Cell culture

HEK293T cells (ATCC, CRL-1126) and SW982 cells (ATCC, HTB-93) are cultured in DMEM supplemented with 15% FBS, GlutaMAX and NEAA. BJ fibroblasts (MEISEN CELL, CTCC-400-0144) are cultured in DMEM/F12 supplemented with 15% FBS, GlutaMAX, and NEAA. The synovial sarcoma cell line HS-SY-II was purchased RIKEN BRC (RCB22316). HS-SY-II cell line was cultured using standard protocols in DMEM low glucose medium (Gibco) supplemented with 10% FBS. Each cell line was grown in a humidified incubator at 37 °C with 5% CO2. We used PRT4165 (TargetMol T3110) to block the H2AK119ub modification. All the cell lines have been confirmed as mycoplasma-free with Beyotime C0297M.

Stable gene expression and gene knockout sgRNA constructs

Constitutive expression of SS18, SS18-HA, SS18-SSX1 and SS18-SSX1 AR-mutations was obtained using an EF1alpha-driven expression vector (pKD-EF-1a-MCS-EGFP/mCherry-IRES2-Puro or pKD-EF-1a-MCS-EGFP/mCherry-IRES2-Blast) expressed in cells by lentiviral infection and selected with puromycin (2 μg/ml) or blasticidin (10 µg/ml) after 48 h. Constitutive expression of sgRNA targeting the region of C-SSX of the SS18-SSX fusion (5′-gCATGCCCAAGAAGCCAGCAG-3′) or a NT non-targeting control (5′-GGTAGCGAACGTGTCCGGCGT-3′) was obtained using lentiviral infection of the Lenti-Cas9 -Blast vector with blasticidin (2 μg/ml) selection.

Lentivirus generation

Lentivirus production was obtained from PEI (Polysciences, #23966) transfection of HEK293T cells with co-transfection of the packaging vectors pSPAX2 and pMD2.G along with the gene delivery vector. Viral supernatants were collected 48 h after transfection. For infection, the viral pellets were added to cells in a dropwise manner in the presence of polybrene. After 8 h, medium containing the lentivirus was replaced. Infected cells were selected by the addition of puromycin (2 μg/ml) or blasticidin (10 µg/ml) after 48 h.

qRT–PCR

Total RNAs were prepared with TRIzol. For quantitative PCR, cDNAs were synthesized with ReverTra Ace (Toyobo) and oligo-dT (Takara), and then analyzed by qPCR with ChamQ SYBR qPCR Master Mix (Vazyme). The qPCR primer sequences are listed in Supplementary Table S1.

Immunofluorescence

Cells growing on coverslips were washed three times with PBS, then fixed with 4% PFA for 30 min, and subsequently penetrated and blocked with 0.1% Triton X-100 and 3% BSA for 30 min at room temperature. Then, the cells were incubated with primary antibody (H2AK119ub, CST, #8240; SS18-SSX, CST, #72364; SS18, Santa Cruz, sc-374299) for 1.5 hour. After 5 washes in PBS, one hour of incubation in secondary antibodies (Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568, A11011; Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, A-11008; Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, A-11001; Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647, A-21245), cells were then incubated in DAPI (Sigma-Aldrich, D9542) for 2 min. Then, the coverslips were mounted on the slides for observation on the confocal microscope (ZEISS, LSM 900).

1,6-hexadiol stimulation experiment

SS18-EGFP and SS18-SSX1-EGFP proteins were overexpressed in BJ cells, respectively, and then planted at the bottom of a glass culture dish of about 0.16 mm. Cultivate overnight in a 37 °C, 5% CO2 incubator to allow complete adhesion to the wall. Relevant experiments were filmed using a Zeiss 900. Open the temperature control module of the Zeiss 900 instrument to provide a suitable environment for the cells. Take the Zeiss 900 laser channel and set the appropriate laser intensity before starting. Carefully remove the supernatant with a pipette, quickly add the medium containing 1,6-hexadiol. Processing duration was recorded, and the condensate state was observed. Imaging was performed with Zeiss 900 confocal microscope.

Fluorescence bleaching recovery experiment

SS18-EGFP and SS18-SSX1-EGFP proteins were overexpressed in BJ cells, respectively, and then planted at the bottom of a glass culture dish of about 0.16 mm. Cultivate overnight in a 37 °C, 5% CO2 incubator to allow complete adhesion to the wall. FRAP experiments were performed using a Zeiss 900 equipped with a fluorescence bleach recovery module. Turn on the temperature control module and CO2 in advance before use to provide suitable growth conditions for living cells. Before fluorescence bleaching, take two consecutive photos to make sure that the selected shooting laser intensity will not bleach the EGFP protein. Bleaching is performed using high-energy laser photography, with a laser wavelength of 488 nm. The diameter of the bleached area is ~1 μm. After bleaching, a picture was taken approximately every 1 s until the fluorescence intensity no longer changed. After the experiment is completed, Zeiss’ own software is used to process the image and collect fluorescence intensity values. After normalization, prism software was used to perform curve fitting to draw a fluorescence bleaching recovery curve, and prism software was used to calculate the average recovery half-life value.

Detection sensitivity of condensates to high temperatures

The temperature sensitivity of phase-separated proteins can be considered as one of the characterized properties of their phase separation capabilities. We first seeded BJ cells overexpressing the SS18-EGFP and SS18-SSX1-EGFP proteins in 24-well plates lined with glass slides. Set the incubator temperature to 40 °C and 42 °C. The 24-well plates were then transferred to the corresponding incubator. Cells were fixed with 4% paraformaldehyde immediately after the appropriate duration. The state of the condensates was observed under an inverted fluorescence microscope. Cells were permeabilized with 0.1% Triton X-100 and stained with DAPI. Then, the coverslips were mounted on the slides for observation on the confocal microscope (ZEISS, LSM 900).

EdU cell proliferation assay

The proliferation of cells was detected using EdU cell proliferation assay according to the reagent instructions. About 3 × 104 cells were seeded in 48-well plates and maintained for 24 h before the assay. A total of 200 µL EdU (10 µM) reagent (Beyotime, C0078S) was added to each well and incubated for 6 h to label the cells. After two times wash with PBS, cells were fixed in a 4% paraformaldehyde solution (Sangon, E672002-0500) for 30 min, permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, 9036-19-5) for 15 min, and then incubated with the click-reaction reagent for 30 min at room temperature in the dark environment. After the end of each of the above steps, wash three times with PBS containing 3% BSA for 3–5 min each time. In all, 1× Hoechst33342 reagent was used to counterstain the nucleus. The result of staining was observed with a fluorescence microscope system ZEISS, and the data were collected by the ImageJ software.

Co-immunoprecipitation experiment

First, EGFP (Control), SS18-SSX1-EGFP-Flag (SS-Flag), and SS18-SSSX1(AR Mut)-EGFP-Flag (AR Mut-Flag) proteins were overexpressed in 293T cells, respectively. Ten million cells were harvested three days later for the co-immunoprecipitation experiments. Then, resuspend cell pellet in 200 µl ice-cold lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1% SDS, 1% Triton X-100) supplemented with protease inhibitor cocktail and 1 mM PMSF. Sonasonic with an ultrasound instrument (Bioruptor plus, SIA-UH008) for 12 cycles. Place the tube on ice for 30 min and extensively pipette the suspension every 10 min. Centrifuge cell lysate at 12,000 × g for 10 min at 4 °C. Transfer cleared lysate (supernatant) to a pre-cooled tube and add 300 µl Wash buffer(10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA) supplemented with 1 mM PMSF and protease inhibitor cocktail. 100 µl of the diluted lysate was saved for further analysis (input). Add 5x SDS-sample buffer into input sample, after 99 °C for 10 min, and set aside. After, Preparation of the beads and the protein binding. Resuspend the Flag beads (DYKDDDDK tag Nanoselector Magnetic beads, Code: 016-101-003) by gently pipetting up and down or by inverting the tube. Transfer 25 µl of bead slurry into 1.5 ml reaction tube. Add 500 µl ice-cold wash buffer. Separate the beads with a magnet until the supernatant is clear. Discard the supernatant. Add diluted lysate to the equilibrated beads. Incubate 4 °C spin for 1 h. At last, Washing and Elution with 1× SDS-sample buffer. Separate the beads with a magnet until the supernatant is clear. Discard the remaining supernatant. Washed four times with 500 µl wash buffer. During the last washing step, transfer the beads to a new tube. Remove the remaining supernatant. Resuspend beads in 100 µl 1× SDS-sample buffer. Boil beads for 10 min at 99 °C to dissociate immunocomplexes from beads. Separate the beads with a magnet. Supernatants were subjected to SDS–PAGE and incubated with corresponding primary antibody(Anti-DYKDDDDK tag, AlpHcAbs® Rabbit antibody, Code: 016-203-001; H2AK119ub, CST, #8240) and secondary antibodies(HRP-labeled Goat Anti-Rabbit IgG(H + L), Beyotime, A0208).

CUT&Tag and data analysis

The CUT&Tag experiments of SS18, SS18-SSXand H2AK119ub were constructed with NovoNGS CUT&Tag 3.0 High-Sensitivity Kit for Illumina (novoprotein N259-YH01) according to the manufacturer’s instructions. Reads form ChIP-seq experiments were mapped to the human genome (hg38) using Bowtie2 (–very-sensitive), and only those reads that mapped once were retained for further analysis. Peaks were called using MACS2 software with the default parameters. The antibodies used were SS18 (CST, #21792), SS18-SSX (CST, #72364), H2AK119ub (CST, #8240), HA (CST, #3724S), Goat anti-Rabbit IgG (Proteintech, B900210) and Rabbit anti-Mouse IgG (Proteintech, B900120).

Statistical information

Data are presented as mean±s.d. as indicated in the figure legends. Unpaired two-tailed student t test, The P value was calculated with the Prism 6 software. A P value < 0.05 was considered as statistically, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. No statistical method was used to predetermine the sample size. The experiments were not randomized. The investigators were not blinded to allocation during the experiment and outcome assessment.