Figure 2.

TFEB is required for normal osteoclast function in vitro and in vivo. (A) Expression pattern of Tfeb and Ctsk in mouse tissues and cell types by qPCR. (SM) Skeletal muscle; (WAT) white adipose tissue; (Liv) liver; (OSB) osteoblasts; (OCL) osteoclasts. (B) qPCR expression analysis in RAW 264.7 cells stably transfected with empty vector or TFEB-Flag and treated for 72 h with RANKL (30 ng/mL). (C) Resorption assay. RAW 264.7 cells expressing TFEB-Flag or not were treated for 96 h with RANKL (30 ng/mL), and the percentage of resorbed area over total area was quantified. (D) qPCR expression analysis in RAW 264.7 cells transfected with control nontargeting siRNA (Con siRNA) or siRNA targeting Tfeb (Tfeb siRNA) and treated for 72 h with RANKL (30 ng/mL). (E) ChIP assays performed on RAW 264.7 cells transfected with vector or TFEB-Flag using Flag antibodies demonstrate binding of TFEB to indicated genes but not to the coding region of Actin (see also Supplemental Fig. S1D). (F) Bone histomorphometric analysis of lumbar vertebrae in 6-wk-old control and Tfeb_osc^−/− female mice. (BV/TV) Bone volume over tissue volume; (OcS/BS) osteoclast surface over bone surface; (NOc/Tar) number of osteoclasts per tissue area; (ObS/BS) osteoblast surface over bone surface; (NOb/BPm) number of osteoblasts per bone perimeter; (MAR) mineral apposition rate; (BFR/BS) bone formation rate over bone surface. Fasting serum CTx levels are also included. (G) TRAP staining of control and Tfeb_osc^−/− bone marrow-derived osteoclasts (5× magnification). The number of osteoclasts per well and the relative TRAP staining intensity are indicated. (H) Resorption assay. Control and Tfeb_osc^−/− primary monocytes were differentiated into osteoclasts on Osteo assay for 6 d. The percentage of the resorbed area over the total area is indicated. All experiments were performed at least in quadruplicate. Four to six animals of each genotype were analyzed in F.