Fig 2: The designs display potent and specific activity in cell-based assays.A Dose–response curves of NFS-60 proliferation upon 48 h treatment with Filgrastim (rhG-GCSF), Boskar3, or Boskar4. Datapoints and error bars represent mean ± standard deviations of three biologically independent replicates. B Time-dependent proliferation trajectory of surface-immobilised NFS-60 cells over a 10-day treatment. Data points and shades represent mean and standard deviation values of three biologically independent replicates. Experiments were performed three times in triplicates. Data of one representative experiment is shown. C, D Dose- and time-dependent proliferation trajectories over a 5-day treatment of free-floating NFS-60 cells, under the influence of Boskar3 and Boskar4 treatments, respectively. Data points and shades represent mean and standard deviation values of three biologically independent replicates. E G-CSFR-deficient primary stem cells (G-CSFR KO), show abolished proliferative responses to either rhG-CSF or the designs. Experiment was performed twice in triplicates. Data points and shades represent mean and standard deviation values of three biologically independent replicates. F Intracellular levels of phospho-AKT (Thr308), phospho-ERK1/2 (p44/42 MAPK), phospho-STAT3 (Tyr705), and phospho-STAT5 (Tyr694) in CD34+ HSPCs treated with rhG-CSF or the designs (see the “Methods” section). Geometric mean of the expression intensity of each phospho-protein (GeoMean intensity) is shown on the y-axis. The experiment was performed twice.

Fig 3: The JAK2 p.G993A mutation confers resistance to the type-II JAK inhibitor, CHZ-858.a pMIG Empty Vector Ba/F3 cells and Ba/F3 cells expressing either non-mutant or RuxR-mutant (JAK2 p.Y931C, p.L983F, p.G993A) ATF7IP-JAK2 were incubated for 72 h with either a DMSO vehicle control or a dose response of the type-II JAK inhibitor, CHZ-868. The percentage of cell death was measured following a 20 min incubation with apoptotic markers and analysis by flow cytometry. Linear regression or non-linear regression models were fit to appropriate normalized data. Error bars indicate SEM over the mean of three biological replicates. b Ba/F3 cells expressing either non-mutant or JAK2 p.G993A-mutant JAK2 fusion genes were incubated in 1 µM of CHZ-858 for 1 h. STAT5 phosphorylation was assessed by intracellular flow cytometry in comparison to JAK2 Ba/F3 cells starved of IL3 for 5 h. Histograms are representative of three biological replicates and the GFP mean fluorescence intensities (MFIs) are shown.

Fig 4: Sex Hormones and the GH Signaling Network Are Perturbed in GF Mice(A and B) Serum levels of testosterone (A) and E2 (B) in male and female ConvR and GF mice.(C) Circulating GH total secretion, peak number, and levels in male and female ConvR and GF mice.(D) Representative examples of temporal profiles of GH secretion in ConvR♂, GF♂, ConvR♀, and GF♀ animals.(E) Enrichment analysis of FOXA1, FOXA2, BCL6, CUX2, and STAT5 (male and female specific) target genes for differentially expressed genes of indicated contrasts (♂ = GF♂ versus ConvR♂; ♀ = GF♀ versus ConvR♀; ConvR = ConvR♂ versus ConvR♀; GF = GF♂ versus GF♀). Target genes have been identified by published chromatin immunoprecipitation sequencing (ChIP-seq) data.(F) Phosphorylation of STAT5 (Tyr694) in male and female ConvR and GF mice.(G) Hepatic mRNA and protein levels of the other GH-dependent TFs BCL6 and CUX2 that are involved in sex-dimorphic gene expression for the indicated conditions.(H) Experimental design to mimic male-specific GH pulses in GF mice.(I) Volcano plot of expression difference in mRNA expression between GH-injected and vehicle-injected GF male mice show a rescue in sex-biased gene expression.(J) Enrichment analysis of male- and female-specific genes (♂ biased, ♀ biased) or repressed or induced genes in GF mice (down or up in GF♂) for differentially expressed genes of the contrast GH- versus vehicle-injected GF♂ mice.(K) Experimental design of an in vitro system of primary hepatocytes treated with sera of different sources (ConvR♂, ConvR♀, and GF♀).(L) Volcano plot of expression difference in mRNA expression of female hepatocytes treated with GF♀ serum versus ConvR♀ serum. Color code corresponds to (B).(M) Enrichment analysis of male- and female-specific genes (♂ biased, ♀ biased) for differentially expressed genes of the indicated contrasts (serum treatment: ConvR♂ versus ConvR♀ or GF♀ versus ConvR♀) in female hepatocytes.(N) Cartoon depicting the experimental design to evaluate the effect of a tonic GH treatment.(O) Number of differentially expressed genes in function of an adjusted p value threshold for the indicated contrast. GH treatment diminished the number of differentially expressed genes between female hepatocytes treated with GF♀ serum and ConvR♀ serum.(P) Enrichment analysis of male- and female-specific genes (♂ biased, ♀ biased) for differentially expressed genes between GH-treated versus untreated female hepatocytes.All boxplots represent at least 12 independent biological replicates per condition, except for (G), for which at least six biological replicates were used. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. All boxplots are Tukey boxplots.

Fig 5: Ectopic Runx2 inhibits ELF5 and p-STAT5 during lactation. (A) Prolactin receptor (Prlr) mRNA expression at pregnancy (d8P, d17P) and lactation (d1L) in Runx2 and WT glands; data are means ± s.d., normalised to HPRT and relative to WT virgin. (B) ELF5 and p-STAT5 protein levels are not affected in MMTV-Runx2 glands at late pregnancy (day 17) but, during lactation, Runx2 overexpression causes a reduction in levels of ELF5 and p-STAT5 (C). Each image is representative of n≥3 mice. Boxed areas are shown at higher magnification. Scale bars: 10 μm (5 μm in higher-magnification images).