Pki 402

To carry out the metabolite extraction assay, firstly, equal numbers of MV4-11 and ABT199-R cells (~3 × 10⁶ cells) that were determined to be in the exponential growth phase were collected, and only the resistant cells were treated with PKI-402 (Selleckchem, Radnor, TX, USA, S2739), which is a dual pan-PI3K/mTOR inhibitor; we selected the dose based on the IC50 value (120 nM and 240 nM). Metabolite extraction was performed on the MV4-11 cells, ABT199-R cells (resistant cells), and ABT-199R cells treated with PKI-402. The cells were immediately lysed by a glass homogenizer using the ice-cold solvent acetonitrile: methanol: water at a ratio of 2:2:1 v/v. The lysates were vortexed for 30 s, incubated for 1 h at −20 °C, and then centrifuged for 15 min at 13,000 rpm at 4 °C. The supernatant was injected into the LC-MS/MS system [18 (link),19 (link),20 (link)].

Reagents used in this study include the following: Methylthiazolyldiphenyl-tetrazolium bromide (MTT)(S6821), PKI-402(S2739), cycloheximide (CHX)(NSC-185) and Thapsigargin (Tg) (S7895)were commercially sourced from Selleckchem (Houston, TX, USA), anti-G3BP1 (13057-2-AP), anti-eIF2α (11233-1-AP), anti-4EBP1 (60246-1-Ig), anti-caspase-9(66169-1-Ig), anti-Bcl-2 (26593-1-AP), anti-Bax (50599-2-Ig), anti-Hsp60 (15282-1-AP), anti-Lonp (66043-1-Ig), anti-Clpp (15698-1-AP), anti-Trap1 (10325-1-AP), anti-Actin(HRP-60008), anti-VDAC (10866-1-AP) (Proteintech, Chicago, IL, USA), anti-YB-1(sc-101198), anti-ATF5(sc-377168), anti-p-Akt(Ser473) (sc-101629)(Santa Cruz, CA, USA), p- eIF2α(Ser51)(3398), p-4EBP1(Thr37/46)(2855), p-Akt(Thr308) (13038), p-P70S6K(9204) (Cell Signaling Technology, USA).