Figure 5.

Apoptosis assay and cell death assessment for investigating the therapeutic mechanism of IR700-12A8-mediated NIR irradiation therapy in vitro. (A) Annexin V-FITC and propidium iodide (PI) staining of GIST-T1 cells treated with vehicle alone, IR700-12A8 alone, or IR700-12A8 + NIR laser (100 J/cm²). The cells were stained 1, 6, 12, and 24 h after irradiation and were analyzed by flow cytometry. (B) Cleaved PARP and Bcl-2 expression in GIST cells treated with IR700-12A8 and NIR laser irradiation (mAb + Laser). The cells were treated with IR700-12A8 and NIR laser irradiation (20 J/cm²) or vehicle alone (Ctrl), and subjected to Western blot analysis for cleaved PARP and Bcl-2. β-actin was used as a loading control. (C) Cell death evaluation of GIST cells treated with IR700-12A8 and NIR laser irradiation. GIST-T1 cells pre-incubated with IR700-12A8 were incubated with vehicle, N-acetyl-l-cysteine (NAC), or sodium azide (NaN₃) for 1 h, and then were irradiated (20 J/cm² or 100 J/cm²). Cell viability was evaluated 24 h later by WST-8 assay and the percentage of dead cells was calculated as 100% minus the percentage of live cells. Data represent mean ± SD (n=3; **p <0.01 by Dunnett's test). (D) The effect of NaN₃ on the expression of cleaved PARP. GIST-T1 cells treated with vehicle (no treatment), IR700-12A8 and NaN₃, IR700-12A8 and NIR irradiation, or IR700-12A8 and NaN₃ and NIR irradiation were subjected to Western blot analysis.