Cold shock and rewarming induce H2AX phosphorylation through ROS production.

<p>The levels of phosphorylated H2AX (γH2AX) were analyzed in WI26 cells cultured at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. (A) Representative western blots. (B) γH2AX was quantified by western blotting. Data are expressed in arbitrary units after normalization by ERK1/2, taking T0 as 1. (C) γH2AX in WI26 cells in normal conditions (1D37°C) and during cold shock for 5 days and rewarming for 2 to 24h was evidenced by immunostaining. Actin stress fibers are stained in red and γH2AX in green. Bar: 100µm. (D) γH2AX was measured by western blot in WI26 cells cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h in absence (-) and in presence (+) of NAC 15mM added at T0. After normalization by GAPDH, results are expressed in arbitrary units taking 5D as 1.</p

Phase contrast micrographs of WI26 cells.

<p>Cells were continuously cultured at 37°C (A), at 25°C for 5 days (B) or at 25°C for 5 days followed by a rewarming to 37°C for 24h (C). Arrows point to refringent, apoptotic-like cells. Bar: 100µm.</p

Cold shock and rewarming affect cell viability.

<p>WI26 cells were cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h. Cells kept at 37°C were used as control (T0). FACS analysis was performed after labeling with FITC-annexin V (FITC-A, X-axes) and propidium iodide (PI, Y-axes). 12.000 to 18.000 events were collected for each experiment. (A) Example of dots graphs (I), contour graphs (II) and annexin V curves (III) of control cells and cells maintained 5 days at 25°C and then warmed-up at 37°C for 4h (4h). Alive cells [A] (double negative staining), cells in early apoptosis [EA] (annexin V positive, PI negative), in late apoptosis [LA] (double positive) and necrotic [N] (annexin V negative, PI positive) are indicated on the graphs. Annexin V curves (III) were used to define the gating allowing to discriminate the populations. (B) Percentage of apoptotic cells as measured by FACS analysis. Cells were cultured at 37°C (T0) or 25°C for 5 days and subsequent warming-up at 37°C for 2 to 24h. (C) Percentage of dead cells as measured by trypan blue exclusion assay. Cells were cultured in duplicate for 5 days at 25°C and then rewarmed for 2 to 24h at 37°C.</p

Cold-shock and rewarming affect heat and cold shock genes expression.

<p>The expression of heat shock (HSP70) and cold shock genes (CIRBP and RBM3) was quantified at the mRNA level by RT-PCR in WI26 cells cultured at 25°C for 1 and 5 days before warming up at 37°C for 1h to 24h. (A) Representative gels showing the RT-PCR amplification products. (B) HSP70, CIRBP and RBM3 mRNA levels are expressed as mean ± SD (n=3) after normalization to the 28S rRNA content used as calibrator. Values at T0 were arbitrary taken as 1. Legend for culture schedule is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069687#pone-0069687-g002" target="_blank">Figure 2</a>.</p

Return to normal temperature after a cold shock affects the phosphorylation of p53.

<p>Total and phosphorylated p53 (P-p53) were analyzed in WI26 cells cultured in normal conditions or at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. (A) Representative western blot. (B) Quantifications of the western-blots are expressed as the mean ratio P-p53/total p53, taking T0 as 1. Total protein loading was monitored by ERK1/2.</p

Cold shock and rewarming induce autophagy and a cellular stress response.

<p>The level of LC3 I and II (A), phospho-JNK (P-JNK) and total JNK (D) was analyzed by western blot in cells cultured at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. The levels of ERK1/2 were taken as calibrator and used to monitor protein loading. Results are expressed as the mean ratio of LC3 II/I (B) and of P-JNK/ total JNK (D) taking T0 as 1. (C) Immunostaining of LC3 was performed in WI26 cells maintained at 37°C in the presence or in the absence of serum or at various time points during the cold shock and rewarming. Actin stress fibers appear in red, LC3 in green and nucleus in blue. Bar: 100µm.</p

Rewarming-induced ROS production and <b>ROS-dependant apoptosis.</b>

<p>(A) ROS were measured in WI26 cells cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h in absence (-) and in presence (+) of 15mM N-Acetylcysteine (NAC). Data are expressed in arbitrary units taking T0 as 1. Significant inhibition by NAC versus condition without NAC is indicated # # # (p<0.001). (B) Cell viability was measured by FACS analysis after labeling with FITC-annexin V (FITC-A, X-axes) and propidium iodide (PI, Y-axes) of cells maintained 5 days at 25°C and then warmed-up at 37°C for 4h (4h) in the presence or in the absence of 15mM of NAC added at T0. (C) Percentage of apoptotic cells in the indicated culture conditions.</p