(C-E) HeLa cells transfected with tfGal3 and either One‐STrEP‐FLAG‐tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G‐Count (D). Then, the percent of GFP+RFP+tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm. 