Fig 2: PTEN mediates miR-19 control of B-cell central tolerance at the immature B-cell stage.(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).

Fig 3: mTORC1 hyper-activation induces PHLPPs translation(A and B) Representative western blots of INS-1E cells (A) and isolated human islets (B) pre-treated with 100 nM rapamycin and cultured with 22.2 mM glucose for 2 (INS-1E) and 3 (human islets) days. n = 3.(C and D) Representative western blots of INS-1E cells transfected with siS6K1 (C), siRaptor (D) or siScr and then exposed to 22.2 mM glucose for 2 days. n = 3(E) Representative western blots of INS-1E cells transfected with active S6K1 or GFP control plasmids for 2 days. n = 3.(F) Experimental strategy of the AHARIBO-based isolation of active polyribosomes and associated RNAs.(G and H) qPCR measurement of PHLPP1 or PHLPP2 mRNA associated with polysomes of INS-1E cells treated with high glucose (G) (n = 3) or of isolated islets from mice fed for 16 weeks with a ND or HFD (H) (n = 2 independent experiments; each pooled from 8–10 mice/condition).Data are expressed as means ± SEM. *p < 0.05 compared with untreated controls.

Fig 4: Genetic inhibition of PHLPP1/2 improved insulin secretion and ß-cell survival in human islets from patients with T2D(A and B) Representative western blots (A) and quantitative densitometry analysis (B) of human isolated islets from non-diabetic controls (n = 9) and patients with T2D (n = 8).(C) Representative images of double immunostaining for PHLPP2 in red and insulin in green of pancreatic autopsy sections from non-diabetic controls (n = 4) and patients with T2D (n = 4; scale bar depicts 10 µm).(D–F) Isolated human islets from non-diabetic individuals (D) and patients with T2D (E and F) were transfected with PHLPP1 and/or PHLPP2 siRNA or control siScr for 2 days. (D and E) Insulin secretion during 1 h of incubation with 2.8 mM (basal) and 16.7 mM (stimulated) glucose, normalized to insulin content (n = 3 controls; n = 5 T2D; each from three independent replicates, respectively). (F) Pooled TUNEL analysis (n = 4; each from three independent replicates, an average of 2,515 ß cells were counted from each treatment condition).(G and H) Representative western blots (G) and quantitative densitometry analysis (H) of human isolated islets from patients with T2D transfected with raptor siRNA or control siScr for 2 days (n = 3).Data are expressed as means ± SEM. *p < 0.05 T2D versus control islets. **p < 0.05 siPHLPP1/2-transfected stimulated versus siPHLPP1/2-transfected basal. #p < 0.05 siPHLPP1/2- or siRaptor-transfected compared with siScr-transfected T2D islets.

Fig 5: PHLPP1/2 inhibits pro-survival AKT and activates pro-apoptotic MST1 signaling in pancreatic ß-cells(A–D) Representative western blots of isolated human islets (A) and INS-1E cells (B) transduced with LacZ control or PHLPP1 and PHLPP2 adenoviruses for 48 h or (C) transfected with PHLPP1 and/or PHLPP2 siRNA or control siScr for 2 days. (D) Representative western blots of islets isolated from WT and PHLPP1-KO mice (A–D: n = 3).(E–N) Schematic cartoons and representative western blots of INS-1E cells overexpressed with adenoviruses for LacZ (control) or PHLPP1/2 and transfected with GFP or siScr (control), Myr-AKT1 (E and F), HA-tagged AKT-S473D (G and H), kinase dead MST1-K59R (I and J), siRNA to MST1 (K and L), or MST1-T387E (M and N) plasmids (all n = 2).(O and P) Representative images of triple staining for TUNEL (black), insulin (green) and DAPI (blue) (O; scale bar depicts 10 µm) and pooled TUNEL analysis (P) of isolated islets from MST1-KO mice and their WT littermates after transduction with adenoviruses for LacZ (control) or PHLPP1/2 (n = 5–6; an average of 15,301 ß cells were counted from each treatment condition).(Q) Pooled TUNEL analysis of isolated human islets overexpressed with adenoviruses for LacZ (control) or PHLPP1/2 and transfected with GFP (control) or Myr-AKT1 or HA-tagged AKT-S473D, or MST1-T387E plasmids (n = 3; an average of 14,034 ß cells were counted from each treatment condition).Data are expressed as means ± SEM. *p < 0.05 compared with LacZ control. **p < 0.05 MST1-KO-PHLPP or PHLPP-AKT-S473D or PHLPP- MST1-T387E compared with WT-PHLPP or PHLPP-GFP.