Dual fluorescent reporter for protein transport. (A) Schematic representation of the reporter based on HeLa TAC-GFP cells. (B and C) Surface and total TAC expressions in HeLa TAC-GFP cells were analyzed by flow cytometry (B) and immunofluorescence microscopy (C). Nuclei were stained with DAPI. Scale bars, 50 µm. (D) HeLa TAC-GFP cells were incubated in presence of 500 ng/ml BFA for the indicated times, after which cells were washed with PBS and detached by incubation in PBS containing 0.5 mM EDTA, and surface and total TAC expressions were analyzed by flow cytometry. (E) HeLa TAC-GFP cells were detached after incubation with 0.5 mM EDTA (Control, Ctr [i]) or with trypsin (Try [ii]), and then cells were plated in complete medium in the absence (iii) or presence (iv) of 500 ng/ml BFA. At indicated times, surface and total TAC expressions were analyzed by flow cytometry. In D and E, surface TAC expression was calculated relative to the total expression as the ratio of the mean of fluorescence intensities and normalized to control sample (mean of n = 3 ± SEM). (F) HeLa TAC-GFP dCas9-KRAB cells were infected with lentivirus to express control sgRNA (Gal4) or sgRNAs targeting the TSS of SCFD1, Sec24A, and Sec61A. 7 d after infection, the knockdown efficiency of targeted genes was monitored by RT-PCR. (G) Surface and total TAC expressions (top panels) and surface/total TAC expression ratio (bottom panels) were analyzed by flow cytometry in control HeLa TAC-GFP dCas9-KRAB cells and in cells depleted for Sec61A, SCFD1, or Sec24A. In bottom panels, lower and upper quartiles are set on control cells and applied to the other experimental conditions. (H) Quantification of the enrichment of cells in the lower quartile compared with the upper quartile as presented in G, bottom panels (mean of n = 4 ± SEM; two-tailed Student’s t test for comparison to control condition sgGal4; **, P < 0.01; ns, statistically nonsignificant).