Sublethal Photothermal Stimulation with a Micropulse Laser Induces Heat Shock Protein Expression in ARPE-19 Cells

Figure 3

Changes in the expression of heat shock protein 70 (Hsp70) in cultured cell layers after sublethal photothermal stimulation. (a–c) Micropulse-mode laser irradiation sessions of cells on the glass-based dish were made using the following laser settings: 1000 ms exposure duration and 600 mW laser power. (a) Sublethal laser irradiation led to a transient upregulation of HSPA1A mRNA, which encodes the Hsp70 protein. At the indicated time points, total RNA was extracted, and HSPA1A mRNA levels were assessed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). GAPDH mRNA was used as an internal control. The data presented in the figure were normalized to the amount of HSPA1A mRNA in nonirradiated samples. Data represent mean values of three independent experiments. Error bars represent one standard deviation. The asterisk () indicates statistical significance (), as determined using Student’s -tests. A schematic of the 81 laser irradiation spots on the cultured cell layer is shown in the insert. (b) Three hours following laser irradiation, HSPA1A mRNA upregulation was higher when 477 laser irradiation spots were made than when 81 spots were made. The asterisk () indicates a statistically significant difference between means (, Student’s -test, ). (c) Upregulation of Hsp70 protein at laser irradiation sites 3 h (upper and middle panels) and 24 h (lower panel) after sublethal photothermal stimulation. Cell layers were immunostained with the indicated antibodies and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescent images were captured with a confocal microscope (projected images). Immunofluorescent signals representing Hsp70 expression were detected around laser irradiation sites 24 h after laser irradiation (lower left).

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