Fig 2: SRC-2 and SRC-3 but not SRC-1 are critical for maintenance of ES self-renewal capability.A, representative AP staining images of control and SRC-1-, SRC-2-, or SRC-3-deficient CGR8 ES clones. Knockdown of SRC-1, SRC-2, and SRC-3 was achieved by transfection of CGR8 ES cells with specific shRNAs as indicated. B, working model illustrating how SRC family coactivators enhance Sox2 transcriptional activity and maintenance of ES cell self-renewal. The top panel illustrates that Sox2 interacts with and recruits p300 as well as the SRC family coactivators. These coactivators, together with other coactivators, mediate transcriptional activation by Sox2. The lower panel shows that p300 catalyzes acetylation of Sox2, which in turn enhances the interaction between Sox2 and SRC-2/3 to further augment Sox2 transcriptional activity and function in maintenance of ES pluripotency. AP, alkaline phosphatase; ES, embryonic stem; Sox2, SRY-box 2; SRCs, steroid receptor coactivators.

Fig 3: Sox2 acetylation promotes the interaction between Sox2 and SRC-2/3.A, IP-WB analysis showing that ectopically expressed p300 elevated Sox2 acetylation. B, co-IP assay showing that coexpressed p300 enhanced the interaction between SRC-3 and Sox2. A nonspecific band that overlaps with Myc-Sox2 (∗). C, IP-WB analysis showing that inhibition of both histone de-acetylase and Sirtuin by TSA and NAM elevated Sox2 acetylation. FLAG-Sox2 was transiently expressed in HEK239T cells, and cells were treated with DMSO or TSA (1 μM) plus NAM (5 mM) for 24 h before being harvested for IP-WB analysis. D, co-IP assay showing that TSA plus NAM treatment enhanced the interaction between Sox2 and SRC-3. FLAG-SRC-3 and Myc-Sox2 were coexpressed in HEK239T cells, and cells were treated with TSA (1 μM) plus NAM (5 mM) or sodium crotonate (NaCro, 20 mM) for 24 h before being harvested for co-IP assay using the antibody as indicated. E, co-IP assay showing that TSA plus NAM treatment markedly enhanced the interaction between ectopically expressed FLAG-Sox2 and endogenous SRC-3. F, the right panel showing the experimental scheme. FLAG-Sox2–transfected HEK239T cells were treated with DMSO or TSA (1 μM) plus NAM (5 mM) (T + N) for 24 h before being harvested for preparation of whole-cell extracts, and the resulting extracts were mixed with an equal volume of extracts derived from Myc-SRC-3–expressed HEK239T cells for the co-IP assay using the antibodies as indicated. G, the right panel showing the experimental scheme. Myc-SRC-3–transfected HEK239T cells were treated with DMSO or TSA (1 μM) plus NAM (5 mM) (T + N) for 24 h before being harvested for preparation of whole-cell extracts, and the resulting extracts were mixed with an equal volume of extracts derived from FLAG-Sox2–expressed HEK239T cells, followed by the co-IP assay using antibodies as indicated. H, experiments were performed essentially as (F), except that the Myc-Sox2 expression construct was used and that FLAG-SRC-1– or FLAG-SRC-2–expressed HEK239T cell extracts were used for the co-IP assay using antibodies as indicated. I, co-IP assay showing that TSA (1 μM) plus NAM (5 mM) treatment (12 h) markedly enhanced the interaction between endogenous Sox2 and endogenous SRC-2 and SRC-3 in E14 ES cells. J, IP-WB analysis showing that TSA (1 μM) plus NAM (5 mM) treatment elevated the level of Sox2 acetylation in E14 ES cells. ImageJ was used for quantitation of WBs. co-IP, co-immunoprecipitation; IP, immunoprecipitation; NAM, nicotinamide; Sox2, SRY-box 2; SRCs, steroid receptor coactivators; TSA, trichostatin A.

Fig 4: Expression of NCOA2 is upregulated during viral lytic replication by RTA.(A and C) The expression kinetics of NCOA2 and RTA were analyzed in iSLK.RGB cells (A) or iSLK cells (C) at indicated time points after dox treatment. (B and D) NCOA2 mRNA expression level in cells from (A) or (C). (E) Effect of RTA on NCOA2 expression. 293T cells were cotransfected with 1 μg of NCOA2 expression plasmid and increasing amounts of RTA expression vector (0, 0.5, 1, 2 μg). The protein level of NCOA2 was examined by immunoblotting. (F) NCOA2 mRNA expression level in cells from (E). (G) RTA inhibits the degradation of NCOA2. 293T cells were cotransfected with the indicated expression constructs for 36 h and then treated with 0.5 μM MG132 or 0.1% DMSO for another 6 h. Cells were lysed and used for western blots with the indicated antibodies. The band intensities of NCOA2 were plotted graphically (H). Data were from a minimum of three experimental replicates with a standard deviation. Data were analyzed by unpaired t-test. *, P < 0.05; **, P < 0.01. (I) Measurement of NCOA2 stability in the absence and presence of RTA. 293T cells were transfected with HA-tagged NCOA2 with or without Flag-tagged RTA expression plasmid for 36 h. Cells were treated with 100 μg/ml of CHX and analyzed at indicated time points by immunoblotting for NCOA2. Tubulin was used as a control for equivalent sample loading. (J) The relative levels of NCOA2 from (I) were quantified by densitometry and normalized to the Tubulin level.

Fig 5: NCOA2 stabilizes RTA protein expression.(A) Effect of NCOA2 on RTA expression. First, 293T cells were cotransfected with 1 μg of RTA expression plasmid and increasing amounts of NCOA2 expression vector (0, 0.5, 1, 2 μg). The expression of RTA proteins was examined by immunoblotting with the indicated antibodies. (B) The 293T cells were treated as in (A). RTA mRNA was detected using RT-qPCR with the indicated primers. (C) shRNA-NCOA2 and shRNA-GFP plasmids were transfected into 293T cells for 12 h. Then, cells were transfected with the Flag-tagged RTA expression plasmid. The expression of NCOA2 and RTA was determined by immunoblotting with the indicated antibodies. (D) shRNA-NCOA2 and shRNA-GFP plasmids were transfected into 293T cells for 12 h. Then cells were transfected with the Flag-tagged RTA-△PARS-II expression plasmid. The expression of NCOA2 and RTA-△PARS-II was determined by immunoblotting with the indicated antibodies. (E) The N-terminal truncation of NCOA2 regulates the stability of RTA. First, 293T cells were cotransfected with HA-tagged RTA expression vector with or without NCOA2 and its truncations. Then, RTA expression levels were examined by western blotting using anti-HA antibodies. (F) Effect of NCOA2 on the expression of the RTA mutant with deletion of the PARS II region. First, 293T cells were cotransfected with 1 μg of Flag-tagged RTA-△PARS-II expression plasmid and increasing amounts of NCOA2 expression vector (0, 0.5, 1, 2 μg). Then, the expression of RTA-△PARS-II proteins was examined by immunoblotting with the indicated antibodies. (G) The 293T cells were treated as in (F), and the mRNA level of RTA-△PARS-II was detected using RT-qPCR with the indicated primers. (H) Measurement of RTA stability in the absence and presence of NCOA2. First, 293T cells were transfected with Flag-tagged RTA with or without the NCOA2 expression plasmid for 36 h. Then, cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA. Tubulin was used as a control for equivalent sample loading. (I) The relative levels of RTA from immunoblots (H) were quantified by densitometry and normalized to the Tubulin level. The band intensities on the exposed film were plotted graphically. (J) Measurement of RTA-△PARS-II stability in the absence and presence of NCOA2. First, 293T cells were transfected with Flag-tagged RTA-△PARS-II with or without the NCOA2 expression plasmid for 36 h. Then, cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA-△PARS-II. Tubulin was used as a control for equivalent sample loading. The band intensities on the exposed film are plotted graphically (K). (L) First, 293T cells were transfected with RTA expression plasmid with sh-NCOA2 or sh-GFP. Then, the cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA. Tubulin was used as a control for equivalent sample loading. The band intensities on the exposed film were plotted graphically (M).