Figure 4.

Changes in intracellular [Ca²⁺]_i induced by A. alternata extract had no effect on fibroblast cell death. Control and idiopathic pulmonary fibrosis (IPF) fibroblasts were incubated with Fura‐2‐AM for 1 h and were treated with 100 μg/mL A. alternata extract (lot no. 169626). (A) Changes in intracellular [Ca²⁺]_i obtained from control and IPF fibroblasts in the presence or absence of A. alternata extract stimulation. (B) Corresponding time course tracings of intracellular calcium concentrations in control and IPF fibroblasts in response to A. alternata extract. The black line represents the mean Ca²⁺ response from a total of 60 cells from three control subjects (20 cells/each). The gray shaded region indicates the SEM for each of the mean values that constitute the solid black line. The green trace represents the mean [Ca²⁺] response of 80 fibroblasts obtained from 4 IPF patients (20 cells/each IPF patient) where the cyan shaded area indicates the SEM for the mean values that constitute the solid green line. Same control fibroblasts sensitive to A. alternata extract‐dependent cell death and IPF fibroblasts that are resistant to A. alternata shown in Figure 2B–E were chosen for these experiments. IPF fibroblast that is resistant to A. alternata extract shown in Figure 1 was selected for an additional IPF cell line. (C) Control and IPF fibroblasts (n = 3 each, 1.0 × 10⁴ cells/well) preincubated with calcium chelator, BAPTA‐AM for 0.5 h were stimulated with 400 μg/mL of A. alternata extract (lot no. 276920) for an additional 7 h, and cell viability was measured. (D) Control and IPF fibroblasts (n = 3 each) preincubated with thapsigargin for 1 h were stimulated with 400 μg/mL of A. alternata extract (lot no. 276920) for an additional 7 h, and cell viability was measured. DMSO was used as a vehicle control (VH) for BAPTA‐AM or thapsigargin. Values are the average ± SEM as the percentage against their respective non‐A. alternata extract treatment groups, set at 100%. Same control fibroblasts sensitive to A. alternata extract‐dependent cell death and IPF fibroblasts that are resistant to A. alternata extract shown in Figure 2B–E were chosen for these experiments. Fibroblast viability in the absence of A. alternata extract was measured under the same conditions as a control (Nontreatment).