On were acquired using a Leica DM-LB2 microscope equipped with DFC480 camera (Leica Microsystems, Wetzlar, Germany). Cell lengths of 100 control and 100 treated cells were measured using ImageJ software (National Institute of Health), calibrated based on the known size of the hemocytometer grid. Changes in protein contents of the cells after treatment were determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Western Blot Analysis. Anti-human monocarboxylate transporter 4 (MCT4) antibodies were purchased from Santa Cruz Biotech.Inc (Santa Cruz, CA, USA). Lactate dehydrogenase A (LDHA) and Glyceraldehyde 3-phosphate dehydrogenase (GADPH) were purchased from Abcam Inc (Cambridge, MA). Lactate dehydrogenase B (LDHB) was obtained from Proteintech (Chicago, IL, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from GE Healthcare (GE Healthcare Bio-Sciences Corp, USA). HIF1-alpha was obtained from Santa Cruz Biotech.Inc (Santa Cruz, CA, USA). Tumor tissues were homogenized with a PowerGen Model 125 homogenizer (Fisher Scientific) with ice-cold RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 Nonidet P40, 0.5 sodium deoxycholate, 0.1 sodium dodecylsulfate, 2 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonylfluoride, 1 mM NaVO3, 1 mM NaF) and kept on ice 16402044 for 20 minutes. The lysates were clarified by centrifugation at 16,000 g for 10 minutes at 4uC. The protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Cells were harvested and washed twice with PBS and lysed with ice-cold RIPA buffer. To each well, 10 mg of protein from each sample of cell lysate or 15 mg of protein from each sample of tumor tissue lysate was EW-7197 biological activity loaded to 12 SDS-PAGE gel with loading buffer. Separated proteins were transferred electrophoretically from gels to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were incubated for 1 hour at room temperature in blocking buffer (20 mmol/L Tris – pH7.4-, 137 mmol/L NaCl, 5 dry skim milk) followed by 2 hours of incubation with primary antibodies: LDHA (1:2,000), LDHB (1:2,000), MCT4 (1:300), HIF1-a (1:100) and 1 hour with HRP-conjugated secondary antibodies with 1:4,000 dilution. Reactive bands were visualized with ECL Western Blotting Detection Reagents (GE Healthcare Bio-Sciences Corp.).BCX-1777 ResultsHyperpolarized [1-13C]lactate and [1-13C]pyruvate signals were observed in the tumors following injections of pre-polarized [1-13C]pyruvate in the rat MDA-MB-231 xenograft model (Fig. 1). Much higher [1-13C]lactate signal as compared to [1-13C]pyruvate signal was observed in the tumors, primarily due to the larger tipangle used to acquire the lactate images. In axial image slices through the tumors, most of the lactate signal observed was within the tumors while a substantial substrate signal can be observed in the area of major vascular structures. Similar to prior dynamic 13C MRS data acquired from mice tumors [7,8], different time courses were observed for pyruvate and lactate (Fig. 1, upper right). The substrate signal reached a maximum around the time the injection ended (average 11.5 s for control tumors and 9.75 s for the treated tumors, which was not significantly different, P.0.2, Student’s ttest) and the [1-13C]lactate signal continued to increase after the end of the bolus and then decayed due to T1 relaxation and, possibly, efflux.Radiation Therapy Response and 13C Metabolic MRIFigure 1. Representative T2-weig.On were acquired using a Leica DM-LB2 microscope equipped with DFC480 camera (Leica Microsystems, Wetzlar, Germany). Cell lengths of 100 control and 100 treated cells were measured using ImageJ software (National Institute of Health), calibrated based on the known size of the hemocytometer grid. Changes in protein contents of the cells after treatment were determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Western Blot Analysis. Anti-human monocarboxylate transporter 4 (MCT4) antibodies were purchased from Santa Cruz Biotech.Inc (Santa Cruz, CA, USA). Lactate dehydrogenase A (LDHA) and Glyceraldehyde 3-phosphate dehydrogenase (GADPH) were purchased from Abcam Inc (Cambridge, MA). Lactate dehydrogenase B (LDHB) was obtained from Proteintech (Chicago, IL, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from GE Healthcare (GE Healthcare Bio-Sciences Corp, USA). HIF1-alpha was obtained from Santa Cruz Biotech.Inc (Santa Cruz, CA, USA). Tumor tissues were homogenized with a PowerGen Model 125 homogenizer (Fisher Scientific) with ice-cold RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 Nonidet P40, 0.5 sodium deoxycholate, 0.1 sodium dodecylsulfate, 2 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonylfluoride, 1 mM NaVO3, 1 mM NaF) and kept on ice 16402044 for 20 minutes. The lysates were clarified by centrifugation at 16,000 g for 10 minutes at 4uC. The protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Cells were harvested and washed twice with PBS and lysed with ice-cold RIPA buffer. To each well, 10 mg of protein from each sample of cell lysate or 15 mg of protein from each sample of tumor tissue lysate was loaded to 12 SDS-PAGE gel with loading buffer. Separated proteins were transferred electrophoretically from gels to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were incubated for 1 hour at room temperature in blocking buffer (20 mmol/L Tris – pH7.4-, 137 mmol/L NaCl, 5 dry skim milk) followed by 2 hours of incubation with primary antibodies: LDHA (1:2,000), LDHB (1:2,000), MCT4 (1:300), HIF1-a (1:100) and 1 hour with HRP-conjugated secondary antibodies with 1:4,000 dilution. Reactive bands were visualized with ECL Western Blotting Detection Reagents (GE Healthcare Bio-Sciences Corp.).ResultsHyperpolarized [1-13C]lactate and [1-13C]pyruvate signals were observed in the tumors following injections of pre-polarized [1-13C]pyruvate in the rat MDA-MB-231 xenograft model (Fig. 1). Much higher [1-13C]lactate signal as compared to [1-13C]pyruvate signal was observed in the tumors, primarily due to the larger tipangle used to acquire the lactate images. In axial image slices through the tumors, most of the lactate signal observed was within the tumors while a substantial substrate signal can be observed in the area of major vascular structures. Similar to prior dynamic 13C MRS data acquired from mice tumors [7,8], different time courses were observed for pyruvate and lactate (Fig. 1, upper right). The substrate signal reached a maximum around the time the injection ended (average 11.5 s for control tumors and 9.75 s for the treated tumors, which was not significantly different, P.0.2, Student’s ttest) and the [1-13C]lactate signal continued to increase after the end of the bolus and then decayed due to T1 relaxation and, possibly, efflux.Radiation Therapy Response and 13C Metabolic MRIFigure 1. Representative T2-weig.
