Fig 1: (A) ATP production rate in MCF7/IGF2 cells silenced for DDR1 and in control cells treated with scramble siRNAs. Glycolytic ATP (red columns-glycoATP) and the mitochondrial (blue columns—mitoATP) production rates were evaluated according to the manufacturer’s instructions (Agilent ATP test). OCR and ECAR were first measured in basal conditions. Injection of oligomycin resulted in inhibition of mitochondrial ATP synthesis and decrease in OCR, allowing the mitoATP production rate to be quantified. Complete inhibition of mitochondrial respiration with rotenone plus antimycin A accounting for mitochondrial-associated acidification allowed the calculation of the glycoATP production rate. The presented histogram (left panel) and the energetic map (right panel) show the mean and range from three independent experiments. (B) Glycolysis related markers: MCF7/IGF2 cells transiently transfected with an siRNA for DDR1 (green columns) or scramble siRNAs (black columns) were processed for mRNA expression of glycolysis-related transporters: MCT1, MCT4, GLUT1-4 (left panel), and enzymes LDHA, PKM1, PKM2, and EK2 (right panel). (C) OxPhos related markers in MCF7/IGF2 cells silenced for DDR1 (green columns) or treated with scramble siRNAs (black columns). Markers of mitochondrial biogenesis (PGC1α, PGC1β, PRC) (left panel), and mitochondrial markers (ARALAR, MFN1, NRF-1, NRF-2a, TFAM, and PNC1) (middle panel) were measured by qRT-PCR analysis. Mitochondrial activity was evaluated by mRNA expression levels of COX1 and cytoB (middle panel), and mitochondrial mass was assessed by measuring mRNA levels of mitochondrial outer membrane protein (TOMM20) (right panel). GAPDH was used as housekeeping control gene. Values are expressed as the means ± SEM of three separate experiments. (D) Western blot of selected glycolysis related molecules (PKM2, LDHA, MCT1, MCT4, and EK2) in MCF7/IGF2 cells silenced for DDR1 or treated with scramble oligonucleotides (left panel) and densitometric analysis of results obtained in three independent experiments (right panel). (E) Western blot with OxPhos antibodies cocktail against mitochondrial complexes showed a decrease of mitochondrial complex IV and I. NRF1 protein was also markedly decreased, whereas ARALAR did not show significant changes. Graphs represent the mean ± SEM of densitometric analysis of three independent experiments, where values were normalized to β-actin. (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student’s t-test).
Fig 2: Glucose consumption, lactate production and expression of glucose and lactate transporters and glycolytic enzymes. Glucose (A) and lactate (B) concentrations were measured in media conditioned from MCF7/IGF2 and MCF7/EV control cells after 48 h using colorimetric assays (see Methods). Values are mean ± SE of three separate experiments (** p < 0.01; * p < 0.05). Cells were processed to evaluate mRNA expression for (C) glucose transporters (GLUT)1–4, (D) monocarboxylate transporter (MCT)1 and MCT4, (E) glycolytic enzymes lactate dehydrogenase A (LDHA), pyruvate kinase M1 (PKM1), pyruvate kinase M2 (PKM2), hexokinase-2 (EK2), by qRT-PCR analysis. MCF7/EV cells were used as control and GAPDH used as the housekeeping control gene. Values are means ± SEM of three separate experiments. (F) MCF7/IGF2 cells and the corresponding control cells (MCF7/EV) were grown in medium containing 10% charcoal stripped- fetal bovine serum (FBS) for 48 h. Cells were then lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies to evaluate the expression of glycolytic enzymes LDHA and PKM2. A representative blot of three independent experiments is shown. Graphs represent the mean ± SEM of densitometric analysis of three independent experiments, where LDHA and PKM2 were normalized to ß-actin. (G) LDHA, PKM2, pIGF1R/pIR, IR, IGF1R, pAKT, AKT, pERK and ERK were then evaluated in MCF7/IGF2 cells before and after exposure to two different IGF1R/IR tyrosine-kinase inhibitors (TKI) (a pyrrolo(2,3-d) pyrimidine derivative, NVP-AEW541, and a selective and orally bioavailable dual IGF1R/IR inhibitor, OSI-906) at the concentration of 0.5 µM every 24 h, for 48 h. ß-actin was used to control for protein loading. Blots are representative of three independent experiments. Graphs represent the mean ± SEM of densitometric analysis of three independent experiments.
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