hES cells engage rapid apoptosis after DNA damage. H9 hES cells were either left untreated or treated with 20 μM etoposide in the absence or presence of the caspase inhibitor z-VAD.fmk (50 μM). Shown are phase contrast images after 5 hrs of treatment (A) and quantification of cell survival. Error bars represent ±SD for triplicate experiments. (B). (C) Untreated and 3 hrs etoposide treated (20 μM) hES cells were fixed and stained with anti-cleaved caspase-3 antibody and Hoechst 33258 (nuclei). Immunofluorescence images show that the untreated hES cells do not have active caspase-3 but it becomes activated as early as 3 hrs after the initiation of the treatment. (D) Quantification of cell death (using nuclear fragmentation) of hES cells treated with Tunicamycin (5 μM), Taxol (1 μM), Etoposide (20 μM), or Doxorubicin (1 μM) as indicated in the figure. Error bars represent ±SD for triplicate experiments. (E) Upper panel: wildtype (wt) and p53 knock-down (shp53) hES cells were treated with 20 μM etoposide (Etop) and cell survival was quantified at various times after etoposide treatment. Lower panel: wildtype (wt) and p53-knock-down (shp53) hES cells were treated with 20 μM etoposide for 3 hrs and immunoblotted with an anti-p53 antibody. Wildtype hES cells were also treated with zVAD.fmk to prevent any cell death. Error bars represent ±SD for triplicate experiments. Western blot shows the efficient downregulation of p53. (F) Wildtype hES cells were treated with 20 μM etoposide for 2 hours and immunolabeled for p53, cyt c and the nuclear stain Hoechst 33258. The immunofluorescence images show the upregulation and nuclear localization of p53 after etoposide treatment.

Bax is constitutively active in hES cells. (A) Wildtype (wt) and Bax knock-down (shBax) hES cells were treated with 20 μM etoposide for 5 hrs and immunoblotted for total Bax. Western blot shows efficient knock down in Bax levels in shBax expressing cells. (B) Quantification of cell survival in wt and Bax knock-down hES cells at various times after etoposide (20 μM) treatment. Error bars represent ±SD for triplicate experiments. (C) hES cells were immunostained with anti-Bax 6A7 and anti-cleaved caspase-3 antibodies. Images show active Bax but no caspase-3 activation in untreated hES cells. (D) Wildtype (wt) or Bax knockdown (shBax) hES cells were immunostained for active Bax with the 6A7 antibodies. Representative image shows no 6A7 positive staining in Bax-deficient hES cells demonstrating that the 6A7 staining is Bax-dependent. (E) Immunocomplexes were precipitated from hES and CCL-151 (human lung fibroblast cell line; untreated or treated with 200 nM staurosporine) extracts (prepared in CHAPS buffer) with the anti-Bax 6A7 antibody and immunoblotted with a total Bax antibody (N20). The input lane was loaded as 1/10 dilution of the pre-IP extract, and IgG served as a negative control.

Constitutively active Bax is localized to the Golgi in hES cells. (A) Untreated hES cells were immunostained with anti-Bax 6A7 and anti-TGN46 antibodies. Hoechst 33258 was used to label the nuclei. Immunofluorescence images show the localization of active Bax at the Golgi (arrows). (B) Active Bax is localized at the trans-Golgi (anti-TGN46 antibody) and not at the cis-Golgi network (anti-GM130 antibody). (C) Upper panels: Ectopically expressed GFP-Bax shows a distinct subcellular localization in hES cells in contrast to the cytoplasmic localization seen in HeLa cells. Lower panels: GFP-Bax immunofluorescence (GFP antibody) shows the localization of Bax to the Golgi (TGN46) in hES cells. (D) Subcellular fractionation of Golgi and mitochondria (Mito) in hES and HeLa cells. Representative Western blots demonstrate that Bax (N20 antibody) can be detected in the Golgi of hES but not of HeLa cells. TGN46 and VDAC were used as markers of Golgi and mitochondrial, respectively. (E) A panel of hES cells (H9, H7, CHB6, H1) and human non-ES cells (HeLa, U87-MG, IMR-90, HDF) were probed for the status of active Bax by immunostaining with 6A7 and TGN46 (for Golgi) antibodies. Images show that active Bax localized to the Golgi was seen in the H9, H7 and CHB6 hES cells.

Active Bax is present predominantly in hES cells in the S phase ; Bax is no longer active in hES cells after differentiation. (A) Left panel: Untreated hES cells were stained with EdU, anti-Bax 6A7 antibody and DAPI and subjected to FACS analysis as described in the Materials and Methods. Analysis showed that Bax 6A7 positivity was seen almost exclusively (greater than 95%) in cells going through the S phase of the cell cycle (~60% of the total cells are in S phase). Right panel: Representative fluorescent images show that cells that are actively replicating (EdU positive; white arrows) have Bax in its active state (6A7 positive), whereas cells that are not in the S phase (EdU negative; yellow arrowhead) do not have active Bax (6A7 negative). Error bars represent ±SD for triplicate experiments. (B) hES cells after one and two days of differentiation were immunostained with anti-Bax 6A7 and anti-TGN46 antibodies. Immunofluorescence images show that 2-day differentiated hES cells are no longer positive for Bax 6A7. (C) Phase contrast images show that embryoid bodies (2 days of differentiation) are no longer sensitive to rapid apoptosis induced by etoposide. (D) Quantification of cell death by nuclear morphology after treatment with 20 μM etoposide. Error bars represent ±SD for triplicate experiments.

p53 is required for Bax translocation to mitochondria after DNA damage. (A) Triple labeled confocal images of untreated hES cells demonstrate active Bax (6A7) colocalizing to the Golgi (TGN46) but not the mitochondria (Tom20). (B) hES cells were either left untreated or treated with etoposide for 5 hours (in the presence of caspase inhibitor 25 μM QVD-OPH). Immunostaining with anti-Bax 6A7 and anti-TGN46 antibodies (and Hoechst 33258 to label nuclei) shows no colocalization of active Bax with Golgi after etoposide treatment. Bottom panels show distinct sites of Bax 6A7 co-localization with mitochondria (anti-Tom20 antibody) after etoposide treatment. (C) hES cells were left untreated or treated with etoposide (20 μM) for 5 hours (in the presence of caspase inhibitor 25 μM QVD-OPH). Immunostaining with anti-Bax 2D2 and anti-Tom20 antibodies shows translocation of Bax to the mitochondria after etoposide treatment. (D) p53 knock-down (shp53) hES cells were either left untreated or were treated with etoposide for 5 hours and immunostained with anti- Bax 6A7 and anti-TGN46 antibodies. Immunofluorescence images show that Bax 6A7 remains colocalized with TGN46 at the Golgi, even after DNA damage. (E) Quantification of results in (D), indicating the number of wild-type (wt) and p53 knock-down (shp53) hES cells with active Bax at Golgi before and after etoposide treatment. Error bars represent ±SD for triplicate experiments.

Inactivation of Bcl-2 and Bcl-XL does not change Bax status at the Golgi in hES cells (A) hES cells were treated with 5 μM ABT-737 for 18 hrs and immunostained with anti- Bax 6A7 and anti-TGN46 antibodies. Immunofluorescence images show that ABT-737 treatment did not affect localization of active Bax to the Golgi. (B) hES cells were immunostained with anti-Bcl-XL or anti-Bcl-2 and anti-TGN46 antibodies. Immunostaining shows no localization of Bcl-2 and Bcl-XL to Golgi. (C) Quantification of survival (with nuclear morphology) in hES cells treated with ABT-737 (5 μM) or etoposide alone (20 μM) or a combination of ABT-737 and etoposide. hES cells treated with ABT737 alone did not die but cells treated with etoposide in combination with ABT-737 died faster than cells treated with etoposide alone. Error bars represent ±SD for triplicate experiments.

Ku70 is constitutively acetylated and does not interact with Bax in untreated hES cells. (A)hES and HeLa cells were either left untreated or treated with etoposide either in the presence of Bax Inhibiting Peptide (200 μM BIP) or a negative control peptide at the same concentration. Results show that while BIP inhibited cell death in HeLa cells, it failed to do so in hES cells. (B) Quantification of cell death (propidium iodide staining) after 5 hrs (hES cells) or 16 hours (HeLa cells) of etoposide treatment (20 μM). Error bars represent ±SD for triplicate experiments. (C) Immunocomplexes were precipitated from untreated hES and untreated or etoposide treated HEK293T cells with the anti-pan acetylated lysine (AcK) antibody and immunoblotted with an anti-Ku70 antibody. For untreated hES cells, a reverse IP with anti-Ku70 antibodies and immunoblot for acetylated lysine are also shown. The input lane was loaded as 1/10 dilution of the pre-IP extract, and IgG served as a negative control. (D) Immunocomplexes were precipitated from untreated hES and untreated or etoposide treated HEK293T cells with the anti-Ku70 antibody and immunoblotted with an anti-Bax (N20) antibody. For untreated hES cells, a reverse IP with anti-Bax antibodies and immunoblot for Ku70 are also shown. The input lane was loaded as 1/10 dilution of the pre-IP extract, and IgG served as a negative control. (E) Model showing the unique mechanisms engaged by hES cells where Bax is present in its constitutively active form at the Golgi, thus priming these cells for rapid apoptosis after DNA damage.