Knockdown of MISSL affects the localization of ERES and ERGIC components.
A, top panel, HeLa cells were transfected with control siRNA (NC1) or two different siRNAs for MISSL (siMI#3 and siMI#4). After 48 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then immunostained with indicated antibodies. Merged images of Sec31A (green) and ALG-2 (magenta) are also shown (Merge). Insets show magnified images of the region indicated by white squares. Bars, 5 μm. Bottom panel, Mander's correlation coefficient between ALG-2 and Sec31A was graphed as mean ± S.D. (n = 15). B, top panel, HeLa cells were treated as in A and immunostained with Sec16A and γ-tubulin for the ERES and the centrosome, respectively. Magnified images of the region indicated by a white square are also shown. Bars, 5 μm. Bottom panel, individual distances of Sec16A-positive dots from the centrosome were measured from 15 cells for NC1- and siMISSL#4-treated cells and from 14 cells for siMISSL#3-treated cells, and they are represented by box and whisker plots. The boxes denote the 25th to 75th percentile with a line at the median, and whiskers denote the 10th to 90th percentile. *, p < 0.05. C, top panel, HeLa cells were treated as in A. Bar, 10 μm. Bottom panel, cis-Golgi area relative to the cell volume was calculated and represented as mean ± S.D. from four independent experiments. **, p < 0.01. D, HeLa cells were treated as in A and immunostained with an antibody for p230, a trans-Golgi marker. Insets show magnified images of the region indicated by white squares. Bars, 5 μm. E, HeLa cells that were transfected with siRNA for MISSL (siMISSL#4) for 24 h were then transfected with the expression plasmid for GFP or GFP-MISSL for 24 h, treated as in A, and immunostained with anti-Sec16A and anti-GM130 antibodies. Knockdown with siMISSL#4 targets the sequence in the 3′-untranslated region (UTR) of human MISSL and therefore does not affect expressions of GFP-MISSL constructs, which lack 3′UTR. The transfected cells are shown by arrows. Bar, 10 μm. The percentages of the recovery of the perinuclear localization of both Sec16A and GM130 (recovered) or that of either protein (partially recovered) upon expression of GFP, full-length GFP-MISSL (FL), or GFP-MISSL deletion mutants are also shown (GFP, n = 51; GFP-MISSL FL, n = 45; GFP-MISSLΔ1–90, n = 53; GFP-MISSLΔ1–138, n = 43; GFP-MISSLΔ147–245, n = 44; GFP-MISSLΔ93–245, n = 39).