1

Rad54 and Lig4 in Chromatid and Chromosome Stability Mills, et al.

Mills et al. Supplemental Material

Supplementary Figure S1. Spontaneous H2AX focus formation is reduced by loss of p53

Immunofluorescent detection of spontaneously occurring H2AX foci was performed as in Figure 2. The high level of spontaneously occurring -H2AX foci in RL cells was significantly reduced by additional deficiency for p53, which also restores proliferation potential to these cells (Figure 1A).

Supplementary Figure S2. Rad54 Lig4 double mutant cells are non-apoptotic

The absence of spontaneous -H2AX foci in RLP cells (Figure S1), raised the possibility that focus formation in these cells was secondary to a p53-mediated response, such as damage-stimulated apoptosis. Moreover, it has been previously noted that phosphorylated H2AX foci, in addition to marking spontaneously occurring or IR-induced DSBs, can also appear in apoptotic cells (Rogakou et al., 2000). Therefore, to determine if the spontaneous -H2AX foci observed in RL cells resulted from the induction of apoptosis, cells were stained with FITC conjugated Annexin V, a marker of apoptosis, and analyzed by flow cytometry (Figure S2A-C). Cells were counterstained with the DNA binding dye TOPRO3. The percentage of cells in the apoptotic gate for each sample is indicated.

(A) Schematic representation of flow cytometry analysis of AnnexinV/TOPRO3 stained cells. The population of cells that occupy each gate are indicated.

(B) As controls for Annexin V staining, WT MEFs were subjected to IR, UV irradiation, or prolonged serum starvation. While UV irradiation or serum starvation result in a high rate of apoptosis in MEF cultures, ionizing radiation has been shown to strongly induce growth arrest, but only weakly induced apoptosis in MEFs (Chernov and Stark, 1997; Dikomey et al., 2003; Lackinger et al., 2001; Li and Ho, 1998; Matsui et al., 2001; Naderi et al., 2002; Radford, 1994; Shaulian et al., 2000; Siu et al., 1999). Thus, as expected, untreated WT cells exhibited a low level Annexin V labeling, while 5 Gy of gamma-irradiation produced a moderate increase in Annexin V labeling, consistent with published results, but both UV-irradiation and serum starvation produced a substantially increased population of Annexin V-positive cells, indicative of apoptosis. Cultures of cells deficient for p53 exhibited few Annexin V positive cells under all conditions. These results confirm the apoptotic potential of the MEFs used in these assays.

(C) Analysis of Annexin V stained, untreated WT, Rad54, Lig4, and RL cells revealed that cultures from all genotypes exhibited similarly low levels of Annexin V staining. These results are consistent with the previously documented premature senescence phenotype exhibited by NHEJ-deficient MEFs (Frank et al., 1998; Gao et al., 1998b), and additionally demonstrate that RL cells did not undergo an increased rate of apoptosis.

(D,E) As an independent assay for apoptosis, cleaved caspase-3 was detected by immunofluorescence. As controls, wild type or p53-/- MEFs were subjected to UV irradiation prior to fixation (Figure S2D, E). By this assay, RL cells showed a small increase in the fraction of spontaneously apoptotic cells. However, consistent with the Annexin V results, cultures representing all genotypes exhibited low overall fractions of cleaved caspase-3 positive cells (Figure S2E). These findings demonstrate that, while a small fraction of RL cells may exhibit apoptosis-related -H2AX foci, in most RL cells the foci are not a secondary consequence of an apoptotic program, but likely represent spontaneously occurring DSBs.

Supplementary Methods

For analysis of Annexin V staining, cells were cultured and trypsinized above. Cells were rinsed in PBS and resuspended in 1X Binding Buffer (10 mM Hepes pH 7.4; 150 mM NaCl; 2.5 mM CaCl2; 1 mM MgCl2; 4% BSA) and incubated in the dark for 15 minutes with FITC-conjugated Annexin V. Stained cells were rinsed with PBS and counterstained with the DNA binding dye TOPRO3. Stained cells were analyzed on a Becton Dickinson FACSCalibur machine, using two parameter flow cytometry on the FL1 and FL4 channels. As controls for Annexin V staining, adherent MEFs of appropriate genotypes were subjected to 5 Gy of gamma-irradiation, 2x105J/cm2 of UV irradiation, or 5 days of culture in DMEM supplemented with 0.2% fetal calf serum (serum starvation). Following irradiation, cells were cultured an additional 24 hours prior to analysis. All Annexin V assays were performed in duplicate. All assays to detect cleaved caspase-3 were performed in triplicate. Immunofluorescence was performed as described in Material and Methods.