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investigate a possible cell-cycle defect in the absence of Atrx, we analysed the cell cycle distribution of bromodeoxyuridine (BrdU)-pulsed ES cells by flow cytometry (Figure S1A). Surprisingly, both Atrxnull ES cell clones exhibited a cell cycle profile that was indistinguishable from ES cells bearing a functional Atrx allele (Atrx WT or Atrx flox). We also specifically quantitated the mitotic index within each population by flow cytometry after staining ES cells for phosphorylated (Ser10) histone H3, a specific marker of mitosis (Figure S1B) [13]. Consistent with the normal cell-cycle profile observed above, there was no depletion in the size of the mitotic population in the Atrxnull ES clones, despite their slow growth. Finally, we investigated whether the growth defect in the Atrxnull ES cells was due to an up-regulation of apoptosis by staining cells with Annexin V (Figure S2) and found that the proportion of apoptotic cells was not significantly affected by the absence of full-length Atrx. Thus, the growth defect observed in ES cells lacking Atrx is not due to a specific cell cycle block or significant induction of cell death. While the cause of the proliferative delay is not yet clear, since Atrxnull ES cells appear to undergo higher rates of spontaneous differentiation (unpublished data), it seems likely that the observed growth defect reflects the spontaneous transition from fast-cycling, undifferentiated ES cells into more slowly cycling, differentiated cell types in these cultures."}

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investigate a possible cell-cycle defect in the absence of Atrx, we analysed the cell cycle distribution of bromodeoxyuridine (BrdU)-pulsed ES cells by flow cytometry (Figure S1A). Surprisingly, both Atrxnull ES cell clones exhibited a cell cycle profile that was indistinguishable from ES cells bearing a functional Atrx allele (Atrx WT or Atrx flox). We also specifically quantitated the mitotic index within each population by flow cytometry after staining ES cells for phosphorylated (Ser10) histone H3, a specific marker of mitosis (Figure S1B) [13]. Consistent with the normal cell-cycle profile observed above, there was no depletion in the size of the mitotic population in the Atrxnull ES clones, despite their slow growth. Finally, we investigated whether the growth defect in the Atrxnull ES cells was due to an up-regulation of apoptosis by staining cells with Annexin V (Figure S2) and found that the proportion of apoptotic cells was not significantly affected by the absence of full-length Atrx. Thus, the growth defect observed in ES cells lacking Atrx is not due to a specific cell cycle block or significant induction of cell death. While the cause of the proliferative delay is not yet clear, since Atrxnull ES cells appear to undergo higher rates of spontaneous differentiation (unpublished data), it seems likely that the observed growth defect reflects the spontaneous transition from fast-cycling, undifferentiated ES cells into more slowly cycling, differentiated cell types in these cultures."}

{"project":"2_test","denotations":[{"id":"16628246-206429-85799748","span":{"begin":554,"end":556},"obj":"206429"},{"id":"T34229","span":{"begin":554,"end":556},"obj":"206429"}],"text":"To investigate a possible cell-cycle defect in the absence of Atrx, we analysed the cell cycle distribution of bromodeoxyuridine (BrdU)-pulsed ES cells by flow cytometry (Figure S1A). Surprisingly, both Atrxnull ES cell clones exhibited a cell cycle profile that was indistinguishable from ES cells bearing a functional Atrx allele (Atrx WT or Atrx flox). We also specifically quantitated the mitotic index within each population by flow cytometry after staining ES cells for phosphorylated (Ser10) histone H3, a specific marker of mitosis (Figure S1B) [13]. Consistent with the normal cell-cycle profile observed above, there was no depletion in the size of the mitotic population in the Atrxnull ES clones, despite their slow growth. Finally, we investigated whether the growth defect in the Atrxnull ES cells was due to an up-regulation of apoptosis by staining cells with Annexin V (Figure S2) and found that the proportion of apoptotic cells was not significantly affected by the absence of full-length Atrx. Thus, the growth defect observed in ES cells lacking Atrx is not due to a specific cell cycle block or significant induction of cell death. While the cause of the proliferative delay is not yet clear, since Atrxnull ES cells appear to undergo higher rates of spontaneous differentiation (unpublished data), it seems likely that the observed growth defect reflects the spontaneous transition from fast-cycling, undifferentiated ES cells into more slowly cycling, differentiated cell types in these cultures."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T2251","span":{"begin":26,"end":36},"obj":"GO:0007049"},{"id":"T2252","span":{"begin":62,"end":66},"obj":"PR:000004503"},{"id":"T2253","span":{"begin":84,"end":94},"obj":"GO:0007049"},{"id":"T2254","span":{"begin":111,"end":128},"obj":"CHEBI:472552"},{"id":"T2255","span":{"begin":130,"end":134},"obj":"CHEBI:472552"},{"id":"T2256","span":{"begin":143,"end":151},"obj":"CL:0002322"},{"id":"T2257","span":{"begin":203,"end":207},"obj":"PR:000004503"},{"id":"T2258","span":{"begin":212,"end":219},"obj":"CL:0002322"},{"id":"T2259","span":{"begin":239,"end":249},"obj":"GO:0007049"},{"id":"T2260","span":{"begin":290,"end":298},"obj":"CL:0002322"},{"id":"T2261","span":{"begin":320,"end":324},"obj":"PR:000004503"},{"id":"T2262","span":{"begin":325,"end":331},"obj":"SO:0001023"},{"id":"T2263","span":{"begin":333,"end":337},"obj":"PR:000004503"},{"id":"T2264","span":{"begin":344,"end":348},"obj":"PR:000004503"},{"id":"T2265","span":{"begin":349,"end":353},"obj":"SO:0000359"},{"id":"T2266","span":{"begin":393,"end":400},"obj":"GO:0007067"},{"id":"T2267","span":{"begin":463,"end":471},"obj":"CL:0002322"},{"id":"T2268","span":{"begin":499,"end":506},"obj":"CHEBI:15358"},{"id":"T2269","span":{"begin":499,"end":509},"obj":"PR:000027594"},{"id":"T2270","span":{"begin":532,"end":539},"obj":"GO:0007067"},{"id":"T2271","span":{"begin":586,"end":596},"obj":"GO:0007049"},{"id":"T2272","span":{"begin":663,"end":670},"obj":"GO:0007067"},{"id":"T2273","span":{"begin":689,"end":693},"obj":"PR:000004503"},{"id":"T2274","span":{"begin":698,"end":700},"obj":"CL:0002322"},{"id":"T2275","span":{"begin":794,"end":798},"obj":"PR:000004503"},{"id":"T2276","span":{"begin":803,"end":811},"obj":"CL:0002322"},{"id":"T2277","span":{"begin":826,"end":852},"obj":"GO:0043065"},{"id":"T2278","span":{"begin":876,"end":885},"obj":"PR:000004078"},{"id":"T2279","span":{"begin":931,"end":946},"obj":"CL:0000445"},{"id":"T2280","span":{"begin":1008,"end":1012},"obj":"PR:000004503"},{"id":"T2281","span":{"begin":1050,"end":1058},"obj":"CL:0002322"},{"id":"T2282","span":{"begin":1067,"end":1071},"obj":"PR:000004503"},{"id":"T2283","span":{"begin":1097,"end":1107},"obj":"GO:0007049"},{"id":"T2284","span":{"begin":1142,"end":1152},"obj":"GO:0008219"},{"id":"T2285","span":{"begin":1221,"end":1225},"obj":"PR:000004503"},{"id":"T2286","span":{"begin":1230,"end":1238},"obj":"CL:0002322"},{"id":"T2287","span":{"begin":1441,"end":1449},"obj":"CL:0002322"}],"text":"To investigate a possible cell-cycle defect in the absence of Atrx, we analysed the cell cycle distribution of bromodeoxyuridine (BrdU)-pulsed ES cells by flow cytometry (Figure S1A). Surprisingly, both Atrxnull ES cell clones exhibited a cell cycle profile that was indistinguishable from ES cells bearing a functional Atrx allele (Atrx WT or Atrx flox). We also specifically quantitated the mitotic index within each population by flow cytometry after staining ES cells for phosphorylated (Ser10) histone H3, a specific marker of mitosis (Figure S1B) [13]. Consistent with the normal cell-cycle profile observed above, there was no depletion in the size of the mitotic population in the Atrxnull ES clones, despite their slow growth. Finally, we investigated whether the growth defect in the Atrxnull ES cells was due to an up-regulation of apoptosis by staining cells with Annexin V (Figure S2) and found that the proportion of apoptotic cells was not significantly affected by the absence of full-length Atrx. Thus, the growth defect observed in ES cells lacking Atrx is not due to a specific cell cycle block or significant induction of cell death. While the cause of the proliferative delay is not yet clear, since Atrxnull ES cells appear to undergo higher rates of spontaneous differentiation (unpublished data), it seems likely that the observed growth defect reflects the spontaneous transition from fast-cycling, undifferentiated ES cells into more slowly cycling, differentiated cell types in these cultures."}