Fig 1: Knockdown of C1QTNF6 suppresses tumor growth in vivo. A Xenograft models in nu/nu mice were generated using Cal-27 cells transfected with Ctrl-shRNA (n = 10) or C1QTNF6-shRNA (n = 10). B Tumor volume was measured every 5 days for 35 days. C The average weights of excised tumors are shown. Data represent the mean ± SD (n = 10). *P < 0.05, **P < 0.01. D After injection, luminescence was detected in 10 nude mice in each group and representative images of each group were displayed. E Compared with the Ctrl-shRNA group, the fluorescence expression of the C1QTNF6-shRNA group decreased*P < 0.05
Fig 2: Knockdown of C1QTNF6 induces G2/M phase arrest and cell apoptosis in OSCC cell lines. Cell cycle distribution of Ctrl-shRNA and C1QTNF6-shRNA transfected cells was examined using PI staining and flow cytometry. A The representative cell cycle distribution of PI-stained cells. B The average percentage of cells in the G0/G1, S, and G2/M phases is shown. C Representative flow cytometry images of Annexin V-APC-stained cells are shown. D The percentages of apoptotic cells are shown. All statistics are represented as mean ± SD. *P < 0.05, **P < 0.01
Fig 3: C1QTNF6 expression is upragulated in OSCC. A The mRNA expression levels of C1QTNF6 in 13 pairs of human OSCC tissues and the paired normal tissues were detected by qRT-PCR. B Detection of C1QTNF6 expression in human OSCC and paracancerous tissues by immunohistochemical staining. C Detection of C1QTNF6 protein expression in OSCC and paracancerous tissues by western blot. GAPDH was used as a internal control. D The protein level of C1QTNF6 in OSCC was significantly higher than that in paracancerous tissues E The mRNA expression levels of C1QTNF6 in HioEC, Cal-27 and SCC-9 cells were detected by qRT-PCR. All statistics are mean ± SD.*P < 0.05,**P < 0.01, ***P < 0.001
Fig 4: Knockdown of C1QTNF6 inhibits cell proliferation in OSCC cell lines. mRNA (A) and protein (B) levels of C1QTNF6 in CAL-27 and SCC-9 cells transfected with LV-Ctrl-shRNA or LV-C1QTNF6-shRNA. GAPDH was used as internal control. Cell growth was measured by Celigo assay over five days. Fluorescent images are shown for Cal-27 cells (C) and SCC-9 cells (D) (magnification, × 200,scale bar,100 μm) transfected with LV-Ctrl-shRNA (shCtrl) or LV-C1QTNF6-shRNA (shC1QTNF6). The cell growth curves (C&D right panels) show the cell number fold from days 1 to 5. Knockdown of C1QTNF6 inhibits proliferation of Cal-27 (E) and SCC-9 cells (F) as assessed using MTT assay.All statistics are represented as mean ± SD.**P < 0.01, ***P < 0.001
Fig 5: Knockdown of C1QTNF6 activates the Acute Phase Response signaling pathway and targets TNF. A Hierarchical clustering analysis heat map of differentially expressed genes. Red indicates upregulation, and green indicates downregulation; black indicates no obvious difference. B The TNF network showed the relationships of predicted upstream regulatory factor, TNF and down-stream molecules in the data set. C Signaling pathways enriched among differentially expressed genes. The Y-axis represents the − log (10) P value for enrichment, with the threshold drawn at P = 0.05.
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