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1.
Figure 7

Figure 7. A model for reversible transformation by the SS18-SSX1 oncogenic fusion. From: Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma.

(A) (left) Anti-Brg IPs on 293T, B35, Aska-SS and Yamato-SS cells bearing introduced BAF47-V5; (middle) anti-V5 IPs; (right) Total protein inputs.
(B) Anti-Brg IPs on nuclear extracts of Aska-SS and Yamato-SS cells with stably introduced BAF47-V5 and co-infection with either control vector, SS18FL, or shSS18-SSX.
(C) Proliferation analyses of Aska-SS cells infected with either control vector, BAF47-V5, or BAF47-V5+co-infected SS18FL, BAF47-V5+co-infected shSS18-SSX. Error bars= s.d.
(D) Model for reversible disruption of BAF complex composition and action upon SS18-SSX incorporation.

Cigall Kadoch, et al. Cell. 2013 March 28;153(1):71-85.
2.
Figure 1

Figure 1. SS18 is a dedicated, stable subunit of mSWI/SNF (BAF) complexes. From: Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma.

(A) Composition of BAF complexes isolated from ES cells, MEFs and brain as determined by mass spectrometric analysis. See also Figure S1C.
(B) Immunoprecipitation (IP) using anti-Brg and anti-SS18 antibodies in 293T nuclear extracts (NE). See also Figure 1A,B.
(C) Glycerol gradient (10-30%) analysis on ES cell NE.
(D) Left, Schematic for urea-based denaturation analyses; right, anti-Brg IPs on 293T NE preps treated with 0-5M urea.
(E) Quantitative densitometry on urea denaturation immunoblots.

Cigall Kadoch, et al. Cell. 2013 March 28;153(1):71-85.
3.
Figure 2

Figure 2. mSWI/SNF (BAF) complexes are disrupted in synovial sarcoma cells bearing the SS18-SSX1 fusion protein. From: Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma.

(A) Diagram of SS18-SSX fusion protein resulting from t(x;18) translocation, hallmark to synovial sarcoma.
(B) Anti-Brg IP (left) and total protein (right), in 293T cells as compared to synovial sarcoma (SS) cell lines, Aska-SS and Yamato-SS. See also Figure S2A-C.
(C) Glycerol gradient (10-30%, fractions 1-20) analysis on Aska-SS cell NE. See also Figure S2D,E.
(D) Side-by-side comparison of fractions 3,4 and 15,16 of Aska-SS glycerol gradient analysis.
(E) Immunodepletion studies performed on 293T and Aska-SS cells using anti-BAF155 and anti-SSX1 antibodies. (undepleted= antibody not added)

Cigall Kadoch, et al. Cell. 2013 March 28;153(1):71-85.
4.
Figure 4

Figure 4. SS18-SSX1 induces Sox2 mRNA expression which drives SS cell proliferation. From: Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma.

(A) Sox2 mRNA levels at day 10 post-infection with LV containing either SS18/SS18-SSX variants or shRNAs to BAF complex subunits. (Normalized to GAPDH; ***p<0.005, **p<0.01). See also Figure S4A.
(B) Time course of Sox2, Oct4, and Nanog mRNA levels post-infection with SS18-SSX-containing LV. (Normalized to GAPDH; error bars reflect s.d. in n=5 separate experiments)
(C) shRNA-mediated knock-down of Sox2 in Aska-SS cells: top, immunoblot analysis; bottom, Sox2 mRNA levels.
(D) Proliferative analysis of Aska-SS cells treated with Sox2 shRNA KD LV. Control, shScramble.
(E) Left, Immunoblot analysis on Aska-SS cells treated with shControl or with either shSS18-SSX1 or shSox2-1; Right, Sox2 mRNA relative expression (normalized to GAPDH).
(F) Left, anti-BAF155 ChIP on human primary fibroblasts treated with either empty vector or SS18-SSX1, with subsequent qPCR for regions at the human Sox2 promoter and two Sox2 transcription factor (TF) binding sites within the exon. Right, anti-H3K27me3 ChIP. Error bars, s.d. of n=3 experiments. See also Figure S4B,C.

Cigall Kadoch, et al. Cell. 2013 March 28;153(1):71-85.
5.
Figure 5

Figure 5. Molecular requirements of the 78aa SSX peptide for BAF47 subunit ejection from mSWI/SNF-like BAF complexes. From: Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma.

(A) Left, Immunoblot analysis for BAF47 and Brg on anti-Brg IPs (Top) of 293T cells transfected with various SS18/SS18-SSX constructs (Bottom). Right, quantitative densitometry depicting BAF47/Brg protein ratios in IP studies.
(B) Sox2 mRNA levels in human fibroblasts day 15 post-infection with LV containing SS18 and SS18-SSX variants. Error bars= s.d.
(C) Hydrophobicity determination using Kyte-Doolittle algorithm for 78 C-terminal amino acids (aa) of SSX1-SSX4 proteins. Region of significant difference highlighted in yellow.
(D) Peptide alignment of SSX1- SSX5 C-terminal 78 aa. Pink arrows indicate aa of significant difference between SSX1/2/4 and SSX3 or SSX1/2/4 and SSX5; yellow highlight indicates regions determined to be critical for BAF47 ejection.
(E) Immunoblot analysis for BAF47 and Brg on anti-GFP IPs of 293T cells transfected with constructs as per above as well as SS18-SSX3; and (F) with SS18-SSX1, SS18-SSX1Δaa43,44 (KR→MI), SS18-SSX3, SS18-SSX3Δaa43,44 (MI→KR). See also Figure S5A.
(G) Quantitative densitometry depicting BAF47/Brg protein ratios in IP studies. Error bars= s.d. (H) Sox2 mRNA levels in human fibroblasts day 15 post-infection with LV containing various constructs. See also Figure S5B. Error bars=s.d.

Cigall Kadoch, et al. Cell. 2013 March 28;153(1):71-85.
6.
Figure 3

Figure 3. SS18-SSX1 ejects BAF47 and wild-type SS18 to recapitulate BAF complex phenotype in synovial sarcoma cells. From: Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma.

(A) N-terminal GFP-tagged constructs of SS18 FL, SS18 1-379 (-8aa), and SS18-SSX.
(B) Anti-GFP IP of BAF complexes 72 hrs post-transfection with various pEGFP constructs in 293T fibroblasts. See also Figure S3.
(C) Immunoblot analysis on total protein isolated from transfected 293T cells at time t=0 hrs to t=168 hrs post- transfection with GFP-SS18-SSX.
(D) Top, Cyclohexamide (CH) chase treatment of 293T cells, t=0 to t=24 hours, +/− MG-132 proteosome inhibitor. Bottom, quantitative densitometry of BAF47 protein levels on immunoblot.
(E) Immunoblot analysis for BAF47 protein in Aska-SS cells treated with MG-132 proteosome inhibitor for t=8 and t=16 hours.
(F) Glycerol gradient analyses on 293T cells infected with lentivirus (LV) containing either empty vector (top half) or SS18-SSX (bottom half).
(G) shRNA-mediated knock-down (KD) of SS18-SSX and wild-type SS18 in Aska-SS cells.
(H) Cell proliferation analyses of Aska-SS cells infected with shScramble control vector or delivery constructs containing shRNA KD to BAF subunits. Cells plated in triplicate at 10^5 cells/well per condition. Error bars, s.d. of n=3 experiments.
(I) Cell proliferation analyses of human primary neonatal foreskin fibroblasts infected with shScramble control vector or shRNA KD to BAF complex subunits. Cells plated in triplicate at 10^5 cells/well per condition. Error bars, s.d. of n=3 experiments.

Cigall Kadoch, et al. Cell. 2013 March 28;153(1):71-85.
7.
Figure 6

Figure 6. Reversible integration, gene expression and occupancy by SS18 and SS18-SSX containing mSWI/SNF (BAF) complexes. From: Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma.

(A) Denaturation studies using 0-8M urea with subsequent immunoblot analysis for SS18 in 293T cells and SS18-SSX in Aska-SS cells. See also Figure S6.
(B) Quantitative densitometry of SS18 or SS18-SSX1 protein immunoblots from n=3 experimental replicates of urea denaturation 0<[urea]<8M. Y-axis: band quantitation/ untreated control. Error bars= s.d.
(C) IP using anti-V5 antibody in urea treated nuclear extracts isolated from 293T fibroblasts infected with either V5-SS18 or V5-SS18-SSX with immunoblotting for BAF complex components.
(D) Left, Immunoblot analysis on total protein isolated from Aska-SS cells with either SS18 or SS18 1-379 or SS18-SSX1 introduced via LV. Right, anti-Brg IP of complexes in either empty vector or V5-SS18FL treated conditions.
(E) Introduction of SS18-SSX1 and shBAF47 into 293T cells with subsequent immunoblot analysis on total protein.
(F) Cell proliferation analyses of Aska-SS cells infected with control vector, SS18, SS18 1-379, and SS18-SSX. Error bars= s.d.
(G) Sox2 mRNA relative expression (normalized to GAPDH) 10 days post infection with LV containing either control shScramble or overexpression of SS18, SS18 1-379, or SS18-SSX. Error bars= s.d.
(H) Left, anti-BAF155 ChIP on Aska-SS cells treated with either empty vector or SS18 FL, with subsequent qPCR for regions at the human Sox2 promoter and two Sox2 transcription factor (TF) binding sites within the exon. Right, anti-H3K27me3 ChIP at Sox2 locus in Aska-SS control treated and SS18 FL-treated cells. Error bars= s.d.

Cigall Kadoch, et al. Cell. 2013 March 28;153(1):71-85.

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