P 473ser akt

2-(trifluoromethyl)-10H-phenothiazine (Sigma-Aldrich, St. Louis, MO, USA) was used as the starting material to synthesize trifluoperazine and trifluoperazine derivatives (Fig. 1A, Figs S3–S16). The identity and purity (>95%) of these synthetic derivatives were identified by proton magnetic resonance spectrometry and HR-EIMS (Figs S5–S38). All agents were dissolved in DMSO, diluted in culture medium, and added to cells at a final DMSO concentration of 0.1%. Antibodies against the following biomarkers were obtained from Cell Signaling Technologies (Danvers, MA, USA): Akt, p-⁴⁷³Ser Akt, p-^180/182Thr/Tyr 38, p38, p-²⁴⁴⁸Ser mTOR, ERK, p-^202/204Thr/Tyr ERK, LC3B, Atg5, p-¹⁷²Thr AMPK, AMPK, p-¹⁵Ser p53, p53, p-¹³⁹Ser H2AX, p-⁷⁹Ser ACC, ACC, PARP, procaspase-8, and caspase-9. β-actin was obtained from Sigma-Aldrich. The GFP-LC3 plasmid was purchased from Addgene (Cambridge, MA, USA). The enhanced chemiluminescence system for detection of immunoblotted proteins was from GE Healthcare (Piscataway, NJ, USA). Other chemicals and reagents were obtained from Sigma-Aldrich unless otherwise noted.

Protein was collected from the cells after various treatments. For Western blots, a previously described procedure was applied [20 (link)]. The immunoblotting was performed with primary antibodies recognizing p⁴⁷³Ser-Akt (#9271), Akt (#9272), PARP (#9542), cleaved caspase-9 (#7237), p¹⁸⁰Thr/¹⁸²Tyr-p38 MAPK (#9215), p38 MAPK (#9212), HIF-1α (#3716), Mcl-1 (#39224), survivin (#2802), LC3B(#2775), p¹⁵Ser-p53 (#9284), and p53 (#2527) all from Cell Signaling Technologies (Beverly, MA, USA); procaspase-8 (MAB4708) from Millipore; Bax (ab7977) from Abcam (Cambridge, UK); Bcl-2 (Sc-509) from Santa Cruz Biotechnology (CA, USA); Atg5 (GTX62601) from GeneTex (Irvine, CA, USA); β-actin (A5316) from Sigma-Aldrich (St. Louis, MO, USA). Immunoblotted bands were visualized by an enhanced chemiluminescence reagent (GE Healthcare Bioscience, NJ, USA) and system (FUSION SoLo S, Deutschland, Germany) using secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) was synthesized as described previously^{36 (link)}. All agents were dissolved in DMSO, diluted in culture medium, and added to cells at a final DMSO concentration of 0.1%. Antibodies for the following biomarkers were obtained from Cell Signaling Technologies (Danvers, MA): Akt, p-⁴⁷³Ser Akt, p-³⁰⁸Thr Akt, cytochrome c, NF-κB, Mcl-1, Bcl-2, Bak, Bcl-xL, Bax, p-⁹Ser GSK3β, GSK3β, p-²⁴⁴⁸Ser mTOR, mTOR, LC3B, IKKα/β, survivin, p62, p-^176/180Ser IKKα/β, cleaved caspase-7, PARP, and caspase-9. COX-IV antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); β-actin antibody, Sigma-Aldrich (St. Louis, MO). The GFP-LC3 plasmid was purchased from Addgene (Cambridge, MA). Mcl-1 plasmid was obtained from OriGene Technologies, Inc. (Rockville, MD). The enhanced chemiluminescence system for detection of immunoblotted proteins was from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Other chemicals and reagents were obtained from Sigma-Aldrich unless otherwise noted.