Figure 9.
Differential Golgi phenotype in control and giantin-depleted cells. (A) GRASP65 W-B of the lysate of HeLa cells: treated with a scramble and giantin siRNAs; β-actin was a loading control. Longer exposure for GRASP65-tetramer is presented in the top panel. (B) Quantification of the intensity of bands corresponding to GRASP65: monomer/dimer and tetramer/dimer. Calculations performed within the same exposure, and data represent mean ± SD from three independent experiments; * p < 0.001. (C) Confocal immunofluorescence images of GRASP65 and Golgin-97 or giantin and Golgin-97 in HeLa cells treated with scramble or a mix of GRASP65 and giantin siRNAs; bars, 10 μm. (D) Quantifications of cells with perinuclear Golgi in cells from C; n = 90 cells from three independent experiments, results expressed as a mean ± SD; * p < 0.001. (E) Giantin and GRASP65 W-B of the HeLa cells treated with scramble or mix of GRASP65 and giantin siRNAs; β-actin was a loading control. (F,G) 3D SIM imaging of giantin and GRASP65 in HeLa cells transfected with a scramble and giantin siRNAs, respectively; bars, 1 μm. Red lines indicate the size of intercisternal connections. The representative area of intercisternal connections is highlighted by white boxes and presented on the right side. (H) Statistical analysis of cisternal length, size of intercisternal connections, and their number. Clear separation of observations in control and giantin KD HeLa cells visualized in an XYZ plot where mean and SD were calculated for each parameter (axis) and then used to display an ellipsoid for both conditions. The large ellipsoid represents control data, the small ellipsoid represents giantin KD. Contour (shadow) of ellipsoids presented at Z = 0. Hypothesis testing for different medians was performed via non-parametric Wilcoxon rank-sum test. p-values for length of cisternae, intercisternal distance, and number of connections are 2.2544 × 10−29, 1.2278 × 10−16, 5.2380 × 10−4, respectively, with Wilcoxon.