T0901317

To explore whether oxysterols could protect against pyolysin-induced MAPK activation, 1.5 × 10⁵ HeLa cells/well were cultured in complete medium for 24 h using 6-well plates, and treated in serum-free medium for 24 h with vehicle, 10 ng/ml 27-hydroxycholesterol, or 50 nM T0901317 (Tocris, Abingdon, UK), which is an LXR agonist (32 (link)). Cells were then challenge with control serum-free medium or 100 HU pyolysin for 10 min, the supernatants discarded, and the cells washed with 300 μl ice cold PBS and lysed with 100 μl PhosphoSafe Extraction Reagent (Novagen, Darmstadt, Germany) for Western blotting of the MAPK pathway.

In this study, we used T0901317 (TOCRIS) as an LXR agonist and SR9243 (TOCRIS, china) as a selective LXR antagonist to stimulate and inhibit LXR signaling pathways in the isolated CSCs. The chemicals were dissolved in DMSO (Merck, Germany) to prepare 0.2 M (50 mg/ml) stock solutions.

T0901317 (catalog no. 2373) was purchased from Tocris Bioscience (Wiesbaden-Nordenstadt, Germany), U-18666A (catalog no. BML-S200) from Enzo Life Sciences (Lausen, Switzerland), staurosporine (catalog no. S-9300) from LC Laboratories (Woburn, MA, USA), triparanol (catalog no. T5200) and calcium ionophore A23187 (catalog no. C7522) from Merck (Darmstadt, Germany), desmosterol (catalog no. 14943), peroxide-free arachidonic acid (AA) (catalog no. 17948), eicosapentaenoic acid (EPA) (catalog no. 17949), and docosahexaenoic acid (DHA) (catalog no. 17950) from Cayman Chemical (Ann Arbor, MI, USA), and recombinant human IL-4 (catalog no. 200-04) and IL-13 (catalog no. 200-13) from PeproTech (Hamburg, Germany). Primers were purchased from Biomers GmbH (Ulm, Germany) and their sequences are available on request. All chemicals were of the highest grade of purity and commercially available.

DMSO and 1α,25-dihydroxy vitamin D₃ were purchased from Sigma-Aldrich (Taufkirchen, Germany). CITCO was provided by ENZO Life Sciences (Lörrach, Germany). Rifampicin was purchased from Merck Chemicals (Darmstadt, Germany). SR12813 and T0901317 were obtained from Tocris Bioscience (Bristol, UK). Minimum essential medium (MEM), Dulbecco‘s modified Eagle‘s medium (DMEM), William’s E medium, and Trypsin-EDTA solution were purchased from Thermo Fischer Scientific (Waltham, MA, USA). L-glutamine, nonessential amino acids, sodium pyruvate, and penicillin–streptomycin mixture were provided by Biozym (Hessisch Oldendorf, Germany). Fetal bovine serum (FBS) was obtained from Biowest (Nuaillé, France). Oligonucleotide primers were provided by Biomers (Ulm, Germany). TaqMan probes were purchased from Applied Biosystems (Waltham, MA, USA).