Fig 2: PP2A deletion in LepR+ MSCs inhibits osteogenesis and chondrogenesis but promotes adipogenesis in vitro.MSCs isolated from WT (WT MSCs) and Lepr-cre; Ppp2r1a fl/fl mice (PP2A KO MSCs) were subjected to analysis. a WT and PP2A KO MSCs exhibit no difference in morphology. Scale bar, 50 μm. b PP2A KO MSCs exhibit decreased proliferation. n = 3 independent experiments. c Represented flow cytometry plots of Annexin V-FITC/PI assay for apoptosis analysis of WT and PP2A KO MSCs. After FSC/SSC gating (left), MSCs are categorized into necrosis, late apoptosis, early apoptosis and live stages (right). The corresponding percentage are shown in bar chart. d WT and PP2A KO MSCs were induced for osteogenic differentiation and stained with Alizarin Red S at different time points. Scale bar, 1000 μm. e WT and PP2A KO MSCs were induced for adipogenic differentiation and stained with Oil red O at different time points. f WT and PP2A KO MSCs in micromass were induced for chondrogenic differentiation and stained with Alcian blue & nuclear fast red at different time points. Scale bar in e, f, 100 μm. g Quantification percentage of Alizarin red S staining, Oil red O staining and Alicante blue staining according to different differentiations respectively at each time points. n = 3 independent experiments. h qPCR analysis of transcript levels for genes associated with osteogenesis (Runx2, Alp), adipogenesis (Pparg, Adipoq) and chondrogenesis (Sox9, Col2a1) differentiation in MSCs induced by differentiation medium at each time points. Transcript levels were normalized based on β-actin amplification. n = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 as determined with Student’s t-test. Data are mean ± s.d.

Fig 3: PP2A deletion in LepR+ MSCs increases Runx2 phosphorylation at Ser472 and reduces chondrocyte hypertrophy.MSCs isolated from WT (WT MSCs) and Lepr-cre; Ppp2r1a fl/fl mice (PP2A KO MSCs) were subjected to analysis. a Representative western blot analysis of WT MSCs after osteogenic induction. b Representative western blot analysis of WT MSCs after adipogenic induction. c Western blot analysis of WT and PP2A KO MSCs. d Co-Immunoprecipitation study of cell lysates from WT MSCs with indicated induction for osteogenesis. IP with PP2A-specific or phospho Ser 472 Runx2-specific antibody and precipitates were probed for phospho Ser472 Runx2 and PP2A. e and f IHC analysis of inactive form phospho Ser472 Runx2 expression in hypertrophic chondrocyte of POC (E18.5) and SOC (P15) from Lepr-cre; Ppp2r1a fl/fl mice and relative WT littermate mice. g Quantification of phospho Ser472 Runx2 percentage in hypertrophic chondrocyte of POC (E18.5) and SOC (P15) from Lepr-cre; Ppp2r1a fl/fl mice and relative WT littermate mice. ***p < 0.001 as determined with Student’s t-test. Data are mean ± s.d. h Scheme representing regulation of MSC differentiation by PP2A and its substrates protein. Scale bar, 50 µm.

Fig 4: Knock-down of PPP2CA inhibits aggressiveness of SH-EP-MYCN-ER neuroblastoma (NB) cells, enhanced by MYCN activation. SH-EP-MYCN-ER cells with (+) or without (−) activation of MYCN by 4-hydroxytamoxifen (4-OHT) were treated with doxycycline to induce expression of lentivirally transduced shRNAs. NS, non-silencing control; sh1, PPP2CA-silencing shRNA1; sh2, PPP2CA-silencing shRNA2. (A) Knock-down of PPP2CA and activation of MYCN. Western blotting of PPP2CA and β-actin (left panel). Full-length Western blotting is shown in Supplementary Figure 2A . qRT-PCR of ODC1 normalized to HPRT (right panel). (B) Inhibition of cell growth. Viable, i.e., Hoechst stain-negative, cells were enumerated by flow cytometry. Cell growth as percentage of cells compared to non-silencing control is shown for different time points after induction by doxycycline. (C) Induction of apoptosis. Apoptosis was determined by enumerating hypodiploid propidium iodide-stained nuclei using flow cytometry. Shown is the specific percentage of apoptotic nuclei. (D) Inhibition of clonogenicity. Cells were grown in plastic dishes for 1 week and stained with crystal violet, and colonies were counted. (E) Inhibition of anchorage-independent growth. Cells were grown in soft agar for 1 week and stained with MTT, and colonies were counted. Experiments were repeated three times. Data are expressed as means ± SEM of triplicates and analyzed by two-way ANOVA test. *p < 0.05, ***p < 0.001; ns, not significant.

Fig 5: Deletion of PP2A in LepR+ MSCs inhibited proliferation and hypertrophic differentiation of SOC in early postnatal stage.Sections of P15 distal femur from Lepr-cre; Ppp2r1afl/fl mice were subjected to immunostaining. a–d Immunohistochemistry (IHC) and immunofluorescence reveal LepR+ MSCs express Ppp2r1a and unphosphorylated (Y307) PP2AC in SOC. (a) LepR IHC. b Ppp2r1a IHC. c Double immunofluorescence of LepR and Ppp2r1a. d Unphosphorylated (Y307) PP2AC IHC. e IHC reveals successful deletion of Ppp2r1a in LepR+ MSCs in SOC. IHC reveals that deletion of Ppp2r1a in LepR+ MSCs leads to decreased expression of Ki67 (f) and hypertrophic markers, such as Runx2, Osterix, collagen X, MMP13 and alkaline phosphatase (g), and increased expression of Perilipin (h) in SOC at P15. HZ = Hypertrophic zone; POC = Primary ossification center; ColX =collagen X; ALP = alkaline phosphatase. Scale bar, 100 µm.