Skip to main content
. 2016 Aug 2;7:12207. doi: 10.1038/ncomms12207

Figure 3. PTEN mediates miR-19 control of B-cell central tolerance at the immature B-cell stage.

Figure 3

(a) Western blot analysis of PTEN, Phlpp2 and Bim protein levels in in vitro-cultured immature B cells from ∼7-week-old WT and TG mice. Expression levels were normalized to Actin. (b) Reporter assays of Rluc gene expression containing the Pten or Phlpp2 3′-UTR fragments harbouring predicted miR-19-binding sites at indicated locations. HeLa cells were co-transfected with luciferase reporter and miR-19-expressing or control vector. The Rluc/Luc activity ratio was arbitrarily set as 1 for control vector for each reporter. The miR-19-binding site was mutated in Pten-3′-UTR-mut. (c) Western blot analysis of PTEN and Phlpp2 protein levels in HeLa cells transduced with miR-19-expressing (miR-19) or control lentiviruses (vector). Protein levels were normalized to Actin. (d) Representative flow cytometry plots showing spleen B-cell compartment of recipient mice. Donor genotypes are indicated above each plot. (e) Numbers of splenic B cells in IgMb-macroself mice reconstituted with bone marrow cells from mice of indicated genotypes. (f) Outline of experimental strategy. HSCs enriched from WT donors were transduced with lentiviruses expressing miR-19 and GFP (miR-19) together with lentiviruses expressing Pten and Ametrine (Pten) or Ametrine alone (Control), and used to reconstitute IgMb-macroself mice, which were analysed for splenic B cells 12 weeks later. (g) Representative flow cytometry plots of escaped B cells and bar graphs summarizing the frequencies of GFP+Ametrine− and GFP+Ametrine+ cells among lentivirus-transduced B cells. (h) Numbers of splenic B cells in IgMb-macroself recipient mice. GFP+Ametrine− cells were from HSCs transduced with miR-19-expressing lentiviruses alone, whereas GFP+Ametrine+ cells were from HSCs transduced with miR-19-expressing lentiviruses together with lentiviruses expressing Pten and Ametrine (Pten) or Ametrine alone (Control). Data are representative of 3 (a,g) or 2 (c) or 3 (d) or pooled from 4 (b,e), 2 (c) or 3 (g,h) independent experiments (mean±s.e.m. in a,b,c,e,g,h) with n=3 (CD19Cre), 5 (Ptenfl/+CD19Cre) or 6 (Ptenfl/flCD19Cre) in d,e and n=7 (miR19+Control) or 8 (miR-19+Pten) in g,h. B-cell numbers above the dash line indicates break of tolerance (e,h).