Figure 1. Loss of CASP2 upregulates endogenous levels of autophagy in unstressed cells. (A) Western blot analysis of LC3 (an increase in LC3-II indicates an increase in autophagosome formation) and SQSTM1 to determine autophagic flux in cell lysates prepared from 4 different batches of WT and casp2^−/− MEFs (loaded in separate lanes 1 to 4) cultured in complete medium in the absence of stressors. The same blots were reprobed with tubulin, α (TUBA) as a loading control. Shown are the representative blots. The experiment was repeated at least 3 times. (B) Densitometry was performed to determine the level of expression using ImageJ analysis. A ratio of expression levels of LC3-II/SQSTM1 as a % of tubulin, α as determined by densitometric analysis demonstrates autophagic flux. Error bar represents ± SEM. Statistical significance was determined by the Student t test. n = 4, **P ≤ 0.01, *P ≤ 0.05. (C and D) The Sqstm1 mRNA levels were determined in the cell lysates prepared from 3 different batches of WT and casp2^−/− MEFs (loaded in separate lanes 1 to 3); Gapdh served as the experimental control. (D) Densitometry was performed to determine the level of expression as % of the control using ImageJ. Error bar represents ± SEM. Statistical significance was determined by the Student t test. n = 3, **P ≤ 0.01, *P ≤ 0.05. (E–H) Cells (WT and casp2^−/− MEFs) were treated with PepA (1 µM) and EST (10 µM) (inhibits lysosomal proteases) for the indicated times to determine autophagic flux. (E and F) Cells were stained with LC3 antibody (green), LysoTracker Red (red, to detect lysosomes) and Hoechst 33258 (blue, to detect nuclei) and analyzed by confocal microscopy. (E) Confocal microscopy analysis of LC3 puncta (determines autophagosomes) and colocalization with LysoTracker Red (stains lysosomes, yellowing due to colocalization of red and green) (determines autolysosomes) in WT and casp2^−/− MEFs. Shown are the representative confocal images (60x with 2 times optical zoom). (F) Quantification of the confocal images for the cells carrying more than 10 LC3 puncta per cell (both autophagosomes and autolysosomes) as a % of the total number of cells counted. At least 150 to 200 cells were counted for each set; n = 3. Error bar represents ± SEM. Statistical significance was determined by performing 2-way ANOVA. **P ≤ 0.01, *P ≤ 0.05. The experiment was repeated at least 3 times. (G) Western blotting for LC3 to detect autophagic flux as described earlier. LC3-II/LC3-I ratios were determined by probing the blot with an antibody specific to LC3, GAPDH was used as loading control. Shown are the representative blots. (H) Densitometric analysis to determine expression levels indicates LC3-II/LC3-I ratio as % of loading control (GAPDH). Error bar represents ± SEM. Statistical significance was determined by the Student t test. **P ≤ 0.01, *P ≤ 0.05. (I) Long-lived protein degradation assay to determine autophagic flux. WT and casp2^−/− MEFs were untreated, treated with EST+PepA or starved (medium was replaced by HBSS containing calcium and magnesium). Long-lived protein degradation % of control was calculated as described in the Materials and Methods section. Error bar represents ± SEM. Statistical significance was determined by performing 2-way ANOVA. n = 4, **P ≤ 0.01, *P ≤ 0.05.