Fig. 4: NEAT1 attenuates BMSCs pluripotency by scaffolding SOX2 and CDK2.

A Silver staining of NEAT1 pulldown in undifferentiated BMSCs, left panel. Arrows show different bands between the sense and antisense lanes. List of the top ten differentially expressed proteins identified by mass spectrometry, FDR < 0.05, right panel (n = 3). B Expression levels of NEAT1 detected by qRT-PCR after RIP for SOX2 in BMSCs. C Western blotting showed NEAT1 pulldown of the SOX2/OCT4 complex and CDK2. The red arrow indicates the SOX2/OCT4 complex. D CatRAPID signature module prediction of the RNA-binding propensity for SOX2 protein followed by the prediction of RNA-binding regions. Overall interaction scores above 50% indicate the binding propensity. E CatRAPID fragment module prediction of the interaction profile and matrix between SOX2 and NEAT1. F Interaction between CDK2 and NEAT1. G, H Immunoprecipitation of SOX2 and OCT4 using an anti-CDK2 antibody. SOX2 and OCT4 were reduced in BMSCs transfected with si-NEAT1. By contrast, SOX2 and OCT4 expression increased in BMSCs overexpressing NEAT1. I Immunoprecipitation of CDK2 and Flag-OCT4 using an anti-Myc antibody. CDK2 and Flag-OCT4 were reduced in 293T cells transfected with the mut-SOX2 (Ser249/250 mutation) plasmid. J Western blotting showed NEAT1 pulldown of BMSCs transfected with SOX2 small interfering and mut-SOX2 plasmid. K si-NEAT1 and NEAT1 overexpression downregulated and upregulated ser249/250-phosphorylated SOX2, but not CDK2, respectively. l Interaction among CDK2, SOX2, and OCT4 was identified in BMSCs co-transfected with si-CDK2 and NEAT1 plasmid by coimmunoprecipitation. M The interaction among CDK2, SOX2, and OCT4 was confirmed in BMSCs co-transfected with the CDK2 plasmid and si-NEAT1 by coimmunoprecipitation. The results were presented as means ± S.D. **p < 0.01 by Student’s t test and one-way ANOVA.