Cell culture and mESC differentiation
Mouse embryonic stem R1 (ATCC, SCRC1036) and J1 (ATCC, SCRC 1010) cell lines were grown in tissue culture dishes (Corning, Amsterdam, The Netherlands) coated with 0.1% gelatin (Sigma, Munchen, Germany) in a Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Munchen, Germany) supplemented with 15% ESC-qualified fetal bovine serum (Sigma, Munchen, Germ any), 0.1mM MEM non-essential amino acids (Sigma), 0.1 mM 2-mercaptoethanol (Sigma, Munchen, Germany), L glutamin (Sigma), 100 U/ml penicillin-100 ug/ml streptomisin mix and 1000 units/ml of recombinant mouse LIF. mESCs were cultured at 37°C in a humidified atmosphere with 5% CO2. Cells were passaged every 3 or 4 d using trypsin EDTA. Media were changed every 2 d. Spontaneous differentiation was induced with the removal of LIF from media.
Real-time quantitative PCR (qPCR)
Total RNA was isolated using a commercial kit (Thermo Fisher Sci.) and reverse-transcribed using an mRNA to cDNA synthesis kit (Thermo Fisher Sci. Cat.#4387406) according to manufacturer’s directions. mRNA expressions of Pfkfb1, Pfkfb2, Pfkfb3, Pfkfb4, Brachyury (T2), Nestin (Nes), Oct4, Sox2, Nanog, Klf4 mRNA expressions, were determined by qPCR using StepOnePlus (Thermo Fisher Sci., NY, USA) with TaqMan probes (Thermo Fischer Sci., Cat.#s: Pfkfb1, Mm01256237_m1; Pfkfb2, Mm00435575_m1; Pfkfb3, Mm00504650_m1; Pfkfb4, Mm00557176_m1; T2, Mm00436877_m1; NES, Mm00450205_m1; Oct4, Mm03053917_g1; Sox2, Mm03053810_s1; Nanog, Mm02019550_s1; Klf4, Mm00516104_m1; Gapdh, Mm99999915_g1). Gapdh was used as housekeeping gene control for normalization of cDNA. Cycle threshold (CT) values were taken from qPCR reactions and up/down regulation of genes of interest was determined by the ΔΔCT method using undifferentiated mESCs as a baseline (Livak et al. 2001).
SDS-PAGE and Western blotting
SDS-PAGE and Western blotting were performed following standard protocols. Primary antibodies specific to Pfkfb3 (Proteintech Cat.#13763-1-AP), P-Stat3 (Cell Signaling Cat.#9145), Stat3 (Cell Signaling Cat.#9139), Nanog (Cell Signaling Cat.#8822), and Gapdh (Cell Signaling Cat.#97166) proteins were used. Appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, Cell Signaling Cat.#7074 or anti-mouse Cell Signaling Cat.#7076) were used. Signals were developed using Amersham ECL plus chemoluminescent reagent (GE Technologies). Bands on membranes were visualized with ChemiDoc MP (BioRad).
siRNA and plasmid transfection
Transfections of and Pfkfb3-specific (Thermo Fischer Sci. Cat.#100779) siRNA molecules into cells was performed in serum-free and antibiotic-free medium using Lipofectamine RNAiMAX (ThermoFisher Sci.) following the manufacturer’s recommendations when cells reached approximately 50% confluency at the time of transfection. As a negative control, cells were transfected with the universal siRNA molecule, which has no homology in the human genome (Thermo Fischer Sci. Cat.# 4390846). Cells were incubated in complete medium at 37°C for 48 hours before cell harvest or further experiments. The final concentrations of siRNA molecules were 10 nM.
For ectopic expressions, pCMV6-A-BSD expression vector encoding Pfkfb3 cDNA (Origene Cat. #MG227622) was transfected into cells using Lipofectamine 3000 reagent (ThermoFisher Sci.) following the manufacturer’s instructions. Cells were 70–80% confluent at the time of transfection.
Cell proliferation
Pluripotent and differentiated (for 5 d) mESCs were lifted with trypsin post-transfections and stained with trypan blue for 1 min. The numbers of viable cells were counted under an inverted microscope (Accu-Scope, China) using hemocytometer (Neubauer improved) as per standard protocol.
Fructose 2,6-bisphosphate assay
Intracellular fructose 2,6-bisphosphate (F2,6BP) levels of differentiated (for 3 and 5 d) and undifferentiated mESCs were analyzed following a Kinetic spectrophotometric coupled enzyme method described by Van Schaftingen et al. (1982). The protocol briefly as follows; mESCs were centrifuged at 270×g and resuspended in 20 volumes of 0.05 N NaOH and then 1 volume of 0.1 N NaOH vortexed for 10 s, heatted at 80°C for 5 min, and cooled in an ice bath. PH of lysates adjusted to 7.2 with ice-cold 1M acetic acid in the presence of 1M Hepes. Next, samples were incubated at 25°C for 2 min in the assay mixture. The contents of the assay mixture were as follows: 50 mM Tris, 2 mM Mg⁺2, 1 mM Fru-6-P, 10 units/liter PPi-dependent PFK1, 0.15 mM NAD, 5 kilounit/liter triose-phosphate isomerase, 0.45 kilounit/liter aldolase, and 1.7 kilounit/liter glycerol-3-phoshate dehydrogenase (Sigma). Reaction was started adding 0.5 mM Pyrophosphate and the rate of changes in absorbance (OD 339 nm) per min was analyzed in 5 min. F2,6BP levels were calculated based on a calibration curve ranging from 0.1 to 1.0 pmol standart of Fru-2,6-BP (Sigma) and normalized to total cellular protein levels.
Glucose uptake assay
To determine glucose uptake by pluripotent and differentiated (for 5 d) mESCs, a commercial glucose uptake kit (BioVision, Milpitas, CA, USA) was used in accordance with the manufacturer's instructions. The principle of this assay is briefly as follows: 2-deoxyglucose (2-DG) is metabolized to 2-DG-6-phosphate (2-DG6P) which cannot be further metabolized, and thus accumulates in the cells. The accumulated 2-DG6P is directly proportional to 2-DG (or glucose) uptake by cells. 2-DG6P is oxidized to generate NADPH, which can be determined by an enzymatic recycling amplification reaction.
ALP staining
Alkaline phosphatase (ALP) Live Stain kit (Thermo Fischer Sci. Cat.#A14353) was used to determine ALP activity in pluripotent and differentiated mESCS. Staining was performed for 20–30 min according to manufacturer's instructions. The protocol was briefly as follows; R1 and J1 mESCs were washed twice with sterile, fresh, pre-warmed basal DMEM/F-12 media. For differentiated and undifferentiated mESCs, 1X working solution which was prepared by diluting the 500X stock solution of the LIVE AP substrate in basal DMEM/F-12 media, was found to be optimal in providing a potent signal. The ALP live stain dye was immediately applied on to the adherent mESC culture. mESCs were incubated with the substrate for 20–30 min and washed twice with the DMEM/F-12 basal media to remove excess substrate. After the final wash, fresh basal media was added and fluorescent-labeled colonies were imaged on EVOS Imaging System (ThermoFischer Sci.). The most robustly fluorescing colonies were selected for imaging within 30–40 min of staining.
Statistical analysis
The Statistical Package for the Social Sciences version 23.0 (SPSS, Chicago, IL, USA) was used for data analyses. Values were expressed as means ± standard deviation. Statistical analysis was performed using Student’s t-test and results were considered to be significant when p value was < 0.05.