Dactolisib

Trametinib (GSK1120212, Tram) was kindly provided by GlaxoSmithKline (Brentford, Middlesex, UK). Alpelisib (BYL719, Alp), dactolisib (BEZ235, Dact), and Geda (PF05212384) were purchased from Selleck Chemicals (Huston, TX, USA). MK-2206 (MK) was kindly provided by Merck and Co. (Kenilworth, NJ, USA). Alp, Geda, MK, and Tram were dissolved in DMSO as a 1 mM (Geda, MK and Tram) and 5mM (Alp) stock solution, dact was dissolved in DMF as a 1mM. All the drugs were stored at -20°C (Alp, Dact, MK, and Tram) or -80°C (Geda). Eve (RAD001) was kindly provided from Novartis Pharma (Basel, Switzerland) and was dissolved in 100% ethanol as a 10 mM stock solution and stored at -20°C.
The final concentration of drugs was obtained by dilution with culture medium.
Effects on cell growth after 72 h of different treatments were monitored by Crystal Violet assay, as previously described (10 (link)).

H460, A549 and H1975 cell lines were purchased from the European Culture and Tissue Collection. Apitolisib (GDC-0980) was gifted under a material transfer agreement from Genentech for use in this study, and was dissolved in dimethyl sulphoxide (DMSO), aliquoted and stored at −20 °C. Dactolisib (BEZ235) was purchased from Selleckchem, dissolved in DMSO, aliquoted and stored at −20 °C.

Sorted leukemia cells (2 × 10⁵) were transferred into Rag^−/−γc^−/− hosts and animals were monitored daily and minimally bled (<20 µL) for FACS determination of leukemia cells (CD19⁺) at days 7 and 10 after transfer. Upon leukemia detection in all animals (mCD45⁺CD19⁺ > 0.5%), mice were randomized into treated and control groups. The PI3K/mTOR inhibitor Dactolisib (BEZ235, NVP-BEZ235; Selleckchem)—dissolved v/v 10 NMP and 90 PEG300 (Sigma Aldrich)—was administered daily at 30 mg/kg by oral gavage for 15 days, whereas vehicle was administered to the control group. Treatment was then stopped for 12 days and subsequently restarted for 13 days. Animals were sacrificed when reaching humane endpoints. Animals without disease symptoms were sacrificed 120 days after cell transfer. For sphingosine kinase inhibitor treatment, sorted leukemia cells (2 × 10⁵) were transferred into Rag^−/−γc^−/− hosts and 1 day after injection animals were randomized into three groups: compound 49, compound 55, or vehicle (20% cyclodextrin/PBS). Both compounds were administered daily at 5 mg/kg by intraperitoneal injection for 21 days. Animals were monitored daily and bled weekly for FACS detection of leukemia cells (CD19⁺). Animals were sacrificed when humane endpoint was reached. Differences in survival curves were determined by log-rank (Mantel–Cox) test using Prism v8.0.

Human ovarian cancer cell lines ES-2 and SKOV3 were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM medium (Gibco, USA) supplemented with 10% FBS. Cells were treated with dezocine, and DMSO was used as negative control (NC). The cDNA sequence of CRABP2 was cloned into pcDNA3.1 vector (Sigma-Aldrich, Germany), and the blank pcDNA3.1 was used as control. The plasmids were transfected into ovarian cancer cells by Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s protocol. After transfection of CRABP2 overexpression plasmid, cells were treated with dezocine. Dactolisib (BEZ235) is a dual ATP competitive inhibitor purchased from selleck.cn. BEZ235 dissolved in DMSO was added to the medium, and the cells were treated for 48 h, then total protein was extracted and the protein expression level of CRABP2 was determined.

The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA). The AP‐1 inhibitor T5224 (#S8966), MEK inhibitor trametinib (#S2673), and PI3K inhibitor dactolisib (#S1009) were purchased from Selleckchem (USA). The JNK inhibitor SP600125 (#420119) and p38 inhibitor SB202190 (#559388) were obtained from Calbiochem (Germany). Gα_i protein inhibitor PTX (#sc‐200837) was purchased from Santa Cruz (USA). Another AP‐1 inhibitor SR11302 (#2476), PAR2‐AP (SLIGKV‐NH2; #3010), and the control peptide (VKGILS‐NH2; #3392) were obtained from Tocris Bioscience (UK).

Fisetin, dactolisib, GSK1059615, BML-277, and WNK463 were purchased from Selleckchem. Quercetin was purchased from Sigma-Aldrich. Stock solutions of the compounds were prepared on the day of an experiment by dissolving the powder in dimethyl sulfoxide (DMSO). Stock solutions were kept at − 80 °C for long-term storage. Working concentrations were diluted from respective stock solutions using complete growth medium. Dilutions of Fisetin and Quercetin were prepared in phenol red-free media. All solutions were protected from light.