Supplementary Materials & Methods
Reagents
LDH antibody, PKM2, and p-GSK3β(ser9) antibody were purchased from Cell
signaling Technology (Cell signaling Technology, Inc., MA) at 1:800 dilution.
PFKFB 2 and PFKFB 1/4, and GSK3β antibody antibody were purchased from
Bioworlde (Bioworlde, MN) at 1:800 dilution. Glut1 antibody, AKT antibody and
p-AKT antibody were purchased from Santa Cruz (Santa Cruz, CA) at 1:500 dilution.
The siRNAs targeting AKT was purchased from Santa Cruz (Santa Cruz, CA)
Intracellular Lactic Acid Production Assay
After harvested, cells (1×106/ml) were suspended with low permeability salt
solution, and kept at room temperature for 1 h. Then the suspension were sonicated
for 5 min and centrifuged at 4°C. The supernatant were kept and assayed following
the Lactic Acid production Detection kit manufacturer’s instructions (KeyGen,
Nanjing, China).
Measurement of Oxygen Consumption
Fluorometric oxygen measurements using BD Oxygen Biosensor plates were
carried out as previously described(Guarino et al. , 2004). 0.2×106 cells were added to
the individual wells of a 96-well BD Oxygen Biosensor System plate (BD
Biosciences, San Jose, CA, USA), followed by culture medium until a final volume of
200 μl per well was attained. After drug treatments, the plates were then maintained at
37°C in a humidified incubator with 5% CO2. Plates were scanned in a
temperature-controlled (37 °C) plate reader (Thermo, Waltham, MA) with an
excitation wavelength of 485 nm and an emission wavelength of 630 nm at
predetermined hours for a total of 72 h. The fluorescence traces in each well were
normalized according to the signal in the air-saturated buffer. Slopes of fluorescence
signal were calculated in the dynamic range of measurements to compare the
respiratory rates of samples. Normalized relative fluorescence unit (NRFU) represents
the oxygen consumption.
Spectrophotometric assay for enzymes activity
Cells were seeded in 24-well plates and grown to confluence. Then, medium was
removed and fresh medium was added, and cells were returned to the incubator in the
presence of 150 μM oroxylin A for 48 h, or 100 μM CTZ for 24 h, respectively. After
this incubation, cells were removed from the plates by trypsinization and counted
using a hemocytometer. Protein concentrations of cell lysates were measured, and the
glycolytic enzyme activities were evaluated.
Cellular HK activity, PFK activity and PK activity were measured following the
instructions of Hexokinase Activity Assay Kit, Phosphofructokinase activity Assay
Kit, and Pyruvate kinase activity Assay Kit, respectively, obtained from Sigma
(Genmed Scientifics Inc., MA); LDH activity was measured with Lactate
Dehydrogenase Activity Assay Kit obtained from Sigma (Sigma, St. Louis, MO).