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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: TSSC3 promotes autophagy via inactivating the Src-mediated PI3K/Akt/mTOR pathway to suppress tumorigenesis and metastasis in osteosarcoma, and predicts a favorable prognosis

Fig. 3

TSSC3-mediated autophagy contributes to TSSC3-induced inhibition of malignant proliferation of osteosarcoma cells. a Blocking autophagy flux using CQ (8 μM, 24 h) attenuates TSSC3-induced inhibition of cell viability, as determined by a CCK-8 assay, as indicated for 2 days. Cells were plated in 96-well plates at 5 × 103 cells for each well. *P < 0.05, NS: No Significance. b The number of EdU positive cells decreased significantly after transfection with the TSSC3-overexpression plasmid in MTF and SaOS2 cells, but partly recovered after combined treatment with CQ (8 μM, 12 h). *P < 0.05, Scale bars: 50 μm. c Blocking autophagy flux with CQ (5 μM, 4 days) attenuates TSSC3-induced inhibition of colony-formation ability. Cells were plated in 6-well plates at 200 cells per well for MTF cells and 400 cells per well for SaOS2 cells. Photographs of colony formation were taken after 11 days of culture for MTF cells or 12 days of culture for SaOS2 cells. *P < 0.05. d Western blotting analysis showing that ATG5 knockdown blocked TSSC3-induced autophagy. Protein levels on the western blots were quantified by densitometry of ATG5, LC3-II, and P62 are normalized to GAPDH and shown as the fold change relative to the Ctrl groups. Quantification data was in Additional file 3: Figure S2e. e ATG5 knockdown blocked TSSC3-induced inhibition of cell proliferation. The viability of MTF-TSSC3-Scrambled/shATG5–1 or 2 and SaOS2-TSSC3-Scrambled/shATG5–1 or 2 cells was measured using CCK-8 assays. Cells were plated in 96-well plates at 1 × 103 cells per well. *P < 0.05, compared with the TSSC3-Scrambled group. f Photographs of colony formation for the cells mentioned in (e), which were plated in 6-well plates at 200 cells per well and 12 days for MTF cells, or 16 days for SaOS2 cells after plating. These quantitative data show that ATG5 knockdown blocked TSSC3-induced inhibition of the colony-formation ability of osteosarcoma cells. *P < 0.05

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