Fig 2: Prep1 -/- Embryoid bodies (EBs) express altered levels of various Wnt RNAs during differentiation and Prep1 directly targets the Wnt3 promoter.(A) Deficient regulation of Wnt genes during differentiation of Prep1 -/- EB. RT-qPCR analysis of Wnt mRNA expression. Fold change variations of Wnt3, Wnt3a, Wnt4, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b and Wnt9b RNAs in Prep1 WT v. KO1 ES cells at day 0, day 2 and day 5 of differentiation. (B) Chromatin Immunoprecipitation (ChIP) analysis of the Wnt3 promoter. Region 1 contains the Prep1 binding site, while Region 2 does not (see S3 Fig for details on the sequence). The fold enrichment over input obtained by immunoprecipitation with an anti-Prep1 antibody in WT ES cells is shown for region 1 and region 2. As negative controls we used either IgG with WT ES cells chromatin, or anti-Prep1 antibody in the Prep1 -/- KO1 ES cells chromatin (see Methods section). (C) Immunobloting of total cell lysates from the WT ES cells (WT), Prep1 -/- ES cells transfected with empty vector (KO-EV), Prep1-rescued ES cells (KO-Prep1) before (D0) and after 5 days of differentiation of EB (D5) using an anti-active beta-catenin antibody (anti-ABC). Anti-vinculin antibody was used as loading control. The quantification of the immunobloting is shown in the right panel. Relative units (R.U.) are the band intensities normalized to that of the band of ES WT cells before differentiation.

Fig 3: Prep1 -/- ES cells express decreased levels of Pbx1 and Pbx2 proteins but not of their RNA.WT as well as Prep1 KO1 and Prep1 KO2 ES cells were infected either with an empty vector or with a vector containing Prep1 cDNA and were subjected to immunoblotting analysis. (A) Immunoblotting analysis of Prep1 expression in total protein extracts of WT, Prep1 KO1 and Prep1 KO2 ES cells infected either with an empty vector or with a vector containing Prep1 cDNA. An Actin immunoblot was used as loading control. (B) Immunoblotting analysis of Pbx1a and Pbx1b expression in nuclear and cytoplasmic protein extracts of WT, Prep1 KO1 and Prep1 KO2 ES cells infected either with an empty vector or with a vector containing Prep1 cDNA. Actin and Vinculin (Vinc) immunoblots were used as loading control of nuclear and cytoplasmic extracts, respectively. Notice the absence of Pbx1 in the cytoplasmic extract. (C) Immunoblotting analysis of Pbx2 expression in nuclear and cytoplasmic protein extracts of WT, Prep1 KO1 and Prep1 KO2 ES cells infected either with an empty vector or with a vector containing Prep1 cDNA. Actin and Vinculin (Vinc) immunoblots were used as loading control of nuclear and cytoplasmic extracts, respectively. Notice the absence of Pbx2 in the cytoplasmic extract. (D) Immunoblotting analysis of Pbx3a expression in nuclear and cytoplasmic protein extracts of WT, Prep1 KO1 and Prep1 KO2 ES cells infected either with an empty vector or with a vector containing Prep1 cDNA. Actin and Vinculin (Vinc) immunoblots were used as loading control of nuclear and cytoplasmic extracts, respectively. Notice the presence of Pbx3 in the cytoplasmic extracts. (E) Quantification of Pbx1a, Pbx1b and Pbx2 immunoblots normalized to Actin. (F) RT-qPCR analysis of Pbx1, Pbx2 and Pbx3 mRNAs in WT, Prep1 KO1 and Prep1 KO2 ES cells infected either with an empty vector or with a vector containing Prep1 cDNA.

Fig 4: Prep1 down-regulation affects the expression and phosphorylation of key genes and proteins involved in adipogenic differentiation. (A) Immunoblotting analysis of Ppar?, C/EBPa and Vinculin (for loading control) in total cell lysates from C and P cells 3 days after induction of differentiation. For the induction Ins, Dex and IBMX as single agents in the presence of FCS or in various combinations, as indicated, were used. Full-length blots are presented in Supplementary Fig. S3. (B) Immunoblotting analysis of cell extracts from Prep1 down-regulated (P) and control cells (C) at various time points after induction using anti-phospho-Irs1 (Tyr941) Ab. Dex, Ins and IBMX in the presence of FCS were used to induce differentiation. The arrow shows the size of the phosphorylated form (pIrs1). Vinculin Ab was used to assess loading. Full-length blots are presented in Supplementary Fig. S4. (C) Immunoblotting of cell extracts from P and C cells at various time points after differentiation induction using anti-phospho-Akt (Thr308) Ab. Total Akt and Vinculin Abs were used as controls. Full-length blots are presented in Supplementary Fig. S5. qRT-PCR analyses of Srebf1 (D), Klf4 (F), Klf5 (G) and Cebpd (I) expression in P (solid line) and C (dashed line) cells at different time points after induction. Relative gene expression was normalized to the levels of Gapdh expression. Immunoblotting of cell extracts from P and C cells at various time points after differentiation induction using anti-Pref1 (E) and anti-Klf5 (H) Abs. Vinculin Ab was used as loading control. Full-length blots are presented in Supplementary Figs S7 and S8, respectively.

Fig 5: Prep1 down-regulation in 3T3-L1 cells results in an increased tendency towards adipogenic differentiation. (A) Oil-red O staining of 3T3-L1 control (C) and Prep1 down-regulated (P) cells 6 days after induction of adipogenic differentiation. (B) Quantification of Oil-red O staining by absorbance at 490 nm. UN - undifferentiated (n = 4); Dif (6d) - after 6 days of differentiation (n = 4), *p < 0.01. (C) qRT-PCR analysis of Ppar?1 and Ppar?2 RNA transcripts in 3T3-L1 cells before (-2), 2 (2) and 4 days (4) after adipogenic differentiation. Each graph is representative of 3 independent experiments +/- std. deviation. *p < 0.01. (D) Clustering heatmaps of gene expression changes assayed by RNA-seq from P and C cells before differentiation (-2 days) and control cells 1 day after differentiation (1 day), where n = 704 represents the number of the genes that changed their expression. The logarithm of ratios for each normalized RPKM is shown. Gene ontology (GO) terms of 3T3-L1 genes enriched by both differentiation of control cells (from day -2 to day +1) and by Prep1 down-regulation at day -2 are shown.