Fig. 4. Gemin4 regulates COPI subunit expression, proliferation and Golgi morphology.

A LNCaP cells were treated with shRNA targeting GEMIN4 for 48 h. Protein expression levels were assessed by western blotting. Actin was used as loading control. B LNCaP cells were transiently transfected with a Gemin4 expression construct and protein expression levels were assessed by western blotting. Actin was used as a loading control. C Cell proliferation in Gemin4 knockdown and overexpressing LNCaP cells was compared to the respective control: control shRNA and EGFP. Cells were grown for 48 h in media supplemented with 10% charcoal-stripped serum. Mean ± SD, N = 3 independent experiments with 3 technical repeats; **P = 0.0064, ns = 0.72. D Quantification of Golgi area (TGN46-postive) from confocal images (shown in E, F) in Gemin4 knockdown and overexpressing LNCaP cells. Mean ± SD. Twenty-two knockdown cells and 20 overexpressing cells were analysed per group. **P = 0.0030, ns = 0.24. E, F Images showing maximum intensity z-projections of confocal images of LNCaP cells transiently expressing EGFP-tagged shRNA or a Gemin4 expression constructs. Cell were stained with a TGN46 antibody (magenta). Scale bar, 25 µm. G Bar graph showing proliferation of LNCaP cells treated with 25 µM STO-609 for 120 h normalised to the DMSO control. Three independent experiments with three technical repeats were analysed. Mean ± SD; ***P = 0.0002. H LNCaP cells were treated with 25 µM STO-609 for 120 h. Protein expression of COPI components and Gemins were assessed by western blotting of 25 µg cell extracts separated on a SDS-PAGE gel. Actin was used as a loading control. Densitometry was performed on two experiments and normalised to control. Mean ± SD. I Bar graph showing the impact of 25 µM STO-609 treatment for 120 h in LNCaP cells on Golgi area (TGN46-positive) compared to CaMKK2 knockdown (shRNA2) cells. Mean ± SD. N = 31 cells; ****P < 0.0001.