Figure 4.

HBHA induces intracellular Ca²⁺ release in RAW 264.7 cells. RAW 264.7 cells were cultured overnight in 12-well plates over a glass coverslip. (a) Intracellular Ca²⁺ measurements were performed with the fluorescent Ca²⁺ indicator Fura-2/AM (5 μM), using the Diaphot inverted fluorescence microscope as described in the Materials and Methods section. Next, HBHA (10 μg/ml) was treated in the presence (normal line) and absence of Ca²⁺ (dashed line) with HBSS solution. Representative recordings of HBHA-evoked cytosolic Ca²⁺ transients were recorded according to the Fura-2 ratio (F340/F380). The colour of the stable cells was green or blue and the colour of cells releasing Ca²⁺ because of HBHA stimulation changed to orange and red. (b) Representative traces of HBHA-induced Ca²⁺ release from ER in RAW 264.7 cells. Cells were incubated with Mag-Fura-2/AM (10 μM) for 1 h and then permeabilised with β-escin (20 μM) for 80–100 s. Representative changes in Ca²⁺ release were recorded according to the Mag-Fura ratio (F340/F380) in RAW 264.7 cells treated with HBHA (10 μg/ml) and PBS using the TILL Photonics imaging system (Universal Imaging, West Chester, PA, USA). (c) Representative traces of mycobacteria-induced Ca²⁺ release from ER in RAW 264.7 cells. Cells in a nominally Ca²⁺-free HBSS solution were infected with live and 5% formaldehyde-fixed bacteria (MOI, 0.5). Cells were incubated with Fura-2/AM (5 μM), and cytosolic Ca²⁺ transients were recorded according to the Fura-2 ratio as in a. (d) Intracellular Ca²⁺ chelation with BAPTA-AM modulates HBHA-induced ER stress responses and caspase activation. To clarify the effects of BAPTA-AM on HBHA-induced ER stress sensor molecules and caspase activation, RAW 264.7 cells were preincubated with BAPTA-AM (10, 20, 30 μM) for 30 min and then stimulated with HBHA (10 μg/ml) for 24 h. Total cell lysate was analysed with western blotting using specific antibodies for each target protein. A cell lysate treated with 2 μg/ml tunicamycin for 6 h was used as a positive control to induce ER stress. The blots were also probed for β-actin to control for loading. Bands corresponding to each protein were quantified, and the intensities of each protein were normalised to the intensity of actin. (e) Intracellular Ca²⁺ chelation with BAPTA-AM modulates HBHA-induced IL-6 and MCP-1 production. Measurement of IL-6 and MCP-1 levels was performed using the CBA flow cytometric assay. Data are representative of at least three independent experiments with similar results. A significant difference was observed in HBHA-stimulated cells compared with unstimulated control cells (*P<0.05, **P<0.01, ***P<0.001)