Figure 2
From: Identification of RNA-binding proteins that partner with Lin28a to regulate Dnmt3a expression
Lin28a regulates Dnmt3a level through direct binding to its mRNA. (a) Expression profile of Dnmt3a and Lin28a was analyzed by western blot at different time points during the transition from ESCs to EpiLCs. Graph represents relative band intensity expressed as Dnmt3a or Lin28a intensity relative to Gapdh (n = 3 biological replicates). *P < 0.05 (Student’s t-test, two tailed). (b) q-PCR analysis to define the expression profile of Dnmt3a mRNA during the establishment of EpiLCs. Expression of ESC (Rex1 and Nanog) and EpiLC (Nanog and Fgf5) markers is reported. The data are presented as fold changes relative to the highest value reached ± SEM of three independent experiments. (c) Schematic representation of the two regions cloned downstream the luciferase reporter and containing possible Lin28 binding sites. (d) Results of the luciferase assay showing the effects of Lin28a overexpression using the two luciferase constructs. After 1 day of EpiLC transition, the cells were co-transfected with Lin28a-Flag expressing plasmid or empty vector (Mock), and Luc-D1 or Luc-D2 constructs. Data on luciferase activity, normalized to Renilla luciferase, are shown as mean ± SEM of fold changes relative to values measured in Mock transfected cells. P value of biological replicates (n ≥ 4) was calculated using Student’s t-test (two tailed). *P < 0.05; ns: not significant. (e) Cells at 2 day of EpiLC transition were transfected with Lin28a-Flag expressing plasmid or the empty vector and were collected at 3 days (EpiLCs) to perform RNA immunoprecipitation analysis. Binding of Lin28a protein to Dnmt3a mRNA was measured by qPCR on reverse-transcribed immunoprecipitated RNAs. Pri-miR-23a, expressed in both ESCs and EpiLCs, was used as negative control. Data are represented as mean fold change ± SEM of the immunoprecipitated RNA in Lin28 overexpressing sample (n = 4 biological replicates). *P < 0.05 (Student’s t-test, two tailed). (f) Western blot analysis of Dnmt3a protein in 3 days EpiLCs upon transfection of Lin28a-Flag or empty vector at 1 day of transition. Western blot analysis with anti-Flag antibody demonstrates the overexpression of Lin28a-Flag protein. Graph represents relative band intensity expressed as Dnmt3a intensity relative to Gapdh (n = 3 biological replicates). *P < 0.05 (Student’s t-test, two tailed). (g) Western blot analysis of Dnmt3a protein level in 3d EpiLCs transfected with non-targeting (si Ctrl) or Lin28a/b targeting siRNAs at 1 day of transition. Western blot analysis with Lin28a antibody shows Lin28 silencing. Graph represents relative band intensity expressed as Dnmt3a or Lin28a intensity relative to Gapdh (n = 3 biological replicates). *P < 0.05 (Student’s t-test, two tailed).
