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, * P6 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.

A) 实验设计：将从葡萄糖激酶调节蛋白（GKRP）敲除小鼠中分离的肝细胞进行单层培养，以5~30×10^6噬菌斑形成单位每毫升（pfu/ml）的滴度转染携带人源或鼠源GKRP（446P或446L）表达的腺病毒载体，培养24小时后，分别针对GKRP（AG-GKRP）与GK（AG-GK）开展mRNA水平、免疫活性及免疫染色分析。
B) 未转染的GKRP敲除肝细胞中未检测到核GKRP或GK免疫染色信号。
C-E) 以10、20、30×10^6 pfu/ml滴度转染人源GKRP（NM_001486.4；NP_001477.2）h-446P/L后，可观察到核内GKRP与GK染色信号。其中(C) 对比未转染的GKRP+/-肝细胞（杂合子，Het）的GKRP免疫活性；(D) 为GKRP与GK核染色的代表性图像；(E) 通过核/胞质（N/C）荧光强度比值测算，在最低滴度10×10^6 pfu/ml时GKRP的核募集达到峰值，* P6 pfu/ml。
F-G) 转染小鼠Gckr-转录本2（NM_144909.2；NP_659158.1）446P/L的肝细胞中，GKRP与GK仅呈现胞质染色。其中(F) 对比未转染的GKRP+/-肝细胞（Het）的GKRP免疫活性（对应小鼠转录本2）；(G) 免疫染色结果显示GKRP与GK仅存在胞质定位。
H) 以5×10^6和10×10^6 pfu/ml滴度转染小鼠GKRP-转录本1（NM_001374741.1；NP_001361670.1）446P/L后，可观察到核内GKRP与GK染色信号。
I) 转染Gckr-X1（XM-006503882）与Gckr-X2（XM-006503883）的细胞未出现核染色信号。
J) 通过GKRP染色阳性细胞核占4',6-二脒基-2-苯基吲哚（DAPI）染色细胞核的比例测算，人源GKRP（hP、hL）或小鼠T1转录本（mP、mL）在5×10^6与10×10^6 pfu/ml滴度下的细胞转染效率无显著差异，实验共使用3批肝细胞制备样本。
K) 以5或10×10^6 pfu/ml滴度转染人源或鼠源GKRP：446P或L载体时，446P与446L载体的人源GCKR或鼠源Gckr-T1转录本水平（以Gapdh mRNA作为内参归一化）无显著差异。该实验中，人源GKRP组的样本量n=8，鼠源GKRP组n=7，数据以均值±标准误（SEM）表示。