Fig. 3: Depletion of CLCC1 impairs internal Ca²⁺ release and dosage-dependently reduces [Ca²⁺]_ER.

a–c 293FT cells infected with the indicated shRNAs were loaded with Fura-2 and stimulated with ATP in a calcium-free medium (gray rectangle). Representative Ca²⁺ release traces were averaged from at least 50 cells (a). The knockdown of CLCC1 reduced the amplitude (b) but increased the time-to-peak (c) of ATP-induced Ca²⁺ release. d–f ER Ca²⁺ content was estimated by CPA-induced cytosolic Ca²⁺ rise in the calcium-free medium (gray rectangle) in 293FT cells infected with the indicated shRNAs. Shown are representative traces of CPA-induced calcium leak averaged from at least 50 cells (d) and data summary for the amplitude (e) and time-to-half peak (f) of CPA-induced cytosolic Ca²⁺ rise. g, h Steady-state [Ca²⁺]_ER in 293FT cells infected with the indicated shRNAs was measured by fluorescent signals of ER-GCaMP6-210, a previously reported low-affinity Ca²⁺ probe,³² by FACS. Baseline, 1 mM EGTA + 10 µM ionomycin; Steady, normal medium containing 2 mM Ca²⁺; Max, 10 mM Ca²⁺ + 10 µM ionomycin. The data summary (h) is from three independent experiments. ΔFsteady = (Fsteady – Fbaseline); ΔFmax = (Fmax – Fbaseline). Values are presented as mean ± SD. i, j Steady-state [Ca²⁺]_ER in the cultured CGNs infected with a ratiometric [Ca²⁺]_ER probe (ER-GCaMP6-150) was measured by fluorescent signal ratio of ER-GCaMP6-150 to DsRed (i). Data summary of [Ca²⁺]_ER between the indicated genotypes (j). k, l CLCC1 expression level and its relationships with ER ion homeostasis and morphology. In b, c, e, f, h and j, n > 150 cells pooled from three independent experiments. N.S., no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA or t-test. In i, scale bar, 10 µm.