Doxycycline dox

Fibroblasts from M. minutoides were dissociated, washed, and suspended in Opti-MEM (Gibco). The cell suspension was then transferred to a 4-mm cuvette electrode (SE-204; BEX Co., LTD), and 2.5 μg/100 μL of plasmid was added individually for iPSC induction (PB-TRE-OKSM, PB-TRE-GNL, PB2-CAG-rtTA-ires-eGFP pGK-Neo, and pCAG-T3-hyPBase-pA(95)). Electroporation was performed using a CUY21EDIT II electroporator (BEX) with a single 10 ms pulse at 350 V, followed by five 50 ms pulses at 40 V at 50 ms intervals. After electroporation, the cells were reseeded on feeder cells in ESGRO Complete Basal Medium (Merck) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), 20% KnockOut Serum Replacement (KSR) (Gibco), and ESGRO-2i Supplement Kit (Merck), with 2 μg/mL (final concentration) doxycycline (DOX) (Takara). Emerging colonies with iPSC-like morphology were passaged on feeder cells and mitomycin C-treated mouse embryonic fibroblasts and cultured with DOX until the reappearance of the colonies. Subsequently, DOX supplementation was discontinued, and the cells were further cultured. Individual colonies that maintained domed-morphology were selected under a stereomicroscope and single cells from a colony were seeded on feeder cells in 96-well plates to establish multiple M. minutoides iPSC lines.

To analyse the function of ΔNp63α, human mammary epithelial cells (HMECs, Thermo Fisher Scientific, Waltham, MA, USA) were purchased and cultured in HuMEC Ready Medium (1X) (HuMEC, Thermo Fisher Scientific) supplemented with gentamicin/amphotericin solution (Thermo Fisher Scientific). DeltaNp63alpha-FLAG (#26,979; RRID:Addgene_26979) was obtained from Addgene (www.addgene.org), and pRetro-X-Tight-Pur and pRetro-X-Tet-Off Advanced vectors were purchased from TaKaRa Bio Inc. (Otsu, Japan). Polymerase chain reaction (PCR) primers were obtained from Merck (St. Louis, MO, USA) (Table S1). Knockdown experiments were carried out by lipofection with siRNA targeting the DNA-binding domain of TP63 mRNA with siLentFect (Bio-Rad, Hercules, CA, USA). Scramble siRNA (scr) was used as a negative control. All siRNA double strands were synthesized by NIPPON GENE (Tokyo, Japan) (Table S1). G418 sulfate (Nacalai Tesque, Kyoto, Japan) and puromycin (TaKaRa Bio Inc.) were used for cell selection. Doxycycline (Dox, TaKaRa Bio Inc.) was used to regulate gene expression using an inducible Tet-OFF system.

C6 cells expressing the reverse Tet-responsive transcriptional activator (rtTA) were obtained as previously described [40 (link)] and were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium 41965, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum, 50 µg/mL penicillin-streptomycin, and 2.5 µg/mL amphotericin B (ThermoFisher Scientific), in a humidified atmosphere (5% CO₂, 37 °C). C6-rtTA cells were transfected with the pTRE2hyg-AMPK-DN construct through the method of calcium phosphate precipitation [41 (link)]. After selection with hygromycine (300 µg/mL; InvivoGen, San Diego, CA, USA), clones were isolated and tested for AMPK-DN expression by myc detection after supplementing the culture medium with doxycycline (DOX; Takara Bio). The DOX induction protocol was then set at 2 µg/mL for 24 h.

The Fluorescence-based screening assay was described previously [60 (link)]. Briefly, the plasmid EYFP-N1-hAPOBEC3G (0.5 μg) was transfected into TRex-hvif-15 cells (2 × 10⁵ per well) using Lipofectamine 2000 (Life Technology) when the cell confluence was approximately 80%. Doxycycline (Dox) (Clontech, Mountain View, CA, USA) (1 μg/mL) was added to induce Vif expression 6 h post-transfection, also added were serially diluted compounds. Cells were observed with a fluorescent microscope (Leica DMI4000B, Weizlar, Germany) or harvested by trypsin treatment 48 h post-transfection. Dead cells were stained with Fixable Viability Dye eFluor^® 660. Enhanced yellow fluorescent protein (EYFP) positive live cells were analyzed. Data acquisition for at least 50,000 events was performed using a FACSVerse^TM flow cytometer (BD Biosciences, San Jose, CA, USA), and the data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA). The plasmid pcDNA3.1-APOBEC3G-HA (0.5 μg) was transfected into TRex-hvif-15 cells (2 × 10⁵ cells/well) using Lipofectamine 2000 when the cell confluence was approximately 80%. Vif expression was induced with 0.1 μg/mL Dox and the compound to be tested was added 6 h post-transfection. After 48 h, the total protein was collected; A3G-HA, Vif, and beta-actin were analyzed by western blot.

SNCA^WT SH-SY5Y cells and SNCA^A53T SH-SY5Y cells were established using Lenti-X™ Tet-On® 3G inducible expression system and SH-SY5Y cells according to user manual. Doxycycline (Dox; Clontech Laboratories, CA, USA) was used to induce the expression of SNCA^WT and SNCA^A53T. Cells were routinely grown in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% tetracycline-free fetal bovine serum (Clontech Laboratories, CA, USA) and cultured at 37°C under humidified 5% CO2 atmosphere. When cells were subcultured once attaining 70-80% confluency, MANF (PeproTech, State of NJ, USA) and Dox were added for 24 or 48 h, respectively.

Human prostate cancer cells, LAPC4, VCaP, DU145, PC-3, and LNCaP cells were obtained from the ATCC (Manassas, VA), which also provided authentication of all cell lines. The cells were grown in ATCC-recommended medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The authenticated human prostate carcinoma C4-2 was purchased from UroCor. Inc (Oklahoma City, OK) and grown in T medium containing 5% FBS. Cells from each of cell line were frozen at early passages and kept in liquid nitrogen providing a stock for cultured cells that were used within 6 month after thawing. Tetracycline responsive PC-3 cells were maintained in media recommended by ATCC for parental PC-3 cells supplemented with 10% Tet system-approved FBS from Clontech (Mountain View, CA). All culture media were purchased from Life Technologies (Carlsbad, CA); fetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO). Doxycycline (Dox) was from Clontech. Collagen type I, fibronectin, and Matrigel were from Becton Dickinson Biosciences (Bedford, MA). CellTracker™ Green CMFDA was from Life Technologies. Bisindolylmaleimide I (BIM-I) was purchased from AdipoGen (San Diego, CA) and phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich. AZD5363 and LY294002 were purchased from Selleckchem (Houston, TX).