Fig. 2: The 26S proteasome is required for dispersal of the Golgi apparatus.
From: Golgi organization is regulated by proteasomal degradation
a A549 cells treated with LCG or EtOH for 8 h. n = 5900 cells in three independent experiments. GRASP65 (green); Giantin (red); Nuclei (blue). Scale bar = 20 μM (b, c) Golgi size (c) and roundness (d) of cells treated as in a were measured based on GRASP65 staining relative to EtOH, 2 h. n = 3 independent experiments, 6790 cells on average/condition. p = 0.0001 (Dunnet multiple comparisons test). Error bars = SEM. d A549 cells were treated with LCG (200 μM), monensin (2 μM), or control for 6 h and imaged by transmission electron microscopy. e, f HeLa cells expressing shRNA targeting either PSMD6 or luciferase (shLuc) were treated with LCG or EtOH. e Representative images. GM130 (green); Nuclei (blue). Scale bar = 20 μM. f The relative Golgi size compared to control was determined based on GM130 staining. FC: fold change. n = 1687 cells on average/condition. Error bars = SD. **p = 0.0082; ****p < 0.0001 (one way ANOVA with Sidak’s multiple comparison test). g RPMI-8226 cells were treated with cycloheximide (CHX 100 µg/ml) and either LCG (200 µM) or EtOH for the indicated times, and analyzed by WB with the indicated antibodies. Image is representative of three experiments. Provided also as a Source Data file. h HeLa cells were treated with either Monensin (2 µM) or LCG (200 µM) and stained for GM130. Left: representative cell images. The intensity of GM130 per cell was analyzed using Harmony software. n = 500 cells per condition. p < 0.0001 (one way ANOVA). i RPMI-8226 cells were treated with LCG (200 µM), LCG (200 µM) + MG132 (20 µM) or EtOH as control for 4 h, and analyzed by WB with the indicated antibodies. n = 3 experiments. j HeLa cells were fractionated on sucrose cushions and Golgi-enriched fractions were incubated for either 0 or 3 h at 37 °C and supplemented with MG-132 (20 µM) where indicated. Image is representative of three experiments. k Quantification of H. n = 3 independent experiments; Error bars = SD; ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Holm-Sidak’s multiple comparison test). l HeLa cells expressing GalT-YFP were treated with LCG (200 µM) or EtOH for six hours, then washed three times with medium and imaged in 20 min intervals for 4 h.
