Figure 1. PKC translocation is involved in radiation-induced bystander effect.

(a,b) PKCα expression in different fractions of bystander A_L cells. Cells were irradiated with a 50-cGy dose and cells on the aluminum-wrapped half of the dish were collected at time points indicated. Membrane fraction proteins were extracted and separated with SDS-PAGE, transferred to PVDF membrane and probed with PKCα antibody (a). Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). In (b), cells were fixed with 4% paraformadehyde, probed with PKCα antibody followed by detection with Alexa Fluor 488 secondary antibody. (c) Effect of PKC inhibitor chelerythrine chloride on the mutagencity of bystander cells. Exponentially growing A_L cells were irradiated as described above. Chelerythrine chloride (10 μM) was added into the cultures 1 hr before irradiation. Data are pooled from four independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between the treated and untreated control (p = 0.03). (d.e) Effect of chelerythrine chloride on ERK activity and COX-2 expression. Proteins from bystander cells were extracted with RIPA buffer at the indicated time points after irradiation and probed with phosphor-ERK1/2 or COX-2 antibody. Left panels, untreated controls; right panels, cells were treated with chelerythrine chloride before irradiation. Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05).