Figure 8: An SIK2 drug-resistant mutant prevented the effects of HG-9-91-01.

(a) HA-tagged SIK2 wild-type (WT) or a T96Q drug-resistant (DR) mutant were expressed in primary mouse hepatocytes (from 8–12-week-old male C57BL/6) for 16 h using adenoviral vectors (1:5 MOI) prior to cell lysis. HA–SIK2 was immunoprecipitated from 100 μg lysate and kinase activity was assayed. Data is presented as mean±s.d., n=3. Lysates were also immunoblotted with the indicated antibodies. (b) Primary hepatocytes were transduced for 24 h (1:5 MOI) with HA–SIK2 wild-type (Ad-SIK2 WT), HA–SIK2 T96Q drug-resistant mutant (Ad-SIK2 DR) or Ad-GFP (as infection control) adenoviruses prior to cell lysis. The cells were treated for the last 12 h with 1 or 4 μM HG-9-91-01 or 0.1 μM glucagon and lysates were subjected to immunoblotting with the indicated antibodies. (c) Primary hepatocytes were infected (1:2 MOI) with HA–SIK2 adenovirus (Ad-SIK2) WT or DR mutant for 16 h followed by 4 μM HG-9-91-01 treatment for 1 h before fixing the cells. The cells were immunostained with the indicated antibodies. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Images were captured with a fluorescence microscope (Nikon Eclipse Ti) using Nikon NIS-Elements BR 3.1 software. Scale bar is 10 μm in length. (d) Gluconeogenic gene expression was measured by quantitative reverse transcription–PCR in primary hepatocytes under the same conditions as in b other than glucagon treatment. (e) The cell culture medium from the experiment in b was used to measure glucose production. Glucose production was normalized to total protein content and presented relative to hepatocytes without treatment (basal). Data is presented as mean±s.d., n=6. *P<0.01 basal versus each treatment in Ad-GFP and in Ad-SIK2 WT group, basal versus glucagon in Ad-SIK2 DR group. ^$P<0.01 HG-9-91-01 treatment in SIK2 WT versus SIK2 DR and ^#P<0.05 basal versus HG-9-91-01 treatment in Ad-SIK2 DR.