Gsk650394

Bisacodyl (4,4’-diacetoxydiphenyl-2-pyridyl-methane; CAS number: 603-50-9) was purchased from Sigma-Aldrich. DDPM (4,4’-dihydroxydiphenyl-2-pyridyl-methane), the active derivative of Bisacodyl was synthesized in-house based on previously described methods [20 (link)]. Methods for the synthesis of the inactive derivative of Bisacodyl, DDPM, are provided in Supplementary Materials and Methods. Chemical structure of the DDPM inactive derivative and activity profile of the compound on quiescent TG1 GSC are shown in Supplementary Figure 2.
S0859 NBC cotransporter inhibitor (CAS 1019331-10-2), DIDS (4,4’-Diisothiocyanatostilbene-2,2’-disulfonate), a stilbene derivative that can inhibit NBCe1 and all other Na⁺-coupled HCO3⁻ transporters but not NBCn1 (CAS 207233-90-7), Akt activator SC-79 (CAS 305834-79-1) and SGK1 inhibitor GSK 650394 (CAS 890842-28-1) were purchased from Sigma-Aldrich. The Akt inhibitor VIII (CAS 612847-09-3) was from Calbiochem (Merck Millipore).

Corticosterone (CORT), dexamethasone (DEX), Compound C, 9-β-D-arabinofuranoside (Ara-A), 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), aspirin, metformin, fluoxetine, RU38486, GSK650394, SAHA, SB939, Hoechst33258 and poly-L-lysine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-AMPK, anti-p-AMPK (Thr172), anti-GR, anti-FOXO3a, anti-p-FOXO3a (Thr32), anti-LKB1 and anti-HDAC5 antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-p-HDAC5 (S259) and anti-p-HDAC5 (S498) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-GAPDH and anti-GFAP (glial fibrillary acidic protein) antibodies were purchased from Santa Cruz (Dallas, Texas, USA). Dulbecco’s modified Eagle’s medium DMEM/F12 was obtained from Biotium (San Francisco, CA, USA) and Gibco Invitrogen Corporation (Carlsbad, CA, USA). Heat-inactivated fetal bovine serum was purchased from Hyclone (Logan, UT, USA). Other agents were purchased from commercial suppliers. All drugs were prepared as stock solutions and stored at -20°C. The final concentration of DMSO was less than 0.05%. No detectable effect of DMSO was found in all the experiments. The final concentrations of drugs were determined according to clinical usage or physiological levels.

Murine microvascular SVEC4-10EE2 endothelial cell line cells (EE2 ECs; ATCC, Manassas, VA) were cultured as described earlier [22 ]. Previously, MG-sensitive SGK1 signaling was studied in EE2 ECs [22 ] and in the present study we elucidate the role of NHE1 in MG-induced leukocyte recruitment in the context of SGK1-dependent activation of NHE1. We, therefore, used EE2 ECs to corroborate our in vivo findings. Where indicated, Tempol (300 μM; Sigma), cariporide (50 μM) or GSK650394 (20 μM; Sigma) was added at the specified concentrations. Targeted gene silencing was accomplished by a 48-h transfection of EE2 ECs with siRNA specifically targeting SGK (Santa Cruz) and with siRNA transfection medium and reagent (Santa Cruz) as described previously [48 (link)]. The control cells were transfected with negative control scrambled siRNA (Santa Cruz) having no homology to any known RNA sequence.