Fig 2: 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 β-Actin protein levels in NamptOE BMM. β-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

Fig 3: Decreased NAMPT expression attenuates inflammation and bone erosion in collagen-induced arthritis.CIA was induced in 8 weeks old, male DBA/1J Nampt+/+ and Nampt+/− mice. Observable inflammation was observed for 10 weeks. Serum and hind limbs were collected for ELISA and micro-CT analysis, respectively. a Changes in joint inflammation scores in Nampt+/+ (n = 11) and Nampt+/− (n = 13). Arthritic index score is reported as mean ± median absolute deviation. b Serum concentrations of anti-collagen antibody (scale ×100,000) and NAMPT. The results are presented as mean ± SD; Nampt+/+ CIA (n = 3), Nampt+/− CIA (n = 5). c Representative micro-CT images of left hind paws and talus bones. Arrow represents the location of the talus in ankle joint. d Bone volume (BV) and bone volume per total volume (BV/TV) in talus are presented as mean ± SD; Nampt+/+ (n = 5), Nampt+/− (n = 4), Nampt+/+ CIA (n = 6), 7 Nampt+/− CIA (n = 6). *P < 0.05, **P < 0.005

Fig 4: NAMPT expression correlates with tumor grade(A) Analysis of Normal Brain [n = 216], Glioma [n = 577], Pilocytic Astrocytoma [n = 41] and N euroblastoma [n = 45] datasets available through the R2 database indicates that NAMPT is significantly upregulated in tumors derived from the ectoderm, such as pilocytic astrocytoma [*p = 0.01 with ANOVA compared to normal brain] and glioma [*p = 0.01 with ANOVA compared to normal brain]. (B) Analysis of normal brain [n = 23] and glioblastoma [n = 81] datasets from the Sun database available in Oncomine indicates that NAMPT is highly overexpressed in glioblastoma [**p < 0.01 with ANOVA compared to normal brain]. (C) New retrospective analyses of new databases, comparing normal brain GSE13564, GSE11882 [n = 216], Glioblastoma [GSE4290, GSE16011 Glioblastoma series taken together; n = 159], Anaplastic Astrocytoma - glioma grade III [GSE4290, Astrocytoma grade III GSM series; n = 16] and Glioblastoma [n = 9; GSM379855, GSM379856, GSM379857, GSM379858, GSM379870, GSM379871, GSM379872, GSM379873 and GSM379874] cell lines shows that NAMPT is upregulated in the mRNA pools of these cell lines [GSE15209 ****p < 0.0001 with ANOVA compared to normal brain] and that its expression is upregulated at baseline in glioma grade III cells [***p < 0.001 with ANOVA compared to normal brain]. (D–E) The Oncomine glioma dataset shows a correlation between NAMPT expression and tumor grade. (D) Analysis of the Oncomine Glioma dataset stratifying NAMPT according to Glioma grade with Grade II [n = 45] Grade III [n = 31] and grade IV [n = 81] tumors shows a significant difference in NAMPT expression between grade II and grade IV tumors [**p < 0.001]. (E) A second database analyzed in Oncomine strengthens the data previously shown: NAMPT expression correlating with tumor grade: Grade II [n = 50], Grade III [n = 26], Grade IV [n = 77] shows a significant difference in NAMPT expression between grade II and grade IV tumors [**p = 0.0052] (F) Analysis of TCGA Glioblastoma molecular subtypes Proneural, Neural, Classical and Mesenchymal.

Fig 5: Gene expression, Ca2+-homeostasis, and Ca2+-induced mitochondrial swelling are altered by Nampt depletion.A) Principal coordinate analysis (PCoA) of RNA sequencing data from gastrocnemius muscle of WT and SMNKO mice (n = 2–6). B) Upset plot showing overlaps of genes differentially expressed between genotypes at the four ages (n = 2–6). C and D) Expression based on RNA sequencing data of mitochondria permeability transition pore (mPTP) related proteins: Peptidyl-prolyl cis–trans isomerase F (Ppif) (C), and Bcl-2-associated X (Bax) (D), on gastrocnemius muscle of WT and SMNKO mice (n = 2–6). E and F) Protein levels of CYPD (E) and BAX (F) in WT and SMNKO male mice (9–14 weeks of age, n = 8–13). G) Mitochondrial volume measured as basal absorbance at 540 nm in isolated mitochondria from gastrocnemius muscle of WT and SMNKO mice (4–5 weeks of age, n = 7–9). H) Mitochondrial swelling in response to stimulation with a submaximal (100 μM CaCl2) or maximal Ca2+ (400 μM CaCl2) concentration, measured in isolated mitochondria from gastrocnemius muscle of WT and SMNKO mice. A 3-way interaction between treatment, time, and genotype was found (4–5 weeks of age, n = 7–9, p < 0.001). I) Rate of mitochondrial swelling in response to stimulation with a submaximal (100 μM CaCl2) or maximal Ca2+ (400 μM CaCl2) concentration in WT and SMNKO mice, calculated as the slope of the curves in 4H over the entire time course (n = 7–9). J and K) Basal cytosolic Ca2+ levels (J) and ΔCa2+ levels upon addition of Thapsigargin, reflecting Ca2+ replenishment in endoplasmic reticulum (ER) (K) of C2C12 myoblasts transduced with scramble or shRNA against Nampt. The Nampt knockdown cells were treated with vehicle or nicotinamide (NAM) for 24 h before the measurement (n = 3). L and M) Protein levels of cAMP-Responsive Element-Binding Protein (CREB) pS133 (L) and sarcoplasmic and endoplasmic reticulum Ca2+ ATPase 1 (SERCA1) (M) in WT and SMNKO male mice (9–14 weeks of age, n = 8–13). N and O) Expression based on RNA sequencing data of the SERCA1 regulatory proteins: Sarcalumenin (Srl) (N), and Sarcolipin (Sln) (O), in gastrocnemius muscle of WT and SMNKO mice (n = 2–6). P and Q) Ca2+ reuptake measured as the τ value (P) or the high and the low slope (Q) in isolated sarcoplasmic reticulum (SR) vesicles from male WT and SMNKO mice, (13 weeks of age, n = 4). R) Ca2+ release in isolated SR vesicles from male WT and SMNKO mice (13 weeks of age, n = 4). Bar graphs: error bars represent SEM. Box plot: boxes extend from the 25th to 75th percentiles, and the whiskers represent the smallest to the largest value. ∗ Difference to WT/scramble control of the same age/treatment.