Fig 2: C/EBPβ is necessary for optimal cold stress response in BAT(A) Overlapping of PGC1α and C/EBPβ peaks after cold stress.(B) Immunoprecipitation with HA-tag antibody and Western blot experiment showing selective NCOA1 interaction with C/EBPβ.(C) Western blot of C/EBPβ and C/EBPα protein expression in WT and AKOβ BAT.(D) qPCR of Cebpa mRNA levels in WT and AKOβ BAT (mean ± SD, n = 5).(E) qPCR quantification of Elovl3 and (F) Hacd2 mRNA in WT and AKOβ BAT (mean ± SD, n = 5).(G) ChIP-seq peak distribution around the Elovl3 enhancer region. See also Figure S5 and Table S2. Cold induced differentially expressed genes in WT that had reduced change in AKOβ.

Fig 3: SRC‐1 blocks E3 ubiquitin ligase SPOP‐mediated degradation of PD‐L1. A) Knockout of SRC‐1 accelerated IFNγ‐induced PD‐L1 protein degradation in HCT116 cells. B) SRC‐1 inhibited the ubiquitination of PD‐L1 in a dose‐dependent manner in vitro. C) The interaction between SRC‐1, SPOP, and PD‐L1 was detected by Co‐IP assay. D) Overexpression of SPOP inhibited PD‐L1 expression, but did not affect SRC‐1 expression in HCT116 cells. E) SPOP‐mediated PD‐L1 degradation could be prevented by exogenous SRC‐1 in HCT116 cells. F) SRC‐1 prevented the poly‐ubiquitination modification of PD‐L1 by SPOP. G) SRC‐1 and PD‐L1 could interact with the N‐terminal domain of SPOP. H) The combination of SPOP and PD‐L1 could be blocked by exogenous SRC‐1. I) Knocking out SRC‐1 promotedc the interaction between SPOP and PD‐L1 in CRC cells. J) The theoretical pattern of SRC‐1 blocking SPOP‐mediated degradation of PD‐L1. Results are representative of one of three experiments. Data are presented as means ± SEM. *p<0.05, ***p<0.001, based on Student's t test.

Fig 4: Inactivation of the estrogen pathway contributed to BRD9 knockdown-induced inhibition of proliferation in CRC cells. (A) KEGG pathway analysis based on the BRD9-binding and interacting genes based on TCGA databases. (B) The cnetplot for the molecular function data in GO analysis is based on TCGA databases. (C) GSEA plot showing that BRD9 expression is negatively correlated with ESTROGEN_DEPENDENT_GENE_EXPRESS. (D,E) BRD9 expression positively correlated with NCOA1. (F) Overexpression of BRD9 in SW480 cells and downregulation of BRD9 in HCT116 cells. Western blot was used to determine the protein levels of BRD9, p-MEK, MEK, p-ERK, ERK, and NCOA1. The relative levels of BRD9 (G), the ratio of p-MEK/MEK (H), the ratio of p-ERK/ERK (I), and NCOA1 (J) were measured using ImageJ and normalized to GAPDH. Data are presented as mean ± SD from at least three independent experiments. **P<0.01 and ***P<0.001 compared with the indicated groups. CRC, colorectal cancer; GO, Gene Ontology; GSEA, gene set enrichment analysis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-MEK, phosphorylated-MEK; p-ERK, phosphorylated-ERK; SD, standard deviation; BP, biological process; CC, cell component; MF, molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; FDR, false discovery rate; BRD9, bromodomain-containing protein 9; TPM, transcript per million; NCOA1, nuclear receptor coactivator 1.

Fig 5: 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.