Fig 2: DDR-related cytokines derived from glioma cells are synergistic with DDR alterations in vitro and in vivo.(a) Gene expression profiling revealed the associations between DDR-related cytokines and the DDR scores or DDR genes. (b) IHC assays were used to detect the expression level of six cytokines, C5, SAA1, MDK, IL6, VEGFA and TNFSF4 in glioma samples within the different DDR clusters (20×). (c) Western blot validation of DDR-associated protein and cytokine expression levels in LN229, LN229R, HG7, HG7R, GL261 and GL261R cells. (d) ELISA assay revealed differential expression of six cytokines in the medium of LN229, LN229R, HG7, HG7R, GL261 and GL261R cells. (e) Cytokine expression in xenograft gliomas formed by GL261 and GL261R cells was detected by IHC assays (20×).

Fig 3: SAA1 knockdown inhibited arecoline-induced EMT in OSCC cells. RT-qPCR and western blot were used to verify the expression of SAA1 in SAA1-knockdown CAL33 and UM2 cells. B, C, Effect of cell migration (B) and invasion (C) in OSCC cells after SAA1 knockdown. D, E, Effect of cell migration (D) and invasion (E) in arecoline-exposed OSCC cells treated with or without SAA1 knockdown. F, G, Relative mRNA expression levels of EMT-associated molecules in OSCC cells after SAA1 knockdown (F), or in arecoline-exposed OSCC cells treated with or without SAA1 knockdown (G). H, Protein expression levels of EMT-associated molecules in OSCC cells or arecoline-exposed OSCC cells. Data are shown as mean ± SD of 3 independent experiments. *P value < .05, **P value < .01, ***P value < .001. Are, Arecoline

Fig 4: Arecoline enhanced secretion of pro-inflammatory cytokines in OSCC cells. A, Cytokine profiles of CS and CL from CAL33 cells treated with or without arecoline were screened using the Luminex multiplex cytokine assays. B, Most cytokines, such as SAA1, CCL2, S100A8, IL-6 and IL-36G, were significantly increased, while CCL20 was decreased. C, mRNA levels of 6 pro-inflammatory cytokines in OSCC cells were measured using RT-qPCR. Data are shown as mean ± SD of 3 independent experiments. *P value < .05, **P value < .01, ***P value < .001

Fig 5: Arecoline promoted metastasis of OSCC in vivo. CAL33 cells treated with or without arecoline were inoculated into the tongues of nude mice. A, Luminescence was observed in the tongue and cervical LNs using an in vivo imaging system (IVIS) at 3 wk after cell injection. B, Representative photographs of tongue tumors and cervical LNs were shown. C, Expression of SAA1 in metastatic lymph nodules was examined by IHC. D, Representative immunohistochemical images showing the expression of cytokeratin and HLA class I in metastatic lymph nodules