 250 (Molecular Devices Corp., Sunnivale, CA, USA) at 562 nm and compared to a heat-shock inactivated bovine serum albumin (BSA) standard.
Western blot Samples of 150 µg protein from LS-180 cells or 100 µg protein from Caco-2 cells were diluted 1:1 with Laemmle dilution buffer and loaded on a 4% stacking and 7.5% separating acrylamide/bisacrylamide gel. Electrophoresis was performed in a MiniProtean 3 cell (Bio Rad; Reinach, Switzerland) with 80 V in the stacking gel and constant 120 V in the separating gel for 1 h. Running buffer (pH 8.5) contained 0.25 M Tris-HCl, 1.93 M glycine and 1% (w/v) SDS. Proteins were electrophoretically transferred to a 0.45 µm pore size pure nitrocellulose membrane using a Mini TransBlot Transfer Cell (Bio Rad; Reinach, Switzerland). Transfer buffer (pH 8.3) contained 0.025 M Tris-HCl, 0.193 M glycine and 20% (m/v) methanol. Blotting was performed with a constant current of 250 mA for 2.5 h and was controlled by staining the
© Georg Thieme Verlag KG · DOI 10.1055/s-2006-931537 (10.1055/s-2006-931611) · Planta Med 2006; 72: e149–e152 · Netsch MI et al.
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membrane with 0.1% ponceau S in 3% acetic acid. The membrane was blocked overnight at 4 °C with 5% (w/v) skimmed milk powder in PBS containing 0.05% (v/v) Tween 20 (PBS-T).
© Georg Thieme Verlag KG · DOI 10.1055/s-2006-931537 (10.1055/s-2006-931611) · Planta Med 2006; 72: e149–e152 · Netsch MI et al.
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Fig. S1 Relative mRNA expression of (a) CYP1A1 and (b) CYP1A2. Transcriptional expression was determined in Caco-2 cells by quantitative real-time PCR. Cells were treated for 24 h with 10 µM B[a]P and with different concentrations of GTE or EGCG. mRNA expression was relative to the respective expression induced by B[a]P. Data represent the pooled results of 3 separate experiments (n = 3) (* statistically significant difference, p < 0.05). Data represent means ± SEM © Georg Thieme Verlag KG · DOI 10.1055/s-2006-931537 (10.1055/s-2006-931611) · Planta Med 2006; 72: e149–e152 · Netsch MI et al.
