Extended Data Fig. 6: Insulin, 3p-hpRNA and virus infection promotes glucose uptake and RIG-I translocation to the plasma membrane in a GLUT4-dependent manner.
From: UBXN9 governs GLUT4-mediated spatial confinement of RIG-I-like receptors and signaling
a-c, Endogenous exofacial expression of GLUT4 in myocytes stimulated with or without insulin (200 nM) for 20 min. Scale bar, 10 μm. d, Mean fluorescence intensity (MFI) of plasma membrane GLUT4 (pmGLUT4) from cells (n = 5-7 individual images/group for vehicle; n = 11 WT, n = 11 Ubxn9−/− and n = 10 individual images for Slc2a4−/− during insulin) stimulated as in (a-c). Dotted line indicates background fluorescence. e, Colocalization of plasma membrane GLUT4 with ZO-1 (white arrows) in wild-type C2C12 myocytes before or after EMCV infection (MOI: 0.5, 24 h). Scale bar, 5 μm. f, 2-DG uptake in C2C12 cells treated with insulin (200 nM) for 20 min (n = 4 replicates/group), transfected with 3p-hpRNA for 12 h (n = 4 replicates/group), treated with IFN-β for 12 h (n = 6 replicates/group), or infected with EMCV (MOI: 0.5) for 24 h (n = 6 replicates/group). The results are normalized to untreated Slc2a4+/+ cells. g, Confocal images of PM staining, RIG-I and nuclear DNA in C2C12 cells mock treated. Scale bar, 5 μm. h, Cytoplasmic fraction from C2C12 myocytes (left) and 3T3-L1 myc-GLUT4-GFP adipocytes (right) before and after various treatments. Insulin for 20 min (200 nM); 3p-hpRNA for 6 h. i, Endogenous MAVS-RIG-I interaction from C2C12 cells stimulated with 3p-hpRNA for 6 h. j, 2-DG uptake by C2C12 cells (n = 12-13 WT, n = 9 Ubxn9−/− and n = 10-11 Slc2a4−/− biological replicates) before or after insulin for 20 min (200 nM). k, Proteins in the PM and TCL of C2C12 cells treated with insulin as in (j). l, Protein band ratios of GLUT4 to caveolin in the PM compartment of WT C2C12 cells (n = 3 biological replicates/group from 3 experiments) infected with VSV and EMCV as in (Fig. 4l). m, GLUT4-RIG-I interaction in 3T3-L1 myc-GLUT4-GFP adipocytes before or after insulin (200 nM) for various times. The data are representative of 2-3 independent reproducible experiments. Bar: mean ± SEM. P values determined by one-way ANOVA with Dunnett multiple comparisons test (d, f, j,) or two-way ANOVA with Šidák’s multiple comparisons test (l).
