Fig 1: B7-H3 regulates glycolysis through HIF-1a and its downstream targets. QRT-PCR and western blot were used to determine the mRNA and protein expression of HIF-1a and key factors regulated by B7-H3 silencing and overexpression. A Silencing B7-H3 downregulated the mRNA level of PFKFB3 and Glut1 without affecting HIF-1a mRNA level. B Overexpression of B7-H3 upregulated the mRNA of PFKFB3 and Glut1 without affecting HIF-1a mRNA level. C Silencing B7-H3 downregulated the protein level of HIF-1a and its downstream targets PFKFB3 and Glut1. D Overexpression of B7-H3 upregulated the protein level of HIF-1a and its downstream targets PFKFB3 and Glut1. E Western blot analysis of HIF-1a, PFKFB3 and Glut1 expression in B7-H3-overexpressing cells when HIF-1a was silenced by siRNA. Silencing HIF-1a downregulated the expression of PFKFB3 and Glut1 in B7-H3-overexpressing cells. F, G Glucose uptake and intracellular lactate production were detected in B7-H3-overexpressing cells when HIF-1a was silenced. Silencing HIF-1a decreased glucose uptake and lactate production in B7-H3-overexpressing cells.
Fig 2: B7-H3 promotes glucose uptake and tumor growth in OSCC tumor xenografts. Cal27 B7-H3-overexpressing OSCC cells were subcutaneously implanted in athymic nude mice (n=4/group). A Images of the tumor specimens. B Tumor volume was monitored by caliper measurements for 6 weeks after injection. C Average tumor weight measured at 6 weeks after injection. Mice in the B7-H3-overexpressing group had a larger tumor weight. D Glucose uptake was measured in vivo at 6 weeks after tumor cell injection using the fluorescent probe 2-DG-750, as described in Materials and Methods (n=3/group; two of the mice died before fluorescent imaging). IVIS images of 2-DG-750 uptake in each individual mouse. Average of total flux (photons/second) indicates the intensity of 2-DG-750 uptake at the primary site of cell injection. Higher 2-DG-750 uptake in the B7-H3-overexpressing group was detected. E The expression levels of B7-H3, HIF-1a and PFKFB3 in implanted tumors were detected by IHC. Intense B7-H3 staining in the B7-H3-overexpressing group and weak B7-H3 staining in the control group were observed at the end point. Similarly, the expression levels of HIF-1a and PFKFB3 in the B7-H3-overexpressing group were higher than those in the control group.
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