Fig. 2

IFN-γ upregulated LAP3 expression in BMECs. a qRT-PCR was used to detect the transcription level of LAP3 in BMECs after treatment with IFN-γ. b ELISA confirmed the content of LAP3 after treatment with IFN-γ in BMECs. c Western blotting was used to test the expression level of LAP3 in BMECs after treatment with 10 ng/mL IFN-γ for 24 h. Full-length blots are presented in Supplementary Fig. S5. d Leucine aminopeptidase activity was used to detect the change of LAP3 enzyme activity in BMECs before and after IFN-γ treatment. e Analysis of intracellular LAP3 expression in BMECs treated with the LAP3 inhibitor, bestatin, for 12 h using qRT-PCR. f HPLC was used to detect the changes of arginine content in BMECs pretreated with 100 mg/mL bestatin for 12 h and stimulated by IFN-γ for 24 h. g Changes in arginine content upon LAP3 overexpression in BMECs with or without IFN-γ treatment, as determined by ELISA. VC, pLenti-CMV-GFP-Puro. pLAP3, pLenti-CMV-LAP3-GFP-Puro. h-i LAP3 expression in normal and malignant BMECs by qRT-PCR (h) and western blotting analysis (i). 0, no IFN-γ treatment, normal BMECs; 30, malignant BMECs after IFN-γ-treated for 30th generations. The relative level of the target protein was estimated using densitometry and the ratio was calculated relative to the GAPDH control. Full-length blots are presented in Supplementary Fig. S6. Bars represent mean values with error bars to represent SD from three independent replicates. Differences between mean values were assessed by two-tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001, compared to control