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. 2015 Feb 7;107(4):dju505. doi: 10.1093/jnci/dju505

Figure 4.

Figure 4.

Interactions between MYCLo-1/2 and RNP binding proteins (HuR and hnRNPK) and their involvement in the regulation of 5’ promoter activities of CDKN1A and CDKN2B. A and B) Schematics (top) of pGL4 luciferase vectors inserted with distal 5’ promoter regions (blue bars) of CDKN1A (A) and CDKN2B (B). Red bars depict proximal promoter regions where MYC binds (MB). Luciferase reporter assays (bottom) representing transcription activity of the distal 5’ promoter regions of CDKN1A (A) and CDKN2B (B) in HCT116 cells transfected with the pGL4 luciferase vectors and siRNAs as indicated. Data are mean ± SD of three independent experiments and each measured in triplicate (**P = .024; *P < .001). C and D) Luciferase reporter assays to identify specific 5’ promoter regions of CDKN1A (C) and CDKN2B (D), where MYCLo-1 and -2 regulate their promoter activities, respectively. Data are mean ± SD of three independent experiments and each measured in triplicate (**P = .027; †P = .014; *P < .001). E and F) RNA pull-down assays showing the interactions between MYCLo-1 and HuR (E), and MYCLo-2 and hnRNPK (F). Total protein in SDS-PAGE gel was analyzed by liquid chromatography–tandem mass spectrometry. Western blot data are representative of two independent experiments. G and H) RIP assays and quantitative real-time polymerase chain reaction detection demonstrating the specific interactions between HuR and MYCLo-1 (G), and hnRNPK and MYCLo-2 (H). Data are mean ± SD of three independent experiments and each measured in triplicate (*P < .001). I and J) Deletion mapping analyses showing the importance of central region of MYCLo-1 (I, 401~690bp) and of marginal regions of MYCLo-2 (J, 71~170bp and 608~658bp) in the interactions with HuR and hnRNPK, respectively. Data are representative of three independent experiments. K and L) ChIP assays showing that HuR binds to the specific 5’ promoter region of CDKN1A, where MYCLo-1 regulates its transcription activity (K) and that hnRNPK binds to the specific 5’ promoter region of CDKN2B, where MYCLo-2 regulates its transcription activity (L). Data are representative of three independent experiments.