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. Author manuscript; available in PMC: 2020 Jan 15.
Published in final edited form as: Science. 2018 Jun 21;361(6400):eaar2555. doi: 10.1126/science.aar2555

Fig. 4. Combined DNA FISH and EWS/FLI1-Halo imaging show that endogenous EWS/FLI1 forms hubs at GGAA microsatellites.

Fig. 4.

(A) Schematic for GGAA microsatellites in the A673 genome nucleating hubs of endogenously Halo-tagged EWS/FLI1.

(B) Western blot of EWS/FLI1 and β-actin (normalization control) from clonal EWS/FLI1-Halo knock-in (KI), WT and clonal EWS/FLI1 knockout (KO) A673 lines. (C) z-projected 3D image of endogenous EWS/FLI1-Halo in an A673 cell nucleus (stained with 200 nM Halo ligand JF549) taken on the lattice light-sheet microscope. (D) Confocal fluorescence images of 3D DNA FISH targeting GGAA microsatellite-adjacent CAV1 gene (enhanced Cy5 labeled, red) and endogenous EWS/FLI1-Halo (JF549 labeled, green). The zoomed-in views depict the region surrounding one particular CAV1 locus. EWS/FLI1-Halo enrichment at the locus is visible but buried in high background.

(E) Averaged two-color images of five GGAA microsatellite-adjacent gene loci (CAV1, FCGRT, ABHD6, KDSR, and KIAA1797) and two gene loci not containing a GGAA microsatellite (Non-GGAA locus 1 targeting ADGRA3 and locus 2 targeting REEP5). The right column shows average surface plots of EWS/FLI1-Halo.

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