Figure 1. Endogenous Ca_V1.2 is expressed during mouse endochondral ossification and in BMSCs, and pharmacological inhibition of Ca_V1.2 channel activity decreases osteoblast differentiation.

(A) LacZ staining of Ca_V1.2^+/lacZ femur at P10 at 5× magnification. (B–E) Boxed regions shown in A indicate the presence of lacZ⁺ cells in the resting (B) and proliferating (C) chondrocytes, in the perichondrium/periosteum (D), and in the lining cells of trabecular bones (E). (F) LacZ staining of Ca_V1.2^+/lacZ tibia at P18, showing lacZ staining in the endosteum. (G) LacZ staining of Ca_V1.2^+/lacZ BMSCs cultured for 6 days on coverslip in α-MEM (without ascorbic acid) plus 15% FBS and 1% penicillin/streptomycin. (H) von Kossa staining of WT BMSCs after 14 days of differentiation in the presence of the L-type Ca²⁺ channel–specific blockers diltiazem (10 μM) or nifedipine (10 μM), n ≥ 3. (I) Expression analysis of osteoblast markers by quantitative PCR of WT BMSCs after 9 days of differentiation in the absence and presence of diltiazem (10 μM). Bar values are normalized means (normalized to H₂O group) ± SD (n = 3, **P < 0.01). Statistical analysis was performed by 2-tailed unpaired t test.