Fig. 7

Prodigiosin blocks autophagy and inhibits cathepsin activity. A HeLa wt cells were treated with DMSO, 100 nM prodigiosin or 10 nM bafilomycin A₁ (BafA₁) in DMEM or EBSS. After 6 h, the cells were lysed and cellular lysates were immunoblotted for the indicated proteins. One representative immunoblot of three independent experiments is shown. LC3: light chain 3; SQSTM1: sequestosome 1. B HeLa wt cells were treated with different concentrations of prodigiosin or DMSO. After 24 h, the cells were lysed and a cathepsin B assay was performed according to the manufacturer´s instructions. 20 µM Z-Phe-Phe-FMK was used as inhibitor control. The fluorescence of duplicates for each treatment of three independent experiments was measured and the mean of the DMSO control was set as 100%. Bars represent the means + SD. C HeLa wt cells were seeded on cover slips. On the next day, cells were treated with DMSO, 100 nM prodigiosin or 10 nM BafA₁ in DMEM or EBSS for 2 h. After treatment, cover slips were prepared for microscopy. Representative sections are depicted. Scale bar: 10 µm. D-G The relative number per cell and mean area of LC3- and LAMP1-positive structures and H the co-localization (Pearson´s coefficient) after Costes thresholding of LC3 and LAMP1 of 15 representative images from three biological replicates for each treatment were quantified using ImageJ 1.53c. p values were determined by ordinary one-way ANOVA with Dunnett´s post hoc test. **** p < 0.0001; ns, non-significant