## 3 Results
### Intracellular Localization Studies  
Because a net effect on secretion of the FVII variant could only be observed in cells treated with 4-PBA, we next investigated whether the treatment with this compound could have an impact on intracellular trafficking. Cells were stained with markers for different organelles and vesicles and analyzed by immunofluorescence. After treatment with 4-PBA, we observed an increased colocalization with the _trans_-Golgi network marker TGN46 and the vesicle marker COPII, but not with the Golgi marker GM130 (Figure 5A). Analysis of Pearson's correlation coefficient demonstrated a significantly increased colocalization of the rFVII-354V-464 Hs with TGN46 in cells treated with 4-PBA compared to untreated (control) cells, whereas the correlation coefficient for COPII did not reach statistical significance (Figure 5B,C). We also observed an increased but not statistically significant colocalization of FVIIwt with TGN46 and COPII after treatment with 4-PBA (Figure S5). No colocalization with other markers, such as the Golgi reassembly stacking proteins GRASP55 or GRASP65, the endosomal markers Rab-8 or Rab-11 or the autophagosomal marker LC3 II was observed (Figure S6).  
Figure 4: The effect of chaperone treatment on the ER folding machinery. (**A**) Representative Western blot analysis (cropped image adjusted for brightness/contrast) of lysates from cells treated with 4-PBA for the detection of BiP and GRP94. \(\beta\)-actin was used as loading control. (**B**) Densitometric analysis of bands from (**A**), representing BiP or GRP94 normalized to \(\beta\)-actin. (**C**) Representative Western blot analysis (cropped image adjusted for brightness/contrast) of lysates from cells treated with BIX for the detection of BiP and GRP94. \(\beta\)-actin was used as loading control. (**D**) Densitometric analysis of bands from (**C**), representing BiP or GRP94 normalized to \(\beta\)-actin. (**E**,**F**) mRNA levels of sXBP1 and uXBP1 were assessed by SYBR green qRT-PCR. Quantitation was performed using the comparative CT method with 18S as the endogenous control gene and untreated cells as the calibrator. Values are shown as mean \(\pm\) SD of fold change relative to untreated cells from three independent experiments. \(\lx@notemark{e}p\leq 0.0001\), \(\lx@notemark{e}p\leq 0.05\) (one-way ANOVA).  
Figure 5: The effect of 4-PBA treatment on intracellular localization of the rFVII-354V-464 Hs variant. **(A)** Confocal images from cells treated with 4-PBA costained with FVII and membrane organelle markers GM130, COPII or TGN46. Cells with transient expression of rFVII-354V-464 Hs were treated with 4-PBA for 42 h. Confocal images from cells stained with FVII (green, b, e, h) and membrane organelle marker (GM130, COPII or TGN46, red, c, f, i). Colocalized green and red pixels are shown in yellow color (a, d, g). Bar: 5 \(\upmu\)m. **(B)** The colocalization of FVII with TGN46 **(B)** and COPII **(C)** was calculated by [PERSON]’s correlation coefficient in untreated (control) cells and cells treated with 4-PBA. Results are presented as the mean \(\pm\) SD of four independent experiments (\(t\)-test). * \(p\) \(<\) 0.05).