Figure 3.
Figure 3. Identification of new factors for protein transport and secretion. (A) 62 genes with unknown or poorly characterized function that were identified as candidate genes either in the lower (39 genes) or upper quartiles (23 genes) were selected for a secondary screen using a secretion assay based on the detection of ss-HRP. HeLa–ss-HRP cells were plated in a 96-well plate prearrayed with specific smart pool siRNAs. After 3 d, cells were washed and incubated for 8 h with complete medium. HRP secretion was monitored from the supernatant by chemiluminescence and normalized to internal HRP activity (n = 2). HRP quantification from cells transfected with control siRNA (in yellow) and siRNA targeting SCFD1 (in red) was used as negative and positive controls, respectively. Genes selected for further analysis are highlighted in green. Entire data are reported in Table S2. (B) HeLa TAC-GFP cells were transfected with the indicated specific individual siRNA and, after 3 d, knockdown efficiency of targeted genes was monitored by RT-PCR. (C) HeLa TAC-GFP cells were transfected with the indicated specific individual siRNA and after 3 d, surface and total TAC expressions (top panels) and surface/total TAC expression ratio (bottom panels) were analyzed by flow cytometry in living cells. In the bottom panels, lower and upper quartiles are set on control cells and applied to the other experimental conditions. (D) Quantification of the enrichment of cells in the lower quartile compared with the upper quartile as presented in C, bottom panels (mean of n = 4 ± SEM; two-tailed Student’s t test for comparison to control condition siCtr; *, P < 0.05; **, P < 0.01). (E and G) HeLa cells were transfected with the indicated specific individual siRNA and after 3 d, surface MHC-I expression was analyzed on cells fixed with PFA without cell permeabilization by flow cytometry (E) and immunofluorescence microscopy (G; nuclei were stained with DAPI). Scale bars, 20 µm. (F) Quantification of the mean of fluorescence of MHC-I surface expression presented in E. Values were normalized to control sample (mean of n = 4 ± SEM; two-tailed Student’s t test for comparison to control condition siCtr; *, P, < 0.05; **, P < 0.01; ***, P < 0.001).

Identification of new factors for protein transport and secretion. (A) 62 genes with unknown or poorly characterized function that were identified as candidate genes either in the lower (39 genes) or upper quartiles (23 genes) were selected for a secondary screen using a secretion assay based on the detection of ss-HRP. HeLa–ss-HRP cells were plated in a 96-well plate prearrayed with specific smart pool siRNAs. After 3 d, cells were washed and incubated for 8 h with complete medium. HRP secretion was monitored from the supernatant by chemiluminescence and normalized to internal HRP activity (n = 2). HRP quantification from cells transfected with control siRNA (in yellow) and siRNA targeting SCFD1 (in red) was used as negative and positive controls, respectively. Genes selected for further analysis are highlighted in green. Entire data are reported in Table S2. (B) HeLa TAC-GFP cells were transfected with the indicated specific individual siRNA and, after 3 d, knockdown efficiency of targeted genes was monitored by RT-PCR. (C) HeLa TAC-GFP cells were transfected with the indicated specific individual siRNA and after 3 d, surface and total TAC expressions (top panels) and surface/total TAC expression ratio (bottom panels) were analyzed by flow cytometry in living cells. In the bottom panels, lower and upper quartiles are set on control cells and applied to the other experimental conditions. (D) Quantification of the enrichment of cells in the lower quartile compared with the upper quartile as presented in C, bottom panels (mean of n = 4 ± SEM; two-tailed Student’s t test for comparison to control condition siCtr; *, P < 0.05; **, P < 0.01). (E and G) HeLa cells were transfected with the indicated specific individual siRNA and after 3 d, surface MHC-I expression was analyzed on cells fixed with PFA without cell permeabilization by flow cytometry (E) and immunofluorescence microscopy (G; nuclei were stained with DAPI). Scale bars, 20 µm. (F) Quantification of the mean of fluorescence of MHC-I surface expression presented in E. Values were normalized to control sample (mean of n = 4 ± SEM; two-tailed Student’s t test for comparison to control condition siCtr; *, P, < 0.05; **, P < 0.01; ***, P < 0.001).

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