Doxycycline d9891

Doxycycline (D9891) and N-acetylcysteine (NAC, A9165) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1 H)-quinazolinone (Mdivi-1, BML-CM127) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). MK-2206 (S1078) was purchased from Selleck Chemicals (Houston, TX, USA). For gene expression knockdown, cells were transfected with validated small inhibitory RNAs (siRNAs) targeting human HSPA9 (5′-AAACGCAAGUGGAAAUUAA-3′), Drp1 (5′-GAGGUUAUUGAACGACUCA-3′), or IFT88 (5′-CCGAAGCACUUAACACUUA-3′) using Lipofectamine 2000. The siRNAs were synthesized by Genolution (Seoul, Korea). At 48 h post-transfection, the cells were treated with the indicated reagents.

A549, Calu-6, NCI-H1299, NCI-H1703, NCI-H1975, NCI-H23, NCI-H460, HCC2279, NCI-H522, A427, Calu-3, NCI-H358, HCC827 and BEAS2B were obtained from the American Type Culture Collection (Rockville, MD, USA). HCC95 and HCC1588 were obtained from the Korean Cell Line Bank (Seoul, Korea), and Normal human bronchial epithelial (NHBE) purchased from Lonza. A549, Calu-6, NCI-H1299, NCI-H1703, NCI-H1975, NCI-H23, NCI-H460, HCC2279, NCI-H522, NCI-H358, HCC827, HCC95, HCC1588, and BEAS2B were cultured with RPMI1640. Calu-3 and A427 were cultured with Dulbecco’s modified eagle medium (DMEM). NHBE was cultured with bronchial epithelial cell growth medium (BEGM) BulletKit (Lonza, Walkersville, MD, USA). All cell lines were maintained in media supplemented with 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin, and cultured under standard conditions at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Stock solutions of the Wee1 inhibitor MK-1775 (Selleck Chemicals, Houston, TX, USA) were dissolved in dimethyl sulfoxide (DMSO) and added to the media at the indicated concentrations (100 nM). Doxycycline (D9891) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Matrigel was purchased from Corning Inc (Corning, NY, USA).

300, 000 human melanoma cells, lentivirally transduced with either an inducible shYY1 or shCtrl vector, were re-suspended in 100 µl of complete RPMI-1640 medium, mixed 1:1 with Matrigel matrix (356,234, BD Biosciences) and subcutaneously injected into the flanks of 10–14-week old female NUDE (Hsd:Athymic Nude-Foxn1^nu/nu; Envigo) using a 1 ml syringe and 25-gauge hypodermic needle. Tumors were grown to a maximum volume of 1 cm³, which was determined by tumor size measurements and the following formula: V = 2/3 × π × ((a+b)/4))³ with a representing tumor width and b representing tumor length, respectively.
For induction of the inducible YY1 knockdown in vivo, the drinking water of the mice was supplemented with 2 mg/ml doxycycline (D9891, Sigma-Aldrich) and 5% sucrose until the end point of the experiment was reached.

HeLa (CCL-2), MDA-MB-231 (HTB-26), and HEK293T (CRL-3216) cell lines were obtained from ATCC and were maintained in DMEM (Gibco, C11995500BT) with 10% fetal bovine serum (Gibco, 10099141 C) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37 °C within a humidified atmosphere of 20% O₂ and 5% CO₂. Primary embryonic fibroblasts (MEFs) were prepared from E13.5 wild type C57BL/6 embryos using standard protocols³⁵ and maintained in DMEM with 10% fetal bovine serum. MEFs, cultured for no more than six passages, were used for experiments. Since pyruvate could attenuate the accumulation of NADH, we replaced the medium with pyruvate-free DMEM (Merck, D0819) in NADH-related experiments to avoid the interference of pyruvate. To induce hypoxia, cells were cultured under 0.5% oxygen conditions. All the cell lines were tested negative for mycoplasma, and no commonly misidentified cell lines were used in the study. Doxycycline (D9891), N-acetyl-L-cysteine (A9165), glutathione monoethyl ester (353905), and 2-ketobutyric acid (K401) were purchased from Sigma. 6-Mercaptopurine (HY-13677), pelitrexol (HY-14530), lometrexol (HY-14521B), Z-VAD-FMK (HY-16658B), necrosulfonamide (HY-100573), ferrostatin-1 (HY-100579), and menadione (HY-B0332) were purchased from MedChemExpress. A769662 (S2697) and AICAR (S1802) were purchased from Selleck.