Palbociclib

Both human lung cancer cell lines (NCI-H23, and NCI-H2126) and HEK-293 cell line were procured from CAS-China and Microbix Biosystems Inc. respectively (Beijing, China). HEK-293 contains adenovirus (Adv) E1A-region for the replication of CRAd.
Cisplatin-resistant sublines, H23/CPR and H2126/CPR, were developed in our lab as described previously [24 (link)]. Afterward, all cancer cells were maintained in DMEM with 10% FBS and 2% of penicillin-streptomycin (HyCloneTM, Thermo Fisher Scientific, Waltham, MA USA USA). Doxorubicin (hydrochloride), cisplatin, elacridar, palbociclib, novobiocin, and probenecid were purchased from MedChemExpress (Shanghai, China) while puromycin was from Sigma-Aldrich. CRAd (incorporated with survivin promoter to regulate E1A region of Adv) was developed in our laboratory as described previously [35 (link)]. Ad-Luc expressing the firefly luciferase gene was used as a control.

Abemaciclib was purchased from Selleckchem (Houston, TX, USA), palbociclib and ribociclib from MedChem Express (Monmouth Junction, NJ, USA). Daunorubicin, mitoxantrone, propidium iodide, RNAse A, NADP+, glucose-6-phosphate, and HPLC grade solvents were bought from Sigma-Aldrich (St. Louis, MO, USA). ABCB1 inhibitor LY335979 and Daunorubicinol were obtained from Toronto Research Chemicals (North York, ON, Canada), while ABCG2 inhibitor Ko143 was from Enzo Life Sciences (Farmingdale, NY, USA). Opti-MEM was acquired from Gibco BRL Life Technologies (Rockville, MD, USA). All other cell culture media and reagents were purchased from Sigma-Aldrich, Lonza (Walkersville, MD, USA), and PAA Laboratories (Pashing, Austria).

Gemcitabine (GEM), GSK-126, and decitabine (DEC) were purchased from Sigma Chemical Co (St. Louis, MO). Palbociclib (PAL) was acquired from MedChem Express (Monmouth Junction, NJ).

Cells were seeded in 12-well tissue culture plates at 150,000 (Jurkat) or 200,000 (MOLM-13) cells per well. Cells were exposed to 0.1–0.5 µM etoposide (provided by the Jena University Hospital Pharmacy) for 6–48 h or to 25–400 nM palbociclib (MedChemExpress, Monmouth Junction, NJ, USA) for 48 h to induce autophagy. Cells were exposed to 2 mM 3-methyladenine (3-MA; Biomol, Hamburg, Germany) for 24 h or 48 h, or to 10 or 25 µM chloroquine (CQ; Enzo Life Sciences, Lörrach, Germany) for 1–48 h to inhibit autophagy.

Palbociclib was purchased from MedChemExpress (MCE, CA, USA) [11 (link)]. Ten milligrams of Palbociclib was dissolved in 4.1323 ml sterilized triple-distilled water (concentration: 5 mmol/ml) and then was subpacked into 1.5 ml EP tubes and stored at -80°C. When used, the mother liquid was diluted with complete medium to 1 μΜ and 2 μM, respectively. Antagonists of CCR5, TAK-779, was purchased from MCE (CA, USA).

Antibodies against GST (1:1000; Z-5, B-14, Santa Cruz), FLAG (1:1000; M2, Sigma), V5 (1:1000; D3H8Q, Cell Signaling), CAD (1:1000; A301-374A, Bethyl Laboratories), RelA (1:1000; 51-0500, Thermofisher), Pyruvate Carboxylase (1:1000; #66470, Cell Signaling, #49381, Cell Signaling), PDK3 (1:1000; AP7040a, Abcepta), Hexokinase 4 (1:1000; AP7901c, Abcepta), β-Actin (1:2000; 8H10D10, Cell Signaling), Phospho-CAD (1:1000; Ser1859, Cell Signaling), LANA (1:500; LN53, Abcam), vCyclin (1:500; 94B, Abcam), MYC(1:1000; 71D10, 9B11, Cell Signaling), CDK6 (1:1000; D4S8S, Cell Signaling) were purchased from the indicated suppliers. Polyclonal antibodies against KSHV vCyclin (1:500) and Phospho-CAD (Ser1900, 1:500) were generated by Genscript.
The glutamine analog 6-Diazo-5-oxo-L-norleucine (DON) was purchased from Sigma. JHU-083 (Ethyl 2-(2-Amino-4-methylpentanamido)-DON), Palbociclib, and N-(phosphonacetyl)-L-aspartic acid (PALA) were purchased from MedChemExpress. [U¹³C] Glucose and [Amide-¹⁵N] L-Glutamine were purchased from Cambridge Isotope Lab. Luciferin was purchased from Goldbio.
L-Lactate Assay Kit was purchased from Cayman Chemical. Cell proliferation Kit II, and Glutamate Assay Kit were purchased from Sigma. SYBR Green Master Mix was purchased from Bio-Rad and Genesee. Dual-Luciferase Reporter Assay System was purchased from Promega.