Fig. 4: Extracellular ammonia does not induce autophagy but inhibits lysosome-mediated protein degradation of LC3B in a dose-dependent manner.

A Depicted cell lines were cultured for three days in media with indicated concentrations of NH₄Cl. Whole-cell lysates were prepared, and Western blot analysis was performed. Ponceau S was used as a loading and blotting control. Molecular markers and densitometry analysis are shown in the Source Data file. B A549 cells were cultured for 24 h in media with the indicated concentrations of NH₄Cl. Then, they were fixed with MeOH, stained by immunohistochemistry, and analysed by fluorescence microscopy. In the merged panel, green represents CD63 while red represents LC3B. Less magnified images including the nucleus are shown in Supplementary Fig. S5. C A549 cells were cultured for 4 h in the indicated presence of 4 mM NH₄Cl, 250 nM pp242, and 25 μM LY294002. Then, the cells were fixed with MeOH, stained by immunohistochemistry, and analysed by fluorescence microscopy. Red squares represent the area shown with increased magnification in the inserts. Yellow arrows highlight classical autophagosomes. D A549 cells were cultured for one day in media with 4 mM NH₄Cl. Where indicated, cells were subjected to 4 h of starvation (HBSS or EBSS) in the continued presence of NH₄Cl. Then, the cells were fixed with MeOH and stained with an antibody targeting WIPI2. The quantification of WIPI2 dots per cell are shown in the figure while representative images used for the quantification are shown in Supplementary Fig. S7. Error bars represent the standard deviation (n > 20). *(p < 0.01, two-tailed Student’s t test). Source data are provided in the Source Data file. E A549 cells were cultured for 5 h in media with the indicated concentrations of NH₄Cl, either with or without bafilomycin (200 nM). The figure depicts relative LC3B-II levels based on quantitative densitometric analysis of Western blots. Error bars represent the standard error of 5 biological replicates. *(p < 0.05, as determined by two-sided Welch’s t test). Source data are provided in the Source Data file.