Fig 1: Impact of CX-4945 treatment of mice on FBP1 expression in liver and blood sugar level. Mice were treated for 3 days with CX-4945 or an equal volume of DMSO for control. Thereafter, they were sacrificed and liver and blood were retrieved and processed. (A) RNA was extracted from liver tissue, reverse transcribed into cDNA. cDNA was subjected to a qRT-PCR analysis using FBP1-specific primers and actin primers for housekeeping control. The graph shows the normalized value for control (DMSO)-treated and CX-4945-treated animals. (B) An amount of 5 µg each of liver tissue from three different mice was loaded on a 12.5% gel, separated, blotted onto a PVDF membrane and subjected to an immunoblot analysis with FBP1-specific antibody and α-tubulin-specific antibody for loading control. The bar graph shows the relative FBP1 expression after normalizing to tubulin. (C) Blood glucose concentration was determined in the serum of DMSO- and CX-4945-treated mice with the glucose dehydrogenase method. The graphs show the result of at least three independent experiments. * Statistical significance was accepted as p < 0.05.
Fig 2: Impact of CK2 down-regulation on FBP1 activity in McA-RH7777 cells. (A) CK2 activity was inhibited by treatment with 10 µM CX-4945 and an equal volume of DMSO (for control). FBP1 enzymatic activity in McA-RH7777 cells was determined with a colorimetric assay as recommended by the manufacturer. FBP1 activity of cells treated with CX-4945 was set into reference to FBP1 activity of control cells (DMSO, 100%). The graph shows the result of at least three independent experiments. (B,C) CK2 activity was either inhibited by treatment with 10 µM CX-4945 and an equal volume of DMSO (for control) (B) or CK2 expression was down-regulated by transfection with siRNA against the catalytic subunits CK2α or CK2α´ (single or combined) or a scrambled siRNA for control (C). Cells were cultivated under conditions promoting gluconeogenesis. Extracellular glucose was determined in the cell culture supernatant using the GlucoseGloTM assay (Promega, Mannheim, Germany) according to the manual of the producer. Extracellular glucose of the treated cells was set in reference to the amount of extracellular glucose of the DMSO or the scrambled control (100%). The graphs show the result of at least three independent experiments. * Statistical significance was accepted as p < 0.05.
Fig 3: Impact of CK2 down-regulation on FBP1 mRNA expression. (A) McA-RH7777 cells were treated with 10 µM CX-4945 for 24 h. Control cells were incubated with an equal volume of the vehicle DMSO. (B) CK2 expression was down-regulated by transfection with siRNA against the catalytic subunits CK2α or CK2α´ (single or combined) or a scrambled siRNA for control for 72 h. Cells from A and B were harvested, and total RNA was isolated using the QIAzol lysis reagent. The mRNA amount of FBP1 was determined using qRT-PCR. After normalization to actin, the amount of FBP1 mRNA of control-treated cells was set 100% and the amount of treated mRNA cells calculated in reference to it. * Statistical significance was accepted as p < 0.05.
Fig 4: Inhibition of CK2 activity by CX-4945 in INS-1 832/13 cells. INS-1 832/13 cells were treated with 2.5, 5 or 10 µM CX-4945 or an equal volume of the solvent DMSO for control for 24 h. Cells were harvested, protein or RNA extracted and subjected to the following analyses. (A) An amount of 10 µg total protein was used for an in vitro phosphorylation with the CK2-specific substrate peptide RRRDDDSDDD. Incorporation of [32P]-labelled phosphate into the peptide in the presence of the inhibitor was set in reference to the amount of incorporated phosphate of the DMSO control (100%). (B) An amount of 30 or 60 µg (for PARP detection) of total protein was separated on a 12.5% or 7.5% (for PARP) SDS polyacrylamide gel and blotted onto a PVDF membrane. CK2 subunits, PARP and loading control tubulin were detected by specific antibodies and enhanced chemiluminescence. (C) RNA was reverse transcribed into cDNA, which was then subjected to a qPCR analysis using FBP1-specific primers. The message was normalized with actin as housekeeping gene. The graph shows the result of three independent experiments. * Statistical significance was accepted as p < 0.05.
Supplier Page from Abcam for Fructose-1,6-Bisphosphatase Activity Assay Kit (Colorimetric)