FIGURE 5.

Effect of simultaneous depletion of ERj3 and ERj6 on ER lumenal Ca²⁺ in HeLa cells. The FRET-based sensor D1ER was stably expressed in HeLa-D1ER cells and used to image the ER lumenal Ca²⁺ ([Ca²⁺]_ER). The gene silencing was carried out with the indicated siRNAs for 96 h. Just before imaging, cells were loaded with Fura-2 AM to image the cytosolic Ca²⁺ ([Ca²⁺]_cytosol) and incubated in a Ca²⁺-free solution containing EGTA. The Ca²⁺ leakage was unmasked with thapsigargin (1 μm). The time courses of [Ca²⁺]_cytosol (A) and [Ca²⁺]_ER (B) were obtained in the same cells. n = 38 (control siRNA) and 22 (ERJ3 + ERJ6 siRNA). To evaluate the effects of gene silencing, we calculate the Δ[Ca²⁺]_cytosol as (R_peak − R₀)/R₀, where R represents the ratio F₃₄₀/F₃₈₀ of Fura-2 signals (A, inset). The time to 50% decay (τ_½) of [Ca²⁺]_ER was measured in individual cells (B, inset).