Fig 1: Expression and secretion of ENO1 in tumor cells and its regulation of lactic acid release. (A) Relative mRNA expression levels of ENO1 were detected by RT-qPCR in different cell lines. β-actin was used as a reference to normalize the data. (B,C) Relative protein expression levels of ENO1 were detected by Western blot. β-actin was used as a reference to normalize the data. (D) Protein secretion levels of ENO1 in cell culture supernatant were detected by ELISA. (E) The transfection efficiency of ENO1 mRNA expression levels were detected by RT-qPCR (48 h). (F,G) The transfection efficiency of ENO1 protein expression levels were detected by Western blot (72 h). (H) The lactic acid concentration was determined in the medium of untreated (control), scrambled siRNA and ENO1-siRNA-transfected CAL27 cells. β-actin was used as a reference to normalize the data. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments.
Fig 2: ENO1 promotes tumor cell migration and invasion through macrophages. (A,C) Wound-healing assay of CAL27 cells incubated with Macro-CM from macrophages induced by TCM of transfected CAL27 cells. (A) representative pictures; (C) bar charts indicating the wound healing rate. Magnification 40×. Scale bar: 500 μm. (B,D,E) Transwell assay for migration (upper panel) and invasion (lower panel) of CAL27 cells cocultured with macrophages induced by TCM of transfected CAL27 cells. (B) representative pictures; (D,E) bar charts indicating the cell numbers of migrated or invasive cells per area. Magnification 200×. Scale bar: 100 μm. (F,H) Wound-healing assay of CAL27 cells incubated with Macro-CM from macrophages induced by TCM without or with rhENO1. (F) representative pictures; (H) bar charts indicating the wound healing rate. Magnification 40×. Scale bar: 500 μm. (G,I,J) Transwell assay for migration (upper panel) and invasion (lower panel) of CAL27 cells cocultured with macrophages induced by TCM without or with rhENO1. (G) representative pictures; (I,J) bar charts indicating the cell numbers of migrated or invasive cells per area. Magnification 200×. Scale bar: 100 μm. Different symbols (circle/square) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; ** p < 0.01 and *** p < 0.001.
Fig 3: IL-6 promotes the migration and invasion of tumor cells. (A) Transwell assay for migration of CAL27 cells (upper chamber) pretreated without or with IL-6R antagonist tocilizumab (Toc, 5 μg/mL) and cocultured with macrophages (lower chamber) for 24 h. Left panel, representative pictures; right panel, bar charts indicating the cell numbers of migrated cells per area. Magnification 200×. Scale bar: 100 μm. (B) Transwell assay for invasion of CAL27 cells (upper chamber) pretreated without or with the IL-6R antagonist tocilizumab (Toc, 5 μg/mL) and cocultured with macrophages (lower chamber) for 48 h. Left panel, representative pictures; right panel, bar charts indicating the cell numbers of invasive cells per area. Magnification 200×. Scale bar: 100 μm. (C) Graphical abstract. ENO1 promotes tumor cell migration, invasion and EMT by orchestrating macrophage-derived IL-6 via secretion of lactic acid and extracellular ENO1 in OSCC, thus forming a positive feedback loop to promote OSCC progression. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; * p < 0.05 and ** p < 0.01.
Fig 4: ENO1 orchestrates IL-6 secretion of macrophages via tumor cell-derived lactic acid. (A,B) CCK8 assay for cell viability of macrophages treated with indicated doses of lactic acid (A) or α-CHC (B) with TCM for 24 h. (C,D) RT-qPCR analysis of the mRNA levels of IL-6 in macrophages treated with 10 mM lactic acid (C) or 1mM α-CHC (D) with TCM for 24 h. (E) ELISA for detection of IL-6 protein levels in CM from macrophages treated with TCM without or with 10 mM lactic acid for 24 h. (F) ELISA for detection of IL-6 protein levels in CM from macrophages treated with TCM without or with 1 mM α-CHC for 24 h (1%DMSO in TCM was used as a solvent control). Different symbols (circle/square) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.
Fig 5: ENO1 promotes epithelial–mesenchymal transition of tumor cells through macrophages. (A) Western blot representative image of E-cadherin, Vimentin and N-cadherin relative protein levels in CAL27 cells incubated with Macro-CM from macrophages induced by untreated (control), scrambled siRNA and ENO1-siRNA-transfected tumor supernatant, respectively, for 48 h. (B) Western blot representative image of E-cadherin, Vimentin and N-cadherin relative protein levels in CAL27 cells cocultured with Macro-CM from macrophage-induced tumor supernatant without or with rhENO1 for 48 h. (C–H) Statistical analysis of Western blot. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; ns, no significance, * p < 0.05 and ** p < 0.01.
Supplier Page from Abcam for Recombinant human ENO1 protein (Active)