MolMet Supp 1: Optimization of adenoviral vector titres for comparing human and mouse GKRP.

<p>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).</p> <p>B) No nuclear GKRP or GK immunostaining in un-transfected GKRP deficient hepatocytes.</p> <p>C-E) Nuclear GKRP and GK staining in transfections with human GKRP (<a href="https://www.ncbi.nlm.nih.gov/nuccore/NM_001486.4" target="_blank">NM_001486.4</a>; <a href="https://www.ncbi.nlm.nih.gov/protein/NP_001477.2" target="_blank">NP_001477.2</a>) 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.</p> <p>F-G) Exclusive cytoplasmic staining for GKRP and GK in hepatocytes transfected mouse Gckr-Transcript-2 (<a href="https://www.ncbi.nlm.nih.gov/nuccore/NM_144909.2" target="_blank">NM_144909.2</a>; <a href="https://www.ncbi.nlm.nih.gov/protein/NP_659158.1" target="_blank">NP_659158.1</a>) 446P/L. (F) GKRP immunoreactivity (mouse transcript-2) compared with untransfected GKRP^+/- hepatocytes (Het). (G) Immunostaining showing exclusive cytoplasmic staining for GKRP and GK.</p> <p>H) Nuclear GKRP and GK staining in transfections with mouse GKRP-Transcript-1 (<a href="https://www.ncbi.nlm.nih.gov/nuccore/NM_001374741.1" target="_blank">NM_001374741.1</a>; <a href="https://www.ncbi.nlm.nih.gov/protein/NP_001361670.1" target="_blank">NP_001361670.1</a>) 446P/L at 5x10⁶ and 10x10⁶ pfu/ml.</p> <p>I) Lack of nuclear staining in cells transfected with Gckr-X1 (<a href="https://www.ncbi.nlm.nih.gov/nuccore/XM_006503882" target="_blank">XM-006503882</a>) and Gckr-X2 (<a href="https://www.ncbi.nlm.nih.gov/nuccore/XM-006503883" target="_blank">XM-006503883</a>).</p> <p>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.</p> <p>K) Similar Human <em>GCKR</em> or mouse <em>Gckr</em>-T1 transcript levels (normalized to <em>Gapdh</em> 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.</p>