A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans
Figure 3
Workflow used to identify changes in CPL-1W32A;Y35A::YFP accumulation after exposure to different RNAi treatments.
(A–C) Synchronized animals were collected in the COPAS Biosort sample cup (A) and passed through a flow cell, where L4 staged animals were gated by a combination of extinction coefficient and time of flight (TOF) (B). A subset of the gated L4 animals was selected on the basis of red fluorescence and TOF (sorted region) through the flow cell (C). (D) Selected animals were dispensed onto NGM plates seeded with E. coli expressing double stranded RNAs. (E) After 48 hours, animals were collected and dispensed into a 384-well optical bottom plate for fluorescence quantification using the ArrayScan VTi automated microscope and analysis system. (F) The number of animals in each well were counted by using the mCherry head marker (red) to identify individual animals while the GFP channel was used to identify the number, intensity and size of the CPL-1W32A;Y35A::YFP accumulations (green). The total area of CPL-1W32A;Y35A::YFP accumulations per worm was calculated by dividing the total area of YFP fluorescence by the total number of mCherry heads identified in each well. Fold-increases values were determined by normalizing to the vector RNAi in order to account for day-to-day variations in transgene expression levels. The experiments were performed in triplicate and displayed as an average of the three trials ± the standard error of the mean (SEM).