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. 2022 Jul 18;206(12):1480–1494. doi: 10.1164/rccm.202110-2358OC

Figure 4.

Figure 4.

NKX2-1 interactome in chromatin of small-cell lung cancer (SCLC) subtype SCLC-Aα cells includes SOX1 (SRY-box transcription factor 1). (A) Experimental strategy to identify binding partners of NKX2-1 (NK2 homeobox 1) in SCLC-Aα. Chromatin prepared from the nuclear fraction of SCLC-Aα cell lines NCI-H187 and NCI-H1436 stably expressing FLAG-NKX2-1 was solubilized using micrococcal nuclease digestion. Coimmunoprecipitated proteins with anti-FLAG antibody from solubilized chromatin were identified using liquid chromatography–tandem mass spectrometry (LC-MS/MS). (B) Venn diagram showing the number of transcription factors identified in each of the two SCLC-Aα cell lines and the 27 overlaps found in both cell lines. The proteins indicated in yellow were also found in core transcriptional regulatory circuitry (CRC) analysis in Figure 4C. (C) CRCs commonly identified in eight SCLC-Aα cell lines. The genes in yellow were also found in LC-MS/MS analysis in Figure 4B. (D) Genome view tracks of H3K27ac signal at the SOX1 locus in four clusters of SCLC cell lines. (E) Top: Immunoblots with anti-SOX1 antibody on endogenous coimmunoprecipitates by anti–NKX2-1 antibody. Bottom: Immunoblots with anti–NKX2-1 antibody on endogenous coimmunoprecipitates by anti-SOX1 antibody as the reciprocal experiment in SCLC-Aα (DMS 79 and NCI-H187) cells. (F) In situ proximity ligation assay signals (red fluorescence) targeting endogenous SOX1 and NKX2-1 in NCI-H841, DMS 79, NCI-H209, NCI-H187, and NCI-H345 cells. Nuclei are counterstained with DAPI (blue). See Figure E5G for three SCLC-Aσ cell lines and two additional controls. ADNP2 = ADNP homeobox 2; AHDC1 = AT-hook DNA binding motif containing 1; AKAP8 = A-kinase anchoring protein 8; ARID2 = AT-rich interaction domain 2; ATF4 = activating transcription factor 4; BCL6 = BCL6 transcription repressor; CASZ1 = castor zinc finger 1; CREB1 = CAMP responsive element binding protein 1; CUX1 = cut like homeobox 1; E2F2 = E2F transcription factor 2; ELF3 = E74 like ETS transcription factor 3; ETS2 = ETS proto-oncogene 2, transcription factor; ETV6 = ETS variant transcription factor 6; FOX = forkhead box; HES1 = Hes family BHLH transcription factor 1; HMBOX1 = homeobox containing 1; HOXD8 = homeobox D8; IP = immunoprecipitation; KDM = lysine demethylase; NFIB = nuclear factor I B; NR2F2 = nuclear receptor subfamily 2 group F member 2; PBX1 = PBX homeobox 1; PLA = proximity ligation assay; POU2F3 = Pou domain, class 2, transcription factor 3; RFX2 = regulatory factor X2; RREB1 = Ras responsive element binding protein 1; RUNX1 = RUNX family transcription factor 1; SKI = SKI proto-oncogene; SP3 = Sp3 transcription factor; ST18 = ST18 C2H2C-type zinc finger transcription factor; TCF = transcription factor; TEAD11 = TEA domain transcription factor 1; TGIF1 = TGFB induced factor homeobox 1; TP73 = tumor protein P73; ZBTB = zinc finger and BTB domain containing; ZNF = zinc finger protein.