Fig. 5 | Nature Communications

Fig. 5

From: A requirement for STAG2 in replication fork progression creates a targetable synthetic lethality in cohesin-mutant cancers

Fig. 5The alternative text for this image may have been generated using AI.

The intra-S-phase cell cycle arrest but not the loss of SMC3 acetylation induced by STAG2 inactivation is p53 dependent. a Immunoblot of total lysate from RPE cells following lentiviral STAG2 shRNA depletion either alone or in combination with TP53 shRNA depletion. b Flow cytometry plot of RPE cells following lentiviral transduction with STAG2 shRNA in combination with TP53 shRNA demonstrating bypass of the S-phase arrest induced by STAG2 depletion alone. c Phase contrast image of RPE cells following lentiviral transduction with STAG2 shRNA in combination with TP53 shRNA demonstrating cellular proliferation and abrogation of the senescence induced by STAG2 depletion alone. d Sequence alignment of representative STAG2 knockout clones that were readily obtained following ectopic expression of Cas9 and STAG2 gRNA in RPE cells subsequent to TP53 shRNA depletion. e Results of STAG2 knockout clone generation following ectopic expression of Cas9 and STAG2 gRNA in multiple human cell lines with wildtype TP53, TP53 shRNA depletion, or mutant TP53 alleles. f Immunoblots of total lysate from several of the STAG2 knockout clones generated by Cas9 cleavage in RPE cells with TP53 shRNA depletion. g Immunoblots of total lysate from the STAG2 knockout clone generated by Cas9 cleavage in TC-106 Ewing sarcoma cells harboring TP53 splice site mutation. h TP53 inactivation via lentiviral shRNA depletion in STAG2-depleted RPE cells restores the interaction of cohesin with the replication factors PCNA, PolE, and PolD, as well as blocking the enhanced binding of cohesin with MCM3, MCM5, CDT1, and Geminin. However, TP53 inactivation does not block the enhanced interaction of cohesin with RPAp34 or CDC6. i Combined STAG2 and TP53 shRNA depletion enables bypass of S-phase cell cycle arrest independent of SMC3 acetylation. Shown are immunoblots using antibodies against acetylated-lysine following SMC3 immunoprecipitation, as well as immunoblots of total SMC3 and STAG2 on whole cell lysates of RPE cells after lentiviral shRNA transduction. j Impaired SMC3 acetylation is present in H4 glioblastoma cells with STAG2 truncating frameshift mutation and TC-106 Ewing sarcoma cells after STAG2 knockout by targeted Cas9 cleavage. Source data are provided as a Source Data file

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