Fig 3. Absence of TRIC-B dysregulates ER Ca²⁺ homeostasis.

(A) Basal levels of [Ca²⁺]_i in control (C) and proband fibroblasts (P1, P2) and osteoblasts (P2). (B) Decreased Ca²⁺ mobilization in TRIC-B deficient fibroblasts (red lines). ATP- and Ionomycin-stimulated Ca²⁺ release, as well as the return to baseline levels, are decreased in proband (P2) cells. (C) Decreased Ca²⁺ mobilization in TRIC-B deficient osteoblasts (red lines). (D) The intracellular Ca²⁺ stores available for IP₃R-mediated release are more rapidly depleted in Proband 1 fibroblasts (right) versus normal control cells (left). ATP-stimulated Ca²⁺ release is abrogated within 10 minutes in proband cells compared to 30 minutes in control cells following inhibition of SERCA channels with thapsigargin (TG). (E) Measurement of ER luminal Ca²⁺ using ER-localized Ca²⁺ indicator. Each point represents the average of one measurement containing 1–7 cells. The difference between the steady-state and Ca²⁺-depleted FRET signal emitted by the D1ER chameleon class Ca²⁺ sensor was equivalent in normal control and proband cells. (F) Quantitative RT-PCR in control (C) and proband (P1, P2, P3) cells. There is no significant difference in expression levels of SERCA2 (ATP2A2) and IP3R1 (ITPR1) in fibroblasts, but transcripts are significantly reduced in proband (P2) osteoblasts. (G) Immunoblots of control (C) and proband (P1, P2, P3) cell lysates demonstrate equivalent levels of SERCA2b and IP3R1 Ca²⁺ channels. *, p < 0.05; **, p < 0.01.