Fig 2: CHC22 is localized at the ERGIC in HeLa GLUT4 and human myotubes. (A and B) Representative SIM of a HeLa-GLUT4 cell (HeLa-G4; A) and the human skeletal muscle cell line hSkMC-AB1190-GLUT4 (hSkMC; B) stained for CHC22 (red) and p115 (blue). The gray circles delineate the nuclei (N). Muscle cell staining with each antibody is shown in black on white below the color images. Scale bars: 10 µm. (C and D) Representative SIM of the perinuclear region of HeLa-GLUT4 cells and hSkMC-AB1190-GLUT4 stained for CHC22 (red), p115 (C), and ERGIC-53 (blue; D). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment for which fluorescence intensities for GLUT4 (green), CHC22 (red), and p115 or ERGIC-53 (blue) were plotted. Arrowheads indicate areas of peak overlap. Scale bars: 1 µm. In A–D, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 or immunostained with anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white.

Fig 3: Depletion of CHC22 or p115, but not GM130, sortilin, or IRAP, disrupts perinuclear targeting of GLUT4. (A) Immunoblotting for CHC22, CHC17, p115, GM130, and ß-actin after transfection of HeLa-GLUT4 cells with siRNA targeting CHC17, CHC22, p115, GM130, sortilin, IRAP, or nontargeting control siRNA (siControl). The position of MW markers is indicated in kilodaltons at the left. (B) Representative IF staining for CHC22 (red) and p115 (blue) in HeLa-GLUT4 cells after siRNA transfection as in A, with GLUT4 detected by GFP tag (green). N, nuclei. Arrows point to a CHC22-depleted cell. Scale bars: 10 µm. (C) Representative IF staining for GM130 (yellow), p115 (red), and CHC22 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting GM130 or with control siRNA, with GLUT4 detected by GFP tag (green). Individual antibody staining is shown in black and white, while the merged image shows all four colors with overlap indicated as below. Scale bars: 25 µm. (D and E) Representative IF staining for GLUT4 (green), CHC22 or sortilin (red), and p115 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting sortilin or IRAP, or with nontargeting control. Scale bars: 5 µm. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white.

Fig 4: HeLa-GLUT4 cells have a functional GLUT4 trafficking pathway that requires CHC22. (A) Representative images of GLUT4 (exofacial HA tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by IF after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GSC. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left: Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle: Quantification of surface-to-total GLUT4 (HA-to-GFP MFI signals). Data expressed as mean ± SEM, n = 3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t test with equal variances: **, P < 0.01. Right: Representative immunoblot for phosphorylated AKT (p-AKT), phosphorylated AS160 (p-AS160), total AS160, and ß-actin in HeLa-GLUT4 cells before and after insulin treatment. The migration position of molecular weight (MW) markers is indicated at the left in kilodaltons. (C) Representative images of total GLUT4 (GFP tag, green) and MHCI (blue) before (basal) or after insulin treatment in HeLa-GLUT4 cells transfected with nontargeting control siRNA (siControl) or siRNA targeting CHC22 (siCHC22). Scale bars: 8 µm. (D) Representative FACS histograms of surface GLUT4 fluorescence intensity (signal from anti-HA labeling) in HeLa-GLUT4 cells transfected with siControl or siRNA siCHC22 before (red) or after (blue) treatment with insulin. Histograms are extracted from the experiment quantified in Fig. 8 A. (E) Representative IF staining for internalized surface-labeled GLUT4 (HA tag, blue) and STX-6 (red) for HeLa-GLUT4 cells at 0, 10, or 30 min after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 7.5 µm. (F) Pearson’s overlap quantification for labeling of STX-6 and HA tag. Data expressed as mean ± SEM, n = 3, 14–19 cells per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test, ****, P < 0.0001. (G) Left: Representative SIM image of a HeLa-GLUT4 cell stained for STX-6 (red). Total GLUT4 (green) was detected by GFP tag. The gray circle delineates the nucleus (N) and the white square delineates the magnified area displayed in the right image. Scale bars: 10 µm; magnified image: 1 µm. Right: The white dashed line in the magnified area spans the segment for which fluorescence intensities for GLUT4 and STX-6 are plotted below, in green and red, respectively. Arrowheads indicate areas of overlap. (H) Representative IF staining for CHC17 (red) and CHC22 (blue) in HeLa-GLUT4 cells transfected with nontargeting siControl or siRNA targeting CHC17 (siCHC17) or siCHC22, with GLUT4 detected by GFP tag (green). Arrows point to a CHC22-depleted cell. Scale bars: 10 µm for siControl and siCHC17 and 7.5 µm for siCHC22. Merged images in A, C, E, G, and H show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white.

Fig 5: GLUT4 pathway markers are variably recruited to L.p.’s replicative vacuole. (A) Representative IF images of single A549 cells from cultures treated with siRNA targeting CHC22 or with nontargeting control siRNA and labeled for Sec22b (red) and CHC22 (green), 1 h after infection with WT L.p. (MOI = 50). Arrows point to L.p. detected with DAPI. Boxed inserts show L.p. region at 5× and 2× magnification for nontargeting control siRNA (siControl) and siCHC22, respectively. Scale bar: 5 µm referring to main images. (B) Quantification of the proportion of L.p. vacuoles staining positive for Sec22b. Data expressed as mean ± SEM, n = 4, 10–20 vacuoles counted per experiment as represented in A. Two-tailed unpaired Student’s t test with equal variances, ***, P < 0.001. (C) Representative images of HeLa cells transiently expressing Fc?RII (needed for L.p. infection; Arasaki and Roy, 2010), infected with WT or mutant ?dotA L.p. (MOI = 50, red) and labeled 1 h after infection with antibodies against Rab1 (green). Hoechst stains the nuclei blue. Arrows point to L.p. Scale bar: 10 µm. (D) Representative images of A549 cells transiently transfected with HA-GLUT4-GFP (green), infected with WT L.p.-expressing mono-DsRed protein (L.p.-DsRed, MOI = 50, red), and labeled 1 h after infection with antibodies against endogenous CHC17 (upper panel) or CHC22 (lower panel; blue). Scale bars: 5 µm. Arrowheads point to L.p. (E) Representative images of HeLa cells transiently expressing Fc?RII, infected with WT or mutant ?dotA L.p. (MOI = 50, red), and labeled 1 h after infection with antibodies against Sec16a (E; green). Hoechst stains the nuclei blue. Arrows point to L.p. Scale bar: 10 µm. (F) Quantification of the proportion of vacuoles staining positive for Sec16a. Data expressed as mean ± SEM, n = 3, 50 vacuoles counted per experiment as represented in E. Merged images in A and D show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white. Merged images in C and E show red/green overlap in yellow.