Fig 2: Mean number of osteoblast cells expressing BMP7 on day 7 and day 14 and osteoclasts expressing NFATc1 on day 7 and day 14.

Fig 3: HFSCs are maintained upon PRC2 loss (A) Schematic illustrating expected tdtomato expression in the HF bulge of K15-CrePGR; RosaCAG-tdtomato mice. (B) Schematic showing the experimental strategy to induce K15-CrePGR activity in telogen II (P50) HFs. (C) IF analyses of tdtomato (red) and DAPI (blue) in K15-CrePGR; RosaCAG-tdtomato mice showing efficient targeting of the HF bulge. (D) IF analysis of H3K27me3 (red), CD34 (green) and DAPI (blue) in HFSCs of P70 control and Eed iKO mice. H3K27me3 single channel is shown in gray. Bulge (Bu) and hair germ (HG) regions of the follicle has been outlined in yellow. (E) Fluorescence intensity quantification of H3K27me3 signal (arbitrary units) in control and Eed-null HFSCs P<0.00001, n = 120 cells from 15 HF sections from three independent biological replicates for each group. (F-J) IF analyses of (F) SOX9 (red) and DAPI (blue), (G) NFATC1 (red) and DAPI (blue), (H) activated-CASPASE3 (red), CD34 (green) and DAPI (blue), (I) Ki67 (red), CD34 (green) and DAPI (blue) and (J) LEF1 (red), CD34 (green) and DAPI (blue) in HFs of P70 control and Eed iKO mice. LEF1 single channel is shown in gray. All IF experiments in this figure was conducted on skin sections collected from three animals for each group from two independent litters. Scale bar: 10µm.

Fig 4: Inhibition of Rankl induced osteoclastogenesis in macrophages from Nampt+/- mice.a Bone marrow-derived macrophages from Nampt+/+ and Nampt+/- mice were treated with M-CSF (20 ng/ml) for 2 days. Non-adherent cells were washed out and cells were further cultured with M-CSF (20 ng/ml) and Rankl (100 ng/ml) for an additional 3 days. TRAP staining was performed 3 days later and cells having 3 or more nuclei were counted as osteoclasts. The frequency of osteoclasts is expressed as mean ± SD (N = 5). An average of 7 fields/well were counted per experimental sample. b Reduced osteoclast target gene expression in Nampt-deficient macrophages from Nampt+/- mice. Osteoclast target Nfatc1, Acp5, Dc-stamp, Cathepsin K mRNA expression were measured using semi-quantitative RT-PCR. ß-Actin was used as a loading control. c Representative western blot of Nampt protein in Nampt-deficient macrophages. Quantification of Nampt protein expression in Nampt-deficient macrophages. Representative images from three Nampt+/- mice with Nampt+/+ littermate controls are presented. Quantification of semi-quantitative RT-PCR of osteoclast target genes expressed as mean ± SD (N = 3). *P < 0.05; **P < 0.005

Fig 5: Overexpression of Nampt promotes osteoclast formation.Bone marrow cells were isolated from male DBA/1J Nampt+/+ and NamptOE mice and differentiated into osteoclasts. TRAP+ cells with >3 nuclei were counted as osteoclasts as described in Materials and methods. a Images of TRAP+ cells were captured under a light microscope (magnification, 10×). b Quantification of osteoclasts. c A representative image of western blot analysis of Nampt, Nfatc1, and ß-Actin protein levels in NamptOE BMM. ß-Actin was included as a loading control. d Quantification of Nampt and Nfatc1 protein levels. Data were presented as relative expression levels based on the value of Nampt+/+ wild type (WT) mice as 1. *P < 0.05 vs. Nampt+/+ mice and ***P < 0.001 vs. Nampt+/+ WT mice