Figure 4

Treatment of kenpaullone or purvalanol A leads in lysosomal biogenesis, independently of cell cycle arrest. (A) Treatment with major CDK inhibitors does not increase cell populations belonging to a particular phase of the cell cycle. HeLa cells were treated with vehicle (DMSO), hydroxyurea (HU), etoposide, nocodazole, kenpaullone, or purvalanol A for 12 h before fixation. Following RNase treatment, cells were stained with propidium iodide and fluorescence intensity was measured by flow cytometry. The percentage of cells in each phase is shown by a stacked bar graph. (B) Major cell cycle inhibitors do not change the red/green ratios of the lysosomal-METRIQ probe. HeLa Tet-On cells expressing the lysosomal-METRIQ probe were cultured with Dox for 48 h followed by treatment with vehicle (DMSO), HU, etoposide, nocodazole, kenpaullone, or purvalanol A for 12 h prior to analysis. The green and red fluorescence intensities were determined by flow cytometry. The red/green fluorescence ratios are shown. The data represent the mean ± SD; *p < 0.05 and **p < 0.01. (C) Treatment with kenpaullone or purvalanol A significantly increases LAMP1-positive structures. HeLa cells were treated with vehicle (DMSO), kenpaullone, purvalanol A, or Torin1 for 12 h before fixation. The cells were stained with antibodies against LAMP1 and analysed by immunofluorescence microscopy. Scale bar, 20 μm. The graph shows the relative ratios of the LAMP1-positive structure areas to the cytoplasmic areas of the cells treated with the indicated compounds (n = 3). The data represent the mean ± SD; *p < 0.05 and **p < 0.01. (D) Endogenous mature cathepsin D in HeLa cells treated with vehicle (DMSO), bafilomycin A₁, brefeldin A, Torin1, kenpaullone, or purvalanol A was analysed by immunoblotting using antibodies against cathepsin D and Hsp90. The mature cathepsin D expression levels were normalised to the HSP90 expression levels (right panel). The data represent the mean ± SD; *p < 0.05. (E) Inhibition of CDKs does not induce nuclear localisation of TFEB. HeLa cells stably expressing TFEB-sfGFP were treated with vehicle (DMSO), kenpaullone, purvalanol A, or Torin1 before fixation and were analysed by immunofluorescence microscopy. Scale bar, 20 μm. (F) Inhibition of CDKs does not decrease mTOR activity. Endogenous phosphorylated 4E-BP in HeLa cells treated with Torin1, kenpaullone, or purvalanol A was analysed by immunoblotting using antibodies against 4E-BP and β-actin (loading control).