Figure 5.

The disruption of the β-tubulin:CCT-β complex using I-Trp evokes capacitative Ca²⁺ entry in HEK-293 cells. (a) I-Trp induces intracellular Ca²⁺ mobilization. HEK-293 cells were pre-incubated with Fluoro-3 (4 μM) for 30 min before the treatment with either I-Trp or IDAM (5 μM of each). The changes in the intracellular fluorescent intensities indicate the increased Ca²⁺ levels and were recorded every 5 s and documented as animation movies (Supplementary Movies) using a confocal microscope. The figures shown represent the alterations in the intracellular Ca²⁺ levels post-treatment with either I-Trp or IDAM at the indicated time courses. (b) The intracellular Ca²⁺ mobilization induced by I-Trp functions via the capacitative Ca²⁺ entry model in HEK-293 cells. The cells were pre-incubated with Fura-2-AM (5 μM) for 30 min, and the intracellular Ca²⁺ levels were subsequently assessed. Next, 5 μM I-Trp (right), 5 μM IDAM (middle) or 1 μM TG (left) was added (arrows) to the cells bathed in Ca²⁺-free focal buffer to detect intracellular Ca²⁺ release after starting the Ca²⁺ level measurements. Calcium chloride (CaCl₂) (100 μM) was then added (arrows) to the external media to determine the extracellular Ca²⁺ influx. The changes in intracellular Ca²⁺ levels are presented with the ratio of Fura-2 fluorescent intensities at 510 nm emitted by dual excitation wavelengths at 340 nm (Ca²⁺-bound) and 380 nm (Ca²⁺-free). (c) The evoked intracellular Ca²⁺ mediates I-Trp-induced apoptosis in HEK-293 cells. The cells were pre-treated with the intracellular Ca²⁺ release inhibitor, DTL, at the indicated doses for 30 min before the treatment with 5 μM I-Trp. Cell apoptosis was assessed 24 h post-treatment. (d) HEK-293 cells were pre-incubated with the cell-permeable Ca²⁺ chelator, BAPTA, at the indicated concentrations for 30 min. Cell apoptosis was then determined 24 h after I-Trp (5 μM) treatment. (e) Cells were pre-treated without or with DTL (50 μM) for 30 min before the treatment without or with I-Trp (5 μM) for 4 h. The mRNA levels of XBP1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were analyzed by RT-PCR using their paired primers. GAPDH was used as an internal control. The results in (c) and (d) are the mean of three independent experiments. The values shown represent the mean±S.D. The symbol ‘*' and ‘***' denote statistical significance at P<0.05 and P<0.001, respectively