Phosphor akt

Cell lysis was prepared on ice and subjected to centrifugation at 4°C, 14 000 g/min for 10 minutes. Proteins were quantified using a BCA Protein Assay Kit (Sigma‐Aldrich) and separated by 10% SDS‐PAGE gel. Subsequently, proteins were loaded on a PVDF membrane (Millipore, Billerica, MA), and incubated with specific antibodies. Antibodies were purchased from Abcam (SELM, PI3K, phosphor‐PI3K, mTOR, phosphor‐mTOR, vimentin, N‐cadherin, and β‐cadherin), Cell Signaling Technology (Akt, phosphor‐Akt, MMP2, MMP9, anti‐rabbit, and anti‐mouse secondary antibodies), and Arigo (GAPDH).

To analyze the cell proliferation marker proliferating cell nuclear antigen (PCNA), autophagy marker LC3-II, the expression of AKT/mTOR, and infiltrates of macrophages (F4/80) and nutrophils (Ly-6G) in tumors, BALB/c nude mice were inoculated subcutaneously ASH-3 (10⁷) and intraperitoneal injected Salmonella (2×10⁶ cfu) or PBS on day 0. Then tumor were excised at day 7 and day 14 then washed with PBS, fix with 3.7% formaldehyde, and embedded in paraffin. Tumor tissues were then processed in 5µm sections and stained with phosphor-AKT (Arigo Biolaboratories), phosphor-mTOR (Arigo Biolaboratories), F4/80 (Arigo Biolaboratories), lymphocyte antigen 6 complex locus G6D (Ly-6G; Arigo Biolaboratories), proliferating cell nuclear antigen (PCNA; Arigo Biolaboratories), and LC3-II (Arigo Biolaboratories) were detected by immunohistochemistry.