Inhibition of autophagy by Spautin-1 prevents cell death and doubles the clonogenic growth.

HEK293 cells were transfected with either the empty vector pcDNA3, NK₁R and exposed or not to SP, or IGF1R as indicated. Either Spautin-1 or vehicle DMSO were added as indicated. Detached cells were collected from the media and cell death was determined by trypan blue exclusion 24 hr after cell death induction. Bars represent standard error, (n = 3, * p<0.05). B, bright field image of the cells that remained attached from the same cultures as in A, that were taken for clonogenic growth assays. C, Spautin-1 increases the clonogenic growth of cells expressing NK₁R and exposed to SP, or expressing IGF1R compared with vehicle. Equal number of cells from cultures shown in B was seeded, and the number of colonies containing more than 10 cells was scored (details in methods section). The plating efficiency was calculated for control cells and included in the calculation of the survival fraction. D, Spautin- 1 promotes the degradation of both Beclin 1 and PI3K-III. Western blot of total protein extracts taken at the indicated times.