Figure 5

CDK6 mRNA is verified as a direct target of ZFP36L1.

(a) Schematic outline of AU-rich elements. (b) Luciferase reporter assays. 293 TN cells were co-transfected with each pGL3-control-based constructs (pGL3-con, VEGFA, CDK6, CDK6-Del) and pcDNA6-ZFP36L1 (or pcDNA6). VEGFA 3′UTR was used as a positive control. Three independent experiments were performed and data are presented as mean ± SD. (c) GFP reporter assay. 293 TN cells were co-transfected with each pll3.7-based constructs (pll3.7, CDK6 and CDK6-Del) and pcDNA6-ZFP36L1 (or pcDNA6).The relative GFP expression was presented as fluorescence pictures (top) and also analyzed by flow cytometry (bottom). (d) The mRNA level of CDK6 was determined in THP-1 cells infected with lenti-ZFP36L1 (or lenti-ctrl) (left) and lenti-shZFP36L1 (or lenti-ctrl) (middle) respectively as well as during the monocytic differentiation of CD34⁺ HSPCs infected with lenti-shZFP36L1 (or lenti-ctrl) (right). (e) The expression of ZFP36L1 and CDK6 was analyzed by western blot in the THP-1 cells that were infected with lenti-ZFP36L1 (or lenti-ctrl) and lenti-shZFP36L1 (or lenti-ctrl) respectively and encountered PMA induction. (f) Western blot analysis of ZFP36L1 and CDK6 expression in the monocytic induction cultures of CD34⁺ HSPCs infected with lenti-shZFP36L1 (or lenti-ctrl). (g) Immunoprecipitation using anti-ZFP36L1 or anti-IgG antibody and extracts of PMA-induced THP-1 cells. ZFP36L1 in immunoprecipitates was analyzed by immunoblot (left: top panel). RNA levels in immunoprecipitates were determined by semi-quantitative PCR (left: bottom panel) and real-time PCR (right panel). The levels of CDK6 and β-actin are presented as fold enrichment in anti-ZFP36L1 relative to anti-IgG immunoprecipitates. *P < 0.05 and **P < 0.01, Student’s t-test.