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

Figure 5. ATP6V1C1 is highly expressed in human breast cancer cells, and ATP6V1C1 knockdown inhibits human breast cancer cell proliferation and mTORC1 pathway activation stimulated by amino acids.

Figure 5

A. Western blotting for ATP6v1c1 from representative cell lines examining relative levels of ATP6v1c1 in C3H10T1/2 (immortalized fibroblast-like), 4T1 (mouse mammary cancer), MCF7, MDA-MB-231, and MDA-MB-435s (human breast cancer) cells. B. Quantification of A (n=3). C. Western blot analysis of the efficacy of various shRNA targeting the human ATP6V1C1 sequence in MDA-MB-435s cells. We used tubulin as a protein loading. Lane C, SHC002 as a control; Lane 1, TRCN0000029564; Lane 2, TRCN0000029565; Lane 3, TRCN0000029566; Lane 4, TRCN0000029567; and Lane 5, TRCN0000029568. D. Quantification of ATP6V1C1 protein expression level (normalized to the tubulin level) in cells treated with lentiviruses expressing different shRNA as indicated (n=3). * P<0.05, ** P<0.01, *** P<0.001, compared with that of cells treated with SHC002. We selected SHC002 (lane C) as the Scramble shRNA control. We selected TRCN0000029566 (lane 3) and TRCN0000029568 (lane 5) as shRNA-1 and shRNA-2 respectively. E-G. ATP6V1C1 knockdown in MDA-MB-231 inhibited cell proliferation and mTOR pathway activation stimulated by amino acid stimulation. (E) Representative data of anti-BrdU staining of MDA-MB-231 cells treated with different lentiviruses as indicated after 3 hours incubation with BrdU (Red arrows show the BrdU positive cells. Blue arrows show the BrdU negative cells). (F) Quantification of percentage of BrdU positive cells per view. (n=10). (G) Representative data of p-p70S6K, p-AKT, p-ERK1/2 and ATP6V1C1 expression in MDA-MB-231 cells treated with different lentiviruses with or without amino acid stimulation as indicated by Western blotting.