JAGGED1-induced mineralization of HBO cells:

Seven HBO cell lines were treated with growth media alone, osteogenic media alone or with Fc (5.7 μM) or JAG1 (5.7 μM). The cells were half-fed every 5 days. On day 21 cells are fixed with 50% ethanol and thereafter, stained with 1% Alizarin Red S. (B) Alizarin Red S dye was extracted from Alizarin Red S-stained cells using a 1:10 dilution of acetic acid and water, and the absorbance was read at 420 nm. Data represents the mean values of three technical replicates per cell line (mean ± SD, one way ANOVA with Tukey post-hoc).

JAG1 delivery in a PEG hydrogel stimulates bone regeneration in a critical-sized bone defect mouse model:

HBO cells alone or in the presence of JAG1-Dynabeads complex (20 μM) ± DAPT and BMP2 (2.5 µM)+ Fc-Dynabeads were incorporated in 4% PEG-MAL hydrogels and implanted into 4 mm critical-sized defects in the parietal bones of 6-8-week old NOD SCID mice (n = 4 – 6 per HBO-cell donor, 13 – 15 total) as 2 separate doses (Initial dose, Week 4). After 8 weeks, we quantified differences in regenerated bone volume, as seen in B, within the defect and compared them between experimental groups by μCT analysis. μCT reconstructions of defects are shown in A. Data are presented as mean (n = 13 −15) ± SD with p-values reported. C shows representative sections of the defect area on skulls from mice from all experimental groups stained with Masson Trichrome stain.

Transcriptional profiling reveals genes and pathways stimulated by non-canonical NOTCH signaling.

(A) RNAseq reveals clusters of genes associated with the non-canonical NOTCH pathway (rows are z-scored, side color bar identifies up- and down-regulated DEGs stimulated by the non-canonical pathway). (B) Overlapping DEGs in the JAG1 vs no-treatment and JAG1+DAPT vs no-treatment comparisons reveal the non-canonical pathway (DEseq2). (C) Overlapping DEGs from JAG1+DAPT vs no-treatment comparison. (D) Gene ontology over-representation test reveals significantly enriched up- and down-regulated pathways (FDR adjusted).

JAGGED1 induces a non-canonical NOTCH pathway in HBO cells.

HBO cells undergo mineralization through non-canonical pathway. Luminex analysis of lysates obtained from three HBO cell lines untreated or treated with Dynabeads-bound recombinant JAG1-Fc fragment (5.7 μM) ± DAPT (15 μM), a NOTCH canonical pathway inhibitor in a time course manner (5, 10, 15 and 30 minutes), revealed that STAT5, AKT, P38, JNK, NF-κB and p70 S6K phosphorylation was increased by JAG1 + DAPT treatment as shown by A) heatmaps and B) Z-scores plotted on graphs. Each data point represents mean n = 3 ± SD per cell line with p-values reported.

p70 S6K is an essential target during JAGGED1-induced mineralization of HBO cells:

HBO cells were treated with growth media alone, osteogenic media alone or with Fc (5.7 μM), S6K18 (a p70 S6K phosphorylation inhibitor) (50 μM), DAPT (15 μM), JAG1 (5.7 μM) alone or in combination with S6K18 (50 μM) or S6K18 (50 μM) + DAPT (15 μM). The cells were half-fed every 5 days. On day 21 cells are fixed with 50% ethanol and thereafter, stained with 1% Alizarin Red S. (B) Alizarin Red S dye was extracted from stained cells using a 1:10 dilution of acetic acid and water, and the absorbance was read at 420nm. (5.7 μM). Data represents mean n = 3 ± SD per cell line with p-values indicated.