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. 2016 Mar 25;7(18):25652–25667. doi: 10.18632/oncotarget.8357

Figure 8. Abolishing the association of Spred2 with LC3 and depletion of LC3 or p62 impedes Spred2-mediated tumor cell death.

Figure 8

(A, B) Cells were infected with adenoviruses expressing Myc-Spred2-WT (AdSpred2-WT), Myc-Spred2-ΔSPR (AdSpred2-ΔSPR), Myc-Spred2-AA (AdSpred2-AA) or vector control (Ad-Vector) at a multiplicity of infection of 250 for 8 h. (A) HeLa cells were cultured in complete medium for 12 days during colony formation assays. The graph represents the number of colonies present in the plate at the end of this time. (B) Cell death was determined by cell counting using trypan blue exclusion staining of HeLa cells. Expression in response to the overexpression of constructs was also examined by IB. (C, D) Cells with a stable knockdown of LC3 (HeLa/shLC3), p62 (HeLa/shp62) or control cells (HeLa/shControl) were infected with adenoviruses expressing Myc-Spred2-WT or vector control at a multiplicity of infection of 250 for 8 h. HeLa/shControl or HeLa/shLC3 cells (C) and HeLa/shControl or HeLa/shp62 (D) were determined by cell counting using trypan blue exclusion staining for cell death analysis. (E, F) Cell lysates of HeLa/shLC3 cells or HeLa/shControl cells (E) and HeLa/shp62 cells or HeLa/shControl cells (F) were analyzed by IB to determine the efficiency of LC3 or p62 knockdown. Data are represented as mean ± S.D from three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).