Figure 5

Constitutive activation of IL7R signaling rescues the cell phenotypes induced by the block of Notch1 signaling pathway in osteoclast progenitors. (A) mRNA expression level of IL7R gene in Notch1-transduced vs control bulk cells as determined by scRNA-seq data. ***p < 0.001 (Student’s t-test). (B) Percentage of UMI counts of CD127 oligo-conjugated antibody in Notch1-transduced vs control bulk cells as determined by Ab-seq assay. ***p < 0.001 (Student’s t-test). (C,D) Flow cytometric analysis of abundance of total cells (C) and CD14⁺RANK^highcell fraction (D) in human CD14⁺CD16⁻ monocytes, isolated from peripheral blood and in vitro RANKL stimulation in presence of recombinant IL7 at the indicated concentration (10 ng/ml and 100 ng/ml) or phosphate-buffered saline (PBS) as mock control. Abundance of total cells (C) and CD14⁺RANK^high cell fraction (D) was tracked over time at the indicated time points by flow cytometry. Alive cells were discriminated using the LIVE/DEAD™ Fixable Near-IR stain. Means ± SD fraction of the initial transduction value are plotted for experiments performed in biological triplicates. **p < 0.01; ***p < 0.001 (Two-way ANOVA with Dunnett’s test, comparing the mock control mean with the other values). (E) Flow cytometric analysis of abundance of total cells after transduction with dnMAM alone (dnMAM) or in combination with IL7R_P2mut (dnMAM + IL7R) construct, harboring the p.Thr244_Ile245insCysProThr mutation to induce constitutive signaling, or empty vector as control. Cell subsets were measured after 3 and 5 days of in vitro RANKL stimulation by flow cytometry. The graphs report the result of two independent experiments performed in biological triplicates. ***p < 0.001 (Student’s t-test). (F) Flow cytometric analysis of cell proliferation by BrdU incorporation in human CD14⁺CD16⁻ monocytes, following transduction with dnMAM alone (dnMAM) or in combination with IL7R_P2mut (dnMAM + IL7R) construct or empty vector as indicated. Cells were measured after 3 days of in vitro RANKL stimulation by flow cytometry. The graphs report the result of two independent experiments performed in biological triplicates. ***p < 0.001 (Student’s t-test). (G,H) Expression level of total and phosphorylated proteins of STAT3 (G) and Akt (H) factors in human CD14⁺CD16⁻ monocytes, following transduction with NOTCH1-ΔE, dnMAM alone (dnMAM) or in combination with IL7R_P2mut (dnMAM + IL7R) construct or empty vector as indicated. Cells were measured after 3 days of in vitro RANKL stimulation by flow cytometry. Each graph reports the ratio of mean fluorescence intensity (MFI) of phosphorylated protein over the MFI of total protein in two independent biological experiments. **p < 0.01; ***p < 0.001 (Student’s t-test). (I) Schematic of the signaling pathways involving NOTCH1 and IL7R in the expansion of osteoclast progenitors at the early stage of human RANKL-induced osteoclastogenesis.