MolMet Supp 1: Optimization of adenoviral vector titres for comparing human and mouse GKRP.
A) Experimental design: hepatocytes isolated from GKRP-deficient mice were cultured in monolayer and transfected with adenoviral vectors for expression of human or mouse GKRP(446P or 446L) at titres of 5 to 30 x 106 pfu/ml and cultured for 24h for analysis of mRNA, immunoactivity and immunostaining for GKRP (AG-GKRP) and GK (AG-GK).
B) No nuclear GKRP or GK immunostaining in un-transfected GKRP deficient hepatocytes.
C-E) Nuclear GKRP and GK staining in transfections with human GKRP (NM_001486.4; NP_001477.2) h-446P/L at titres of 10,20,30 x 106 pfu/ml. (C) GKRP immunoreactivity compared with untransfected GKRP+/- hepatocytes (Het). (D) Representative images of nuclear staining of GKRP and GK. E) Maximum nuclear sequestration of GKRP at the lowest titres of 10x106 pfu/ml, measured from the nuclear / cytoplasmic (N/C) intensity ratio, * P< 0.02 vs 10x106 pfu/ml.
F-G) Exclusive cytoplasmic staining for GKRP and GK in hepatocytes transfected mouse Gckr-Transcript-2 (NM_144909.2; NP_659158.1) 446P/L. (F) GKRP immunoreactivity (mouse transcript-2) compared with untransfected GKRP+/- hepatocytes (Het). (G) Immunostaining showing exclusive cytoplasmic staining for GKRP and GK.
H) Nuclear GKRP and GK staining in transfections with mouse GKRP-Transcript-1 (NM_001374741.1; NP_001361670.1) 446P/L at 5x106 and 10x106 pfu/ml.
I) Lack of nuclear staining in cells transfected with Gckr-X1 (XM-006503882) and Gckr-X2 (XM-006503883).
J) Similar cellular transfection efficiency at 5x106 and 10x106 pfu/ml for human GKRP (hP,hL) or mouse T1 (mP,mL) determined from GKRP stained nuclei % DAPI staining, n=3 hepatocyte preparations.
K) Similar Human GCKR or mouse Gckr-T1 transcript levels (normalized to Gapdh mRNA) for 446P and 446L vectors in transfections with human or mouse GKRP:446P or L (at 5 or 10 x 106 pfu/ml). Means ± SEM for n=8 (human GKRP), n=7 (mouse GKRP) hepatocyte experiments.
MICA: Exploring a new perspective on the mechanism of action of Glucokinase Activators in liver, a preclinical study
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