Extended Data Fig. 3: ENO2 regulates neuroendocrine differentiation, proliferation, and regorafenib sensitivity in CRC cells.
a, Western blot analysis of ENO2, SYP, and TUBB3 in DLD1 and LoVo cells transfected with the ENO2 overexpression plasmid or ENO2 shRNA. Representative of n = 3 independent replicates. b, The mRNA levels of ENO2 and CHGA in CRC cells transfected as indicated were determined by qPCR assay. n = 3 biologically independent experiments. c, Morphological change of CRC cells transfected with the ENO2 overexpression plasmid. White arrows indicated neurite extensions. Scale bar, 100 μm. Representative of n = 3 independent replicates. d, The effect of ENO2 on CRC cell viability was evaluated by CCK8 assay. n = 3 biologically independent experiments. e, The effect of ENO2 on CRC cell proliferation was determined by EdU cell proliferation assay. Scar bar, 100 μm. EdU incorporation was quantified. f. HUVECs were treated with conditioned medium from ENO2-overexpressing or ENO2-silenced DLD1 and LoVo cells, and tube formation assay was performed. Quantification of the number of HUVEC tubular structures is shown. Scale bar, 100 μm. (e, f, representative of n = 3 biological replicates). g, The effect of ENO2 on the viability of DLD1 and LoVo cells treated with regorafenib for 24 h was evaluated by MTT assay. n = 3 biologically independent experiments. h, ENO2 was knocked out by Cas9 system in CRC cells, and Western blotting was conducted to evaluate the expression of ENO2, CHGA, SYP, and TUBB3 in Control and ENO2−/− CRC cells. ENO2−/−, ENO2 knockout. Representative of n = 3 independent replicates. i, CCK8 assay was performed to evaluate the effect of ENO2 on CRC cell viability. j, The viability of Control and ENO2−/− CRC cells treated with regorafenib for 24 h was evaluated by MTT assay. k. Quantification of the number of HUVEC tubular structures after treatment with conditioned medium from Control and ENO2−/− CRC cells is shown. (i–k, n = 3 biologically independent experiments). Data are presented as the mean ± s.e.m. P values were determined by unpaired two-tailed Student’s t-test (b, e-g, ENO2-transfected group; j, k), one-way ANOVA with Tukey’s multiple comparison test (b, e-g, siENO2-transfected group), or one-way ANOVA with Tukey’s multiple comparison test (d and i).
