Effects of HKI and HKII overexpression and knockdown on glucose utilization in ARVM and NRVM. (A) In ARVM, overexpression of HKI dramatically increased the rate of glucose utilization (white) compared with control (gray), whereas overexpressing HKII had no significant effect (black). (B) All the individual half-times of the FRET ratio change associated with glucose utilization obtained for the different conditions in ARVM are represented in the dot plot. The bar plot indicates a fivefold decrease (effect size) in the half-time when HKI is overexpressed, while the values are similar to control for HKII overexpression. The error bars represents the 95% CIs of the effect size. (C) In NRVM, however, overexpression of HKI or HKII did not significantly change the half-time of the FRET ratio change associated with glucose utilization (gray, white, and black traces). (D) However, down-regulation of HKI (white), but not HKII (black), dramatically slowed the half-time in NRVM, which suggests that glucose phosphorylation is performed primarily by HKI and not HKII. Down-regulation of HKI and HKII using lentivirus shRNA were assessed by Western blotting and compared with WT and nontargeting shRNA (scr-shRNA) preparations. (E) The dot plot shows that in NRVM, the half-time of FRET ratio change associated with glucose utilization is only affected (2.3-fold increase, effect size in bar plot) when HKI is knocked down, but not by HKII knockdown (KO), or HKI and HKII overexpression (OE). P-values reported were calculated using bootstrap resampling (see Materials and methods for details).