Sphk1

Fetal bovine serum (FBS), penicillin-streptomycin (PS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen (Carlsbad, CA, USA). Trypsin EDTA was bought from Gibco (Waltham, MA, USA). Isorhapontigenin was purchased from Sigma Chemical (St. Louis, MO, USA). Enzyme-linked immune sorbent assay (ELISA) development kits, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin (IL-1β) were acquired from R&D Systems (Minneapolis, MN, USA). The primary antibodies α-tubulin, β-tubulin, SPHK1, SPHK2, PARP, caspase-3, caspase-9, p38, pp38, JNK, pJNK, ERK, and pERK were purchased from Cell Signaling (Beverly, MA, USA). Secondary antibodies for Sirt1, Bax, Bcl2, cytochrome-C, and GAPDH were purchased from Santa Cruz Technology. MCF7, T47D, and MDA-MB-231 cells were purchased from the Korean Cell Line Bank. 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) powder, RNase-A, propidium iodide, and DCFDA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The annexin V-FITC apoptosis detection kit and trypan blue were purchased from R and D Systems.

GDC-0349 was from Dr. Zhou at Hubei Cancer Hospital^{11 (link)}. Antibodies of phosphorylated (“p”)-Akt (Ser-473) (#9271), Akt (Thr-308) (#13038), Akt1 (#75692), p-S6K1 (#9234), S6K1 (9202), p-JNK1/2 (#9255), JNK1/2 (#9252), SphK1 (#12071), cleaved-caspase-3 (#9664), cleaved-caspase-9 (#20750), cleaved-poly (ADP-ribose) polymerase (PARP) (#5625), and β-tubulin (#15115) were purchased from Cell Signaling Tech (Beverly, MA). All cell culture reagents were obtained from Hyclone Co. (Suzhou, China). N-acetylcysteine (NAC), sphingosine-1-phosphate (S1P) and SP600125, rapamycin, perifosine, AZD-2014, puromycin, and polybrene were purchased from Sigma-Aldrich (St. Louis, Mo). Primers, sequences and all viral constructs were designed and provided by Shanghai Genechem (Shanghai, China) unless otherwise mentioned.

After transfection, cells were washed with phosphate-buffered saline and lysed using a lysis buffer (50 mM Tris-HCL, 150 mM NaCl, 1% NP-40) containing protease and phosphatase inhibitors cocktail tablet. Equal amounts of protein were resolved by SDS–PAGE analysis using 10% polyacrylamide gels and transferred to a nitrocellulose membrane. Membranes were blocked with non-fat milk (5%) in Tris buffered saline (TBS, pH 7.4) containing 0.1% Tween-20 (TBS-T), for 1 h at room temperature. After rinsing, membranes were incubated with antibodies according to the manufacturer instruction against FLAG (Sigma Aldrich F7425), actin (Sigma Aldrich), SphK1 and SphK2 (Cell Signaling; Abcam). After three washing in TBS-T buffer, the nitrocellulose membranes were incubated with a HRP conjugated anti-rabbit IgG antibody (Sigma Aldrich). Detection was accomplished by chemiluminesence using ECL detection reagent and exposed to Hyperfilm-ECL film (GE Healthcare).

Whole cell lysates of CCA cell lines were obtained in Pierce RIPA buffer (Thermo Scientific, Rockford, IL). Protein samples were separated on 8%–12% gradient sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to Immobilon-P (Millipore, Bedford, MA) membranes. Antigen-antibody complexes were detected using the ECL blotting analysis system (Millipore). Primary antibodies against the following targets were used: cleaved poly (ADP-ribose) polymerase (c-PARP, 9541S; Cell Signaling), β-actin (Abcam ab6276), caspase 9 (9508; Cell Signaling), caspase 3 (9662; Cell Signaling), SPHK1 (9252; Cell Signaling), phospho-Akt (4058; Cell Signaling), AKT (9272; Cell Signaling), phospho-p42/44 MAPK (Erk1/2) (4376; Cell Signaling), and ERK (SC-135900; Santa Cruz).

The cells were lysed in RIPA buffer (Cell signaling, USA). The protein extract were separated on SDS–polyacrylamide gels and were electrotransferred to a nitrocellulose membrane (GE healthcare life sciences, UK). The membranes were blocked in 5 % non-fat dry milk and probed with primary antibodies for SPHK-1 (Cell signaling, USA), HIF-1α (Novus Biologicals, USA), AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), GSK-3β (Invitrogen, USA), p-GSK-3β (Cell signaling, USA), VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PCNA (DAKO, USA), PI3K (Millipore, Germany), and β-actin (Sigma-Aldrich, St, Louis, MO, USA) overnight at 4 °C and HRP-conjugated secondary antibodies. Detection of specific proteins was carried out with an enhanced chemiluminescence (ECL) assay (GE Healthcare Life Sciences, UK).