Extended Data Fig. 3: Phenotype of osteoclast-specific PHGDH knockout mice.

a Phgdh mRNA levels in osteoclasts (oc.), skeletal stem/progenitor cells (SSPCs), chondrocytes (chon.), total bone (tot. bone) and bone marrow cells obtained after flushing (BM flush) from wild-type (Phgdh^oc+) and osteoclast-specific PHGDH knockout (Phgdh^oc-) mice (n = 4; unpaired, two-tailed Student’s t-test). b Immunoblot of PHGDH and β-actin levels in cultured osteoclasts isolated from Phgdh^oc+ and Phgdh^oc- mice (n = 4). Two biological replicates are shown. c, d Body weight (c) and tibia length (d) of Phgdh^oc+ and Phgdh^oc- male mice (n = 9 Phgdh^oc+ and 7 Phgdh^oc- mice). e-k MicroCT-based analysis of trabecular thickness (Tb.Th; e), trabecular number (Tb.N; f), trabecular separation (Tb.Sp; g), endocortical perimeter (Ec.Pm; h), medullary area (Ma.Ar; i), periosteal perimeter (Ps.Pm; j) and cortical porosity (Po; k) in tibiae from Phgdh^oc+ and Phgdh^oc- mice (n = 9 Phgdh^oc+ and 7 Phgdh^oc- mice; unpaired, two-tailed Student’s t-test). l Tnfrsf11a (encoding for RANK), Nfatc1 (P = 0.04), Acp5 (P = 0.046), Ctsk (P = 0.007), Atp6v0d2 (P = 0.0008), Tnfsf11 (encoding for RANKL), Opg and Tnfsf11/Opg mRNA levels in total bone from Phgdh^oc+ and Phgdh^oc- mice (n = 4; unpaired, two-tailed Student’s t-test with *p < 0.05 vs Phgdh^oc+). m Schematic overview of the experimental setup for isolation of BM cells that are enriched for CD45 expression, with gating strategy for flow cytometry analysis of BM-derived myeloid subpopulations. BMMCs is bone marrow mononuclear cells. Created with BioRender.com. n-q Quantification of the number of neutrophils (n), macrophages (o), monocytes (p) and B cells (q) as percent of the CD45-positive BM population (n = 4). r-t Quantification of circulating neutrophils (r), lymphocytes (s) and monocytes (t) in Phgdh^oc+ and Phgdh^oc- mice (n = 3). u Quantification of the number of Gr-1^-/lowCD11b⁺ BMMCs as percentage of the CD45-positive BM population of Phgdh^oc+ and Phgdh^oc- mice (n = 4). v-x H&E staining of the tibial metaphysis (v) with quantification of osteoblast surface per bone surface (Ob.S/B.S; w) and the number of osteoblasts per bone surface (N.Ob/B.S; x) in Phgdh^oc+ and Phgdh^oc- mice (n = 9 Phgdh^oc+ and 7 Phgdh^oc- mice). Scale bar is 100 µm. y-z Calcein staining of the tibial metaphysis (y) with quantification of the bone formation rate (BFR; z) in Phgdh^oc+ and Phgdh^oc- mice (n = 9 Phgdh^oc+ and 7 Phgdh^oc- mice). Scale bar is 100 µm. a′-b′ Serum osteocalcin levels (a′; n = 9 Phgdh^oc+ and 7 Phgdh^oc- mice) and Runx2, Col1a1 and Bglap mRNA levels (b′) in flushed bone from Phgdh^oc+ and Phgdh^oc- mice (n = 4). Data are means ± s.d.

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