Figure S6.
Live-cell microscopy of tubular-vesicular ERGIC elements and of control cells. (A) βCOPEN-Halo (green) KI HeLa cells were transfected with a plasmid encoding for SNAP-ERGIC53 (magenta). Live cells were stained with the Halo substrate JF571-CA together with the SNAP substrate JFX650-BG to perform dual-color live-cell STED. Yellow arrows highlight COPI-positive clusters associated with tubular-vesicular ERGIC elements highlighted by white arrows. (B and C) ARF1EN-Halo (B, green) and ARF4EN-Halo (C, green) KI HeLa cells were transfected with a plasmid encoding for SNAP-ERGIC53 (magenta) and labeled with the Halo substrate JF552-CA and the SNAP substrate JFX650-BG. White arrows highlight peripheral ERGICs without any ARF, while yellow arrows highlight structures in which ARFs are co-localizing with peripheral ERGICs. (D) ARF4EN-Halo (cyan) and ARF1EN-SNAP (magenta) double KI HeLa cells were transfected with a plasmid encoding for YFP-ERGIC53 (yellow) and labeled with the Halo substrate JF552-CA and the SNAP substrate JFX650-BG. White arrows highlight peripheral ERGICs positive only for ARF4, while yellow arrows highlight peripheral ERGICs positive for both ARF1 and ARF4. (E and F) ARF1EN-Halo KI HeLa cells were stained with the two Halo substrates JF571-CA (green) and JFX650-CA (magenta) used for-dual color live-cell imaging as a control for chromatic aberrations (E). Line profiles in each panel correspond to the dotted boxes in the cropped images. Scatter dot plot with mean and SD represents the quantification of the distances between the two channels in control ARF1EN-Halo cells stained with both dyes (negative control) or between ARF1 and ARF4 nanodomains on peripheral ERGICs (ARF1 vs. ARF4). Examples are shown in Fig. 4 E. ARF1 vs. ARF4 n = 71; negative control n = 56 (F). n = number of ERGICs from at least three independent experiments. Images were deconvolved, background subtracted (A and E), and smoothed with a Gaussian filter (A–E) as described in Materials and methods. Scale bars are: 5 and 1 µm in the cropped images (A); 10 and 2 µm in the cropped images (B–D); 10 µm and 500 nm in the cropped images (E).

Live-cell microscopy of tubular-vesicular ERGIC elements and of control cells. (A) βCOPEN-Halo (green) KI HeLa cells were transfected with a plasmid encoding for SNAP-ERGIC53 (magenta). Live cells were stained with the Halo substrate JF571-CA together with the SNAP substrate JFX650-BG to perform dual-color live-cell STED. Yellow arrows highlight COPI-positive clusters associated with tubular-vesicular ERGIC elements highlighted by white arrows. (B and C) ARF1EN-Halo (B, green) and ARF4EN-Halo (C, green) KI HeLa cells were transfected with a plasmid encoding for SNAP-ERGIC53 (magenta) and labeled with the Halo substrate JF552-CA and the SNAP substrate JFX650-BG. White arrows highlight peripheral ERGICs without any ARF, while yellow arrows highlight structures in which ARFs are co-localizing with peripheral ERGICs. (D) ARF4EN-Halo (cyan) and ARF1EN-SNAP (magenta) double KI HeLa cells were transfected with a plasmid encoding for YFP-ERGIC53 (yellow) and labeled with the Halo substrate JF552-CA and the SNAP substrate JFX650-BG. White arrows highlight peripheral ERGICs positive only for ARF4, while yellow arrows highlight peripheral ERGICs positive for both ARF1 and ARF4. (E and F) ARF1EN-Halo KI HeLa cells were stained with the two Halo substrates JF571-CA (green) and JFX650-CA (magenta) used for-dual color live-cell imaging as a control for chromatic aberrations (E). Line profiles in each panel correspond to the dotted boxes in the cropped images. Scatter dot plot with mean and SD represents the quantification of the distances between the two channels in control ARF1EN-Halo cells stained with both dyes (negative control) or between ARF1 and ARF4 nanodomains on peripheral ERGICs (ARF1 vs. ARF4). Examples are shown in Fig. 4 E. ARF1 vs. ARF4 n = 71; negative control n = 56 (F). n = number of ERGICs from at least three independent experiments. Images were deconvolved, background subtracted (A and E), and smoothed with a Gaussian filter (A–E) as described in Materials and methods. Scale bars are: 5 and 1 µm in the cropped images (A); 10 and 2 µm in the cropped images (B–D); 10 µm and 500 nm in the cropped images (E).

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