FIGURE 4.
Pharmacological dissection of autophagosome maturation in CbCln3+/+ and CbCln3Δex7/8/Δex7/8 cells. A, representative confocal images are shown for Rab7 (Anti-Rab7, red) immunostaining of wild type (CbCln3+/+) and homozygous CbCln3Δex7/8/Δex7/8 cells stably expressing GFP-LC3 (green), following DMSO or bafilomycin (1 μm, 24 h), thapsigargin (0.1 μm, 24 h), or combined treatment (1 μm bafilomycin and 0.1 μm thapsigargin, 24 h). Settings for confocal image capture were identical across the entire set of images. Enhanced contrast to images on the independent channels was applied uniformly within a given treatment set, but differed for the treatments (DMSO and Thapsigargin images were identically enhanced, and Bafilomycin and Bafilomycin + Thapsigargin images were identically enhanced). This was necessitated by large changes in the overall signal intensities due to the drug treatments and to ensure staining and degree of signal overlap could be visualized in the representative images shown. Scale bar, 5 μm. Note that bafilomycin treatment sensitized CbCln3+/+ cells to thapsigargin, mimicking the effect of the Cln3 mutation on thapsigargin response. To aid in visualization of the extent of co-localization in some panels, white arrows point to examples of structures with signal overlap. B, Pearson's correlation coefficients for automated co-localization analysis (co-loc2, ImageJ/Fiji) of Rab7-GFP-LC3 signal overlap in wild type and homozygous CbCln3Δex7/8/Δex7/8 cells, treated with DMSO, bafilomycin (Baf, 1 μm, 24 h), thapsigargin (Thap, 0.1 μm, 24 h), or combined treatment (Baf + Thap, 1 μm bafilomycin and 0.1 μm thapsigargin, 24 h), are plotted in the displayed bar graph. Bafilomycin significantly increased the Pearson's correlation coefficient compared with the virtual lack of co-localization observed in the DMSO-treated CbCln3+/+ cells (****, p < 0.0001). Intriguingly, however, bafilomycin did not significantly change the Pearson's correlation coefficient in CbCln3Δex7/8/Δex7/8 cells compared with that determined in DMSO-treated CbCln3Δex7/8/Δex7/8 cells, which was significantly higher than DMSO-treated CbCln3+/+ cells (**, p < 0.01). No co-localization of Rab7 signal with GFP-LC3 was observed in thapsigargin-treated CbCln3+/+ cells or CbCln3Δex7/8/Δex7/8 cells, and bafilomycin + thapsigargin co-treated CbCln3Δex7/8/Δex7/8 cells. Error bars represent S.E. n = mean values from 9 to 12 images per genotype/treatment (∼4–8 cells per image), from a representative experiment. *, p < 0.05; NS, not significant. Statistical significance values shown are from Bonferroni post hoc following two-way ANOVA (p < 0.0001 interaction, p < 0.0001 treatment).