Fumitremorgin c

Cell lines were harvested using PBS/EDTA and trypsin/EDTA to obtain single-cell suspensions, as described above. For determination of ABC transporter activity, cell suspensions were incubated with JC-1 (final concentration 1 μM; Sigma-Aldrich, cat. T4069) or mitoxantrone (MTX, final concentration 15 μM; Sigma-Aldrich, cat. T6545) for 1 h at 37C in the dark. Fumitremorgin C (final concentration 10 μM; Sigma-Aldrich, cat. T9054) was used as a positive control for ABC transporter activity inhibition. ALDH activity was determined using Aldefluor Kit (StemCell, cat. 01700), as recommended. Samples, together with autofluorescence controls manipulated the same way were recorded with SP-6800 Spectral Analyzer (JC-1 and Aldefluor 488 nm excitation laser, MTX 640 nm excitation laser).

Both adherent cells and prostatospheres were cultured for 7 days, in their specific conditions, in 48-well plates at 37°C in 5% of CO₂. Following this, the media were changed to include topotecan (Sigma) or daunorubicin (Sigma), drugs that are substrates for ABCG2 transporter, at different concentrations for 48 h. Afterwards, culture media containing the drugs were removed and replaced with 100 μl of MTT (dimethyl-thiazol-diphenyl tetrazolium) solution (Sigma). The incubation was performed for 2 h at 37°C in darkness. After the incubation, the MTT solution was removed and replaced by DMSO and plates incubated with stirring at room temperature. Then, each plate was analyzed in a micro-plate reader at 570 nm (BioTek Instruments, Inc., Winooski, VT, USA). The results were expressed as the percentage of survival respect to the control cells incubated without drugs, which were considered as 100% survival. Furthermore, in parallel experiments, the ABCG2 inhibitor fumitremorgin C (Sigma) was used alone or in combination with both drugs. For topotecan and daunorubicin, dose-response curves for each culture type (adherent and prostatospheres) were carried out. The corresponding half maximal effective concentrations (EC50) for both drugs were determined by analyzing the resulting dose-response curve using GraphPad Prism 6.0 software.

Side population (SP) stem-like cells were isolated from Ahr^+/+ and Ahr^−/− livers essentially as described with some modifications^{22 (link)}. Briefly, liver tissues were finely minced in PBS and rotated for 30 min at 37 °C in digestion solution (PBS containing 0.5 U/ml dispase and 60 U/ml collagenase, Invitrogen). Once digested, tissues were homogenized by passing through a 21-gauge syringe, filtered by a 0.40 μm mesh and centrifuged at 300 × g for 5 min. Pellets were then resuspended in sorting medium (PBS containing 10% FBS). To isolate SP liver cells, cellular pools were stained for 90 min at 37 °C with a solution containing 5 μg/ml Hoechst 33,342 (Sigma Aldrich). Cells were centrifuged and resuspended in HEPES-HBSS buffer and incubated with 50 μM Fumitremorgin C (Sigma Aldrich) to inhibit the ABCG2 extrusion pump. Propidium iodide (10 nM) was used to discriminate dead cells from the purification step. A MoFlo Astrium EQ flow cytometer (Beckman Coulter) was used.
Also, liver cells were stained for the undifferentiation markers CD133 PE and EpCam APC (Milteny Biotech) to quantify more undifferentiated stem cells. Briefly, 5 µl of each antibody were added to 1 million cells and incubated for 30 min at RT, after washing in PBS 0,1% BSA, cells were resupend in 500 µl PBS 0.1% BSA and analyzed in a Cytoflex S flow cytometer (BeckmanCoulter).

Rhodamine 123, probenecid, verapamil, fumitremorgin C, rifampicin, phenobarbital, chenodeoxycholic acid (CDCA) and tert-butylhydroquinone (tBHQ) were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France), whereas carboxy-2,7-dichlorofluorescein (CF) diacetate and Hoechst 33342 were from Life Technologies (Saint Aubin, France) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from Cambridge Isotope Laboratories (Cambridge, UK). [³H(G)] taurocholic acid (sp. act. 5.0 Ci/mmol), [6,7-³H(N)] estrone-3-sulfate (E3S) (sp. act. 54.0 Ci/mmol) and [1-¹⁴C] tetra-ethylammonium (TEA) (sp. act. 3.5 mCi/mmol) were supplied by Perkin-Elmer (Boston, MA, USA). Mouse monoclonal antibodies against P-gp (clone C219), MRP2 (clone M2III-6) and MRP3 (ABCC3) (clone M3II-9) were from Alexis Biochemicals (Lausen, Switzerland), whereas rabbit antibody against the p38 mitogen-activated protein kinase (MAPK) was provided by Santa Cruz Biotechnology (Dallas, TX, USA). Mouse monoclonal antibody against MRP4 (ABCC4) and rabbit polyclonal antibody against MRP5 (ABCC5) were purchased from Abcam (Paris, France). All other chemicals and reagents were commercial products of the highest purity available.