SLC6A14 Depletion Contributes to Amino Acid Starvation to Suppress EMT-Induced Metastasis in Gastric Cancer by Perturbing the PI3K/AKT/mTORC1 Pathway

Figure 4

SLC6A14 depletion mediates amino acid starvation to alleviate the activation of the PI3K/AKT/mTORC1 pathway. (a) MKN28-M cells were transfected withLV-pTZU-SLC6A14 plasmid (); 48 h later, differentially expressed genes (DEGs) in SLC6A14-silenced-MKN28-M, compared to MKN28-M cells, were identified using mRNA microarray analysis and were shown in heat map. (b) KEGG pathway analysis was carried out to clarify the top 10 overrepresented canonical pathways that were enriched by these DEGs in MKN28-M cells following LV-pTZU-SLC6A14 transfection. (c–f) Indicated cells were treated with α-MT (5 mM), LV-pTZU-SLC6A14 (), or LV-pcDNA3.1-SLC6A14 () plasmid for 48 h. Western blot analysis was conducted to detect the expression of signaling proteins in the PI3K/AKT pathway in (c) MGC-803, (d) BGC-823, (e) SGC7901-M, and (f) MKN28-M cells, respectively. (g–j) Indicated cells were treated in accordance with the methods as described in (c–f); the expression of mLST8, total, and phospho-Raptor, phospho-PRAS40, phospho-p70S6K, and phospho-4EBP1 was determined by Western blot analysis. For (c–j), the experiments were repeated for three times, and representative results were described as the representatives (upper panels) and the of relative expression (bottom panels). . (k, l) GC cells were stimulated with α-MT (5 mM), LV-pTZU-SLC6A14 (), or LV-pcDNA3.1-SLC6A14 () plasmid for 48 h. (k) ASNS and (l) CHOP mRNA were examined by Quantitative Real-time PCR assay. Data were displayed as the relative expression that were normalized to HPRT1 with the from three irrelevant experiments. .

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