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. 2017 May 2;8(29):47675–47690. doi: 10.18632/oncotarget.17544

Figure 4. Atp6v1c1 knockdown in the 4T1 cell line inhibits mTORC1 pathway activation stimulated by amino acids by impairing mTOR recruitment to LAMP1+ lysosomes.

Figure 4

A-D. Confocal images of mTOR and lysosomal marker Lamp-1 co-localization in different 4T1 cell lines stimulated by amino acids demonstrating that ATP6v1c1 knockdown impairs mTOR localization with Lamp-1+ lysosomes in response to amino acid stimulation (Green staining is mTOR, red staining is Lamp-1, and yellow staining shows the co-localization of mTOR and Lamp-1). (A) Shows control infected 4T1 cells where amino acid stimulation induces colocalization of mTOR with LAMP1+ lysosomes. (B) ATP6v1c1 knockdown 4T1 cells showing that knockdown impairs colocalization of ATP6v1c1 with LAMP-1+ lysosomes in response to amino acid stimulation. (C) and (D) are ROI figures for A and B, respectively; color coordinated with the boxes indicating the ROI. E. Western blot assessment of ATP6v1c1 modulation of various phosphorylation pathways (p-p70S6K, p-AKT, and p-ERK1/2) in different 4T1 stably infected cell lines with or without amino acid stimulation. F. Quantification of E (n=3). Results are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001. 4T1 cell types are: 4T1 are wild type 4T1 cells, 4T1-LacZ are control vector infected, 4T1-c1s3-1 are ATP6v1c1 knocked down (~90%), 4T1-c1s3-2 are ATP6v1c1 knocked down (~60%).