Depletion of genes encoding various proteasome subunits decreases proteasomal activity in vivo. A) Control cells expressing the proteasome degradation reporter GFP-CL1 are similarly sized as neighboring non-GFP control fat body cells and show diffuse fluorescence. Cell nuclei are labeled with DAPI (blue). B)Rpn2 RNAi cells are smaller than control cells and accumulate large aggregates of GFP-CL1. C) Proteasome RNAi leads to accumulation of GFP-CL1 aggregates relative to control cells shown in panel A. Statistically significant differences are marked by asterisks (Kruskal-Wallis test, n = 5-6 per genotype, ** P < 0.01, * P < 0.05), and error bars denote standard error. The different types of subunits are indicated as 20S core subunits (α and β), and 19S regulatory particle subunits (ATPase and non-ATPase). Pomp is required for assembly of the 20S core. D) Schematic of the clonal expression system. GFP-positive cells of interest are surrounded by wild-type cells in the same tissue of mosaic animals, serving as an internal control in various staining experiments. E) Depletion of Rpn2 leads to accumulation of ubiquitinated protein aggregates in Lamp1-GFP marked fat body cell clones. Note that the numerous small Lamp1-GFP dots representing lysosomes are not ubiquitin-positive. F) Similarly, silencing of Rpn2 leads to cell-autonomous accumulation of p62 aggregates in Lamp1-GFP marked fat body cell clones. Note that Lamp1-GFP dots are not p62-positive either. G) Proteasome RNAi leads to accumulation of endogenous p62 aggregates relative to control cells. n = 5-6 per genotype. Statistically significant differences are marked by asterisks (Kruskal-Wallis test, n = 5-6 per genotype, ** P < 0.01, * P < 0.05), and error bars denote standard error. Boxed areas in E and F are shown enlarged. Scale bar in A equals 20 μm for A, B, E, F.
Lőw et al. BMC Cell Biology 2013 14:29 doi:10.1186/1471-2121-14-29