Fig. 1.

Subtilase cytotoxin induces SG formation through a PERK-dependent pathway.

A. HeLa cells were incubated with catalytically inactive SubA_S272AB or SubAB (400 ng ml⁻¹) for the indicated times at 37°C, and cell lysates were subjected to immunoblotting with the indicated antibodies. α-tubulin served as a loading control.

B. After cells were treated with SubAB for the indicated times at 37°C, the fixed cells were reacted with anti-TIAR antibody (red) and anti-eIF4γ antibody (green) and observed by confocal microscopy. A merged picture shows colocalization in HeLa cells. Cell nuclei were stained by DAPI (cyan). The rate of SG formation is presented as mean ± standard deviation (SD) from four different fields, which included at least 20 cells/field (right panel). Bars represent 50 μm. Experiments were repeated two times with similar results, and significance is *P < 0.05. The white arrows indicate SG, including TIAR and eIF4γ.

C. The cells were incubated with 400 ng ml⁻¹ of SubAB or SubA_S272AB for 3 h at 37°C. The fixed cells were reacted with anti-TIAR antibody (red) observed by confocal microscopy. The arrows indicate SG formation, including TIAR. Bars represent 20 μm. Experiments were repeated three times with similar results.

D. Control (NC) and PERK siRNA-transfected cells were incubated with SubA_S272AB (mt) or SubAB (wt) for 3 h at 37°C. Cell lysates were subjected to immunoblotting with the indicated antibodies. GAPDH served as a loading control. Cells were fixed, immunostained with the indicated antibodies and observed by confocal microscopy as described in Fig. 1. The rate of SG formation is presented as mean ± SD from four different fields, which included at least 20 cells/field (right panel). Bars represent 20 μm. Experiments were repeated three times with similar results, and significance is *P < 0.05. The arrows indicate SG.