Increased p53 protein expression in irradiated <i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- BM-MNCs is independent of mRNA expression.

<p><b>(A)</b> Cell lysates from irradiated Lin–ve enriched BM-MNCs (2 Gy or 6 Gy) at different time points (15’, 30’ and 45’) after radiation exposure were used for immunoblotting for p53. A549 and H358 cell lysates were used as positive and negative controls respectively. (<b>B)</b> Intensities of p53 bands were normalized to β-actin. Values are expressed as densitometric ratios. (<b>C)</b> BM-MNCs were lysed 45’ or 60’ after irradiation (2 Gy or 6 Gy). The RNA extracted from these lysed cells was used to determine p53 mRNA expression by qRT-PCR. (<b>D</b>) Cell lysates from phorbol 12-myristate 13-acetate (PMA) stimulated (30ng/ml) or irradiated Lin–ve encriched <i>Egr1</i>^<i>+/+</i> BM-MNCs (2 Gy or 6 Gy) at different time points (60’) and (45’, 60’) respectively. Lysate of normal splenic wildtype B cells stimulated with PMA (30ng/ml) for 60’ was used as a positive control for EGR1 expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169767#pone.0169767.ref041" target="_blank">41</a>]. RNA extracted from wildtype BM-MNCs in (C) was used to determine EGR1 mRNA expression by qRT-PCR.</p

Radiation activates the DSB DNA response pathway in <i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- BM-MNCs.

<p><b>(A)</b> Left: γ-H2AX protein expression by immunoblotting in irradiated BM-MNCs 30’, 45’ and 60’ after radiation exposure (2 Gy or 6 Gy). Right: Intensities of γ-H2AX bands were normalized to β-actin and are expressed as densitometric ratios. <b>(B)</b> Left: p-Chk2 protein expression by immunoblotting in irradiated BM-MNCs at different times after radiation exposure (2 Gy or 6 Gy). Right: p-Chk2 band intensities were normalized to those of GAPDH and are expressed as densitometric ratios. Results are representative of two experiments.</p

Ionizing radiation induced apoptosis of primary <i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- BM-MNCs equally in vitro.

<p><b>(A)</b> Representative analysis of irradiated lineage negative (Lin–ve) enriched BM-MNCs by flow cytometry. BM-MNCs isolated from WT and <i>Egr-1</i> KO mice were enriched for Lin–ve cells (as described in the methods). After 2 days of cytokine stimulation (mSCF-50ng/ml, mIL3-10ng/ml, hIL6-10ng/ml), Lin–ve enriched BM-MNCs were left untreated, or exposed to 2 Gy or 6 Gy irradiation (1 × 10⁶/ml). 24Hrs after irradiation, cells were stained with c-KIT-APC, Sca-1-PB and streptavidin APC CY7 antibodies, and annexin-V-PE CY7 to identify LSK and apoptotic cells respectively. A minimum of 500,000 cells were collected per sample on the BD LSR II flow cytometer and the data was analyzed using the FlowJo single cell analysis software for the percentage of apoptotic cells (by annexin) in the various cell populations (all cells, lin–ve cells and LSK cells). <b>(B)</b> Annexin +ve cells in the BM-MNC, and gated subpopulations from WT and <i>Egr-</i>1 KO mice are shown. <b>(C)</b> BM-MNC were stained for cleaved caspase-3. Left panel shows representative flow cytometry profile of irradiated WT or KO BM-MNCs (clear histograms) overlaid on untreated BM-MNCs (gray histograms). Summary of data from triplicate cultures is shown in the right panel. Results represent mean ± SE of triplicate cultures. *Indicates p<0.05 comparing untreated cells to cells exposed to radiation. Results from one of two experiments with similar outcomes are shown.</p

<i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- mice have comparable recovery kinetics of blood cells after sub-lethal TBI.

<p>Mice were exposed to 6.5 Gy TBI. Blood cells were enumerated at 3, 9, 15, 22, and 28 days after TBI by the HEMAVet 950FS automatic veterinary hematology analyzer. Baseline (day 0) measurement was done before irradiation. The results are expressed as the mean of blood cells concentration ± SE (n = 8 mice/ group). Results from one of two similar experiments are shown.</p

Radiation induces DNA DSBs in <i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- BM-MNCs.

<p>Immunofluorescence analysis of irradiated BM-MNCs for γ-H2AX foci was performed as described in the methods. Representative immunofluorescence images of γ-H2AX foci in BM-MNCs 4Hrs after exposure to 6 Gy irradiation.</p