Autophagy is the primary process for recycling cellular constituents through lysosomal degradation. lines have accelerated turnover of long-lived proteins labeled with 14C-leucine in a pulse-chase assay as additional validation of our screening assays. Data from this comprehensive autophagy screen point toward novel regulatory pathways that might yield new therapeutic targets for neurodegeneration. (knockdown via siRNA transfection, the cytoplasmic intensity of GFP-SQSTM1 decreased by 60% (Fig.?2B). Immunostaining of LAMP2 allowed us to visualize lysosomes and quantify the extent to which GFP-SQSTM1 and LAMP2 colocalized in the cytoplasm using the Pearson correlation coefficient, (Fig.?2C); a comparable approach has been used in primary T cells to quantify autophagic activity.9 Alternative measurements of colocalization were evaluated, including the Manders overlap coefficient10 and the intensity correlation quotient,11 but the Pearson correlation coefficient yielded the highest Z scores with and siRNA controls during assay development; the greater power of the Pearson correlation coefficient in our assay family member to the Manders overlap coefficient is usually consistent with previous findings.12 Knockdown of (ATPase, H+ transporting, lysosomal 38kDa, V0 subunit deb1), a proton pump subunit required for lysosomal acidification,13 produces a phenotype in which GFP-SQSTM1 is loaded into autolysosomes, but is not fully degraded. This is usually evident in Fig.?2C, where knockdown increases GFP-SQSTM1 and LAMP2 colocalization relative to cells transfected with nontargeting siRNA. Alternatively, knockdown of decreases GFP-SQSTM1 and LAMP2 colocalization as expected (Fig.?2C). Physique 2. Review of primary screen. (A) U2OS cells expressing GFP-SQSTM1 (green, top right) were siRNA-transfected for 96?h and subsequently fixed and stained for nuclear DNA (blue, top left), TUBB (red, bottom left), and LAMP2 (orange, bottom right). Hoechst … We transfected cells with a siRNA library covering 12,037 genes, with 4C8 siRNAs per gene. Each siRNA was plated into a individual well. Cells were stained, imaged, and GFP-SQSTM1 mean ring intensity, GFP-SQSTM1 and LAMP2 colocalization, and cell count were calculated for each Rabbit polyclonal to ZNF238 individual well (Fig.?2D). Results were normalized to control wells where 661-19-8 cells were transfected with nontargeting siRNA; results are reported as percent of control (POC). Roughly 8.3% of the 75,342 siRNAs tested reduced GFP-SQSTM1 mean ring intensity by at least 40% relative to controls (dotted green box in Fig.?2D), and these reductions were associated with a range of GFP-SQSTM1 and LAMP2 colocalization phenotypes. For each phenotype measured, siRNA POC values were condensed to gene-level values using a rank-based orthogonal gene averaging (OGA) algorithm (Fig.?2E).14 In the primary screen, 661-19-8 knockdown of 205 genes (1.7%) resulted in significant reductions in GFP-SQSTM1 intensity with OGA values < 0.05, with having the 16th lowest value. OGA values were used to select primary screen hits with low GFP-SQSTM1 intensity, high cell counts, and both low and high GFP-SQSTM1 and LAMP2 661-19-8 colocalization values. Additional genes were selected for follow-up if 50% or more of their siRNAs resulted in favorable phenotypes. In total, 617 genes were selected for follow-up from the primary screen. OGA values for GFP-SQSTM1 mean ring intensity, GFP-SQSTM1 and LAMP2 colocalization, and cell count for these genes are tabulated in Table?S1, along with values from several other measurements collected throughout the screening campaign. GFP-SQSTM1 and LAMP2 colocalization uphit and downhit values are plotted against GFP-SQSTM1 mean ring intensity downhit values for the 617 genes that were re-evaluated in the confirmation screen (Fig.?2F and G, respectively). Whereas cytoplasmic GFP-SQSTM1 intensity decreased upon activation of autophagy via inhibition or knockdown of (Beclin 1, autophagy related), (phosphatidylinositol 3-kinase catalytic subunit type 3), (phosphoinositide-3-kinase regulatory subunit 4), and (autophagy-related 5) (Fig.?2G). Furthermore, whereas knockdown of or significantly increased GFP-SQSTM1 and LAMP2 colocalization, these autophagy-promoting and autophagy-blocking perturbations, respectively, were distinguished by their GFP-SQSTM1 mean ring intensity downhit values (Fig.?2F). Altogether, the combination 661-19-8 of these 661-19-8 phenotypic measurements, GFP-SQSTM1 mean ring intensity and GFP-SQSTM1 and LAMP2 colocalization, highlighted several autophagy-related genes and provided us with valuable information regarding the direction of autophagy regulation induced by RNAi. For confirmation and profiling assays, we selected 6 new siRNAs for each gene. To reduce potential.