Extended Data Fig. 2: ENO1 upregulates YAP1 expression by recruiting YTHDF3.
From: ENO1 promotes liver carcinogenesis through YAP1-dependent arachidonic acid metabolism
a, Co-IP assay in HEK293T cells transfected with Flag-ENO1 or Hep3B-Flag-ENO1 cells. b, Protein levels of eIF5A, YTHDF3, and YAP1 were measured by immunoblotting analysis in Hep3B cells with eIF5A or YTHDF3 knockdown. Actin served as a loading control. Par: parental; NC: negative control. c, Binding of endogenous YAP1 mRNA transcripts by Flag-ENO1 in Hep3B-Flag-ENO1 cells with or without YTHDF3 knockdown (n = 3 independent assays). Protein levels of ENO1, YTHDF3, and YAP1 were measured. Actin served as a loading control. d, HEK293T cells expressing shRNA targeting endogenous ENO1 were further transfected with plasmids to overexpress Flag-tagged wild-type ENO1, ENO1S40A, or ENO1D245R mutants (n = 1 sample detected at different time points). Then the ENO1 enzyme activity of these cells was measured by the consumption of NADH in an assay buffer that includes pyruvate kinase (PK), lactate dehydrogenase (LDHA), and the required substrates including 2-phospho-D-glycerate (2PG), phosphoenolpyruvate (PEP), and pyruvate. The consumption of NADH is monitored as a decrease of absorbance at 340 nm. e, Flag and YAP1 protein levels were measured in HEK293T cells expressing Flag-tagged wild-type ENO1 and indicated ENO1 mutant variants. Actin served as a negative control. f, g, Photographs showed xenografts described in Fig. 1l (f). ENO1 and YAP1 protein levels were measured (g). Actin served as a negative control. Immunoblots are representative of two independent experiments (a-c,e). Error bars denote the mean ± s.e.m. (c,d). Statistical analyses were performed by two-tailed Student’s t-test (c,d).
