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Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

Abstract

It remains unknown if biophysical or material properties of biomolecular condensates regulate cancer. Here we show that AKAP95, a nuclear protein that regulates transcription and RNA splicing, plays an important role in tumorigenesis by supporting cancer cell growth and suppressing oncogene-induced senescence. AKAP95 forms phase-separated and liquid-like condensates in vitro and in nucleus. Mutations of key residues to different amino acids perturb AKAP95 condensation in opposite directions. Importantly, the activity of AKAP95 in splice regulation is abolished by disruption of condensation, significantly impaired by hardening of condensates, and regained by substituting its condensation-mediating region with other condensation-mediating regions from irrelevant proteins. Moreover, the abilities of AKAP95 in regulating gene expression and supporting tumorigenesis require AKAP95 to form condensates with proper liquidity and dynamicity. These results link phase separation to tumorigenesis and uncover an important role of appropriate biophysical properties of protein condensates in gene regulation and cancer.

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Fig. 1: AKAP95 regulates cancer cell growth and gene expression.
Fig. 2: AKAP95 directly regulates splicing of key transcripts for cancer.
Fig. 3: AKAP95 is dispensable for normal cell growth but required for transformation and suppressing oncogene-induced senescence.
Fig. 4: AKAP95 undergoes phase separation with liquid-like properties in vitro.
Fig. 5: AKAP95 forms dynamic foci in cell nucleus.
Fig. 6: AKAP95 condensation requires tyrosine in 101–210 and regulates splicing.
Fig. 7: YF mutation enhances AKAP95 condensation propensity and renders condensates a more solid-like state.
Fig. 8: Regulation of tumorigenesis and gene expression by AKAP95 requires its condensation with proper biophysical properties.

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Data availability

The RNA-seq data have been deposited in Gene Expression Omnibus database under accession no. GSE122308. Previously published RNA-seq and RIP-seq data that were re-analysed here are available under accession code GSE81916. The human data for 28 cancer types were derived from the TCGA Research Network: http://cancergenome.nih.gov/. Data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank L. Wang for providing the MDA-MB-231 cells, and Y.G. Tusing for technical assistance with mice work. This work was supported by start-up funds from the University of Alabama at Birmingham and the University of Virginia, and a Department of Defense Breast Cancer Research Program Breakthrough Award (BC190343). The confocal microscopy system at the Keck Center of the University of Virginia was supported by a grant from NIH (OD016446; PI: Ammasi Periasamy). H.J. is a recipient of the American Society of Hematology Scholar Award, the American Cancer Society Research Scholar Award (RSG-15-166-01-DMC) and the Leukemia & Lymphoma Society Scholar Award (1354–19). F.P. and M.A.D. were supported in part by a grant from the NSF (MCB-1615701). M.A.D. and E.G. were funded by NIH grant no. P41-GM103540.

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Authors and Affiliations

Authors

Contributions

H.J. conceived, designed and supervised the study, and wrote the manuscript. W.L. designed and performed molecular and functional analyses for tumorigenesis, and most studies on protein condensation in vitro and in vivo, and also participated in manuscript writing and editing. J.H. made most constructs and cell lines, initiated and participated the protein condensation assays in vitro and in vivo, and performed molecular and functional analyses. B.S. conducted all bioinformatic analyses and performed molecular analyses. F.P. performed and analysed the RICS experiments under the guidance of M.A.D. and E.G.

Corresponding author

Correspondence to Hao Jiang.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 AKAP95 regulates cancer cell growth and gene expression.

a, RNA-Seq–derived gene expression levels from TCGA were analysed by UALCAN portal. Box plot analysis shows relative expression of AKAP95 in 28 types of cancer (red box) versus normal (blue box) samples, unless indicated for different cancer stages or tumour grades for certain cancer types. Cancer type in red font has significantly higher AKAP95 expression in cancer than in normal (or in later stage than in earlier stage). Cancer type in blue font has significantly lower AKAP95 expression in cancer than in normal samples (or in later stage than in earlier stage). Numbers on the left of the plot stand for the number of samples. Data are median (line), 25–75th percentiles (box) and minimum-maximum values recorded (whiskers). b, Assays for cell proliferation by BrdU incorporation (top) and apoptosis by Annexin V staining (bottom) for control and AKAP95-KD MDA-MB-231 cells. Images of flow cytometry results are shown (left), and percentages of cells positive for BrdU or Annexin V are shown as mean ± SD from n = 3 independent KD assays. c, MDA-MB-231 cells were infected to express scramble (control) or AKAP95 shRNA #1 (KD) and indicated constructs. Top, immunoblotting of total cell lysates. Middle, images of these cells seeded at high (5 ×104 cells/well in 6-well plate, top) and low (400 cells/well in 24-well plate, bottom) densities and stained with crystal violet. Bottom, cell numbers (seeded at high densities) as mean from 2 independent experiments. d, MCF7 cells were infected to express scramble control shRNA or two AKAP95 shRNAs. Bottom, images of indicated cell colonies stained with crystal violet. Top, cell numbers were quantified and presented in the bar graph as mean from 2 independent experiments. e, Top 10 gene sets enriched in genes down- (top, n = 951 genes) and up- (bottom, n = 294 genes) regulated by AKAP95 KD in MDA-MB-231 cells. NES, normalized enrichment score. P values by two-sided Student’s t-test for a-d and modified Fisher’s exact test for e. Uncropped blots and statistical source data are provided as in Source Data Extended Data Fig 1.

Source data

Extended Data Fig. 2 AKAP95 directly regulates splicing of key transcripts for cancer.

a,b, MDA-MB-231 cells virally expressing control or indicated shRNAs (a) or shRNA combined with indicated constructs (b) were subject to immunoblotting of total cell lysates (top) and colony formation assay. Middle, colony numbers as mean ± SD from n = 3 (a) or mean from 2 (b) independent experiments. Bottom, images of cells stained with crystal violet. c, Top, mRNA-seq profiles for CCNA2 in control or AKAP95 KD 293 cells33. The numbers of exon junction reads are indicated. The red asterisk at the gene diagram indicates a stop codon 57 bp downstream of exon 1 in the intron. The number of reads for the junction of exons 1 and 2, and for the average neighbouring exons, and their ratios are in the tables below for indicated cells. d, Total RNAs were used for RT-PCR for intron 1 region in control and AKAP95-KD MDA-MB-231 cells treated with or without cycloheximide for 6 h. Repeated 3 times. e, Relative mRNA levels of UPF in UPF1-KD samples and BTZ in BTZ-KD samples, respectively, each relative to the control samples, as determined by RT-qPCR and normalized to GAPDH. Mean is from 2 independent experiments. f, Relative expression level of TGF-β pathway genes based on RNA-seq reads from control and AKAP95-KD MDA-MB-231 cells expressing vector or AKAP95. Venn diagram shows numbers of TGF-β pathway genes (from GSEA) downregulated by AKAP95 KD and upregulated by rescue with AKAP95 expression, and the relative expression of the 16 overlapped genes in both categories are plotted. One representative analysis from 2 repeats. g, RIP-seq profiles showing AKAP95 binding to RPUSD3 and PPM1K pre-mRNAs. Track information is the same as in Fig. 2c. Red circles indicate the alternatively included exons (corresponding to the middle exon in the gene diagrams in (h), and red boxes show AKAP95 binding at the introns flanking these exons. h, Sashimi plots showing that the alternative splicing of RPUSD3 and PPM1K pre-mRNAs was affected by AKAP95 KD and rescued by restored expression of AKAP95. The numbers of exon junction reads and PSI are indicated. P values by two-sided Student’s t-test for a and b. Uncropped blots and statistical source data are provided as in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 AKAP95 is required for transformation and overcoming oncogene-induced cellular senescence.

a, Schematic of the “Knockout-first” Akap95 allele (null without further recombination). b, Body weights of mice with indicated Akap95 genotypes. Not significant (n.s.) between any two groups at any time for male, or after week 26 for female. *between Het and KO before week 26 for female. c, Peripheral blood profiles of 8-week mice of indicated genotype. n in b, c refers to the number of mice analysed. d, MEFs were derived from mouse embryos. Images of six embryos from the same litter were shown at the top, followed with genotyping results, Ponceau S staining, and immunoblotting of MEFs. e, Top, heatmap showing relative expression levels of genes and clustered by changes in un-transduced KO MEFs from 2 embryos each. It includes 203 and 20 genes down- or upregulated in KO, respectively. Bottom, heatmap showing relative alternative splicing of genes clustered by PSI changes. It includes 285 and 332 alternative splicing events with decreased or increased PSI in KO, respectively. Also see Supplementary Table 2f and 7. f, Top 10 gene sets enriched in genes down- (left, n = 265 genes) and up- (right, n = 742 genes) regulated in the MYC-transduced KO versus Het MEFs. g, Rescue of the gene expression profile by introduction of human AKAP95 into the MYC-transduced KO MEFs as shown by immunoblotting and heatmap for relative expression of down- or upregulated genes in the indicated cells. Also see Supplementary Table 2b. Repeated 2 times. h, Relative expression of Akap95 and Akap8l in un-transduced (-MYC) and MYC-transduced (+MYC) MEFs from n = 2 Het and two KO embryos, as determined by normalized RNA-seq reads. i, Heatmap showing relative alternative splicing of genes clustered by PSI changes in MYC-transduced MEFs from 2 embryos each. It includes 216 and 252 alternative splicing events with decreased or increased PSI in KO versus Het MEFs, respectively. Also see Supplementary Table 2c. j, Gene ontology analysis for the indicated gene clusters from the heatmap in i. Blue (n = 216 genes) and red (n = 252 genes) show functions significantly enriched in genes with PSI increase or decrease by KO, respectively. k, Sashimi plots showing alternative splicing changes for each gene cluster from the heatmap using two examples, Asb7 for cluster 1, and Xpo4 for cluster 2. ns, not significant, *P < 0.05, by two-sided Student’s t-test for b, one-way ANOVA for c and modified Fisher’s exact test for f, j. Uncropped blots and statistical source data are provided as in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 AKAP95 phase separation in vitro.

a, 293T cells were transfected with either empty vector (vec), or indicated AKAP95 construct with FLAG-HA-tag. Following α-Flag IP, the pulldown proteins were boiled and resolved by SDS-PAGE and detected by immunoblotting with α-HA. Blue and red asterisks indicate monomer and dimer, respectively. b, Identification of 1–100 as a probable prion subsequence on AKAP95. By the PLAAC program, using homo sapiens as background and core length of 30. c, Purified MBP and MBP fused to AKAP95 truncations as indicated or full-length AKAP95 (1–692) were resolved on SDS-PAGE and stained with Coomassie blue. d, MBP fused to AKAP95 truncations as indicated or full-length AKAP95 were resolved on SDS-PAGE and stained with Coomassie blue following treatment with TEV protease. Note that the cleaved MBP serves as a better indicator for cleavage efficiency as staining signal various for protein fragments of different sequences and sizes. e, Another event of fusion of two droplets formed by 50 µM MBP-AKAP95 (101–210) in 30 mM NaCl and 10% of PEG 6000 after treatment with TEV protease for 30 min. Scale bar, 5 µm. Also see Supplementary Video 1. f, Quantification of nuclear AKAP95 concentration by anti-AKAP95 Western blot. Total lysates from indicated number of MDA-MB-231 (M231) and flp-TREx 293 cells (f293, un-induced and dox-induced for FH-AKAP95 expression) were loaded, along with indicated ng of purified MBP-AKAP95. AKAP95 signal of un-induced f293 is similar to that of 25 ng of MBP-AKAP95. All experiments were repeated 2 times. Uncropped blots are provided as in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 AKAP95 partially localizes in nuclear speckles and with actively transcribing Pol II.

Fluorescence microscopy images of HeLa cells transiently expressing AKAP95-GFP. Nucleus DNA was stained by DAPI, and specific proteins were stained with antibodies for SRSF2 (a), Pol II (b), and Pol II-S2P (c). The assays were repeated 10 times for each staining and show similar trend. Right, quantification of the signal intensity of indicated molecules across the dotted lines shown in the images. Quantification by ImageJ. Scale bar, 5 µm for all. Statistical source data are provided as in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 AKAP95 condensation requires Tyrosine in 101–210 and regulates splicing.

a, Amino acid enrichment for AKAP95 (101–210). By Composition Profiler, using SwissProt 51 Dataset as background. b, Purified MBP fused to AKAP95 (101–210) WT and mutants were resolved on SDS-PAGE and stained with Coomassie blue. Repeated 2 times. c, Disorder plot of AKAP95 WT or mutants with indicated mutations in 101–210. d, MBP-AKAP95 (101–210) WT, YS, and YF, all at 35 µM and in 30 mM NaCl, were treated with TEV protease for 2 hrs in 3 independent assays, and subjected to centrifugation. The supernatant and pellets (resuspended in the same volume as the supernatant) were resolved by SDS-PAGE followed with coomassie blue staining. MBP signal in the pellet reflects residual supernatant fraction, and its percentage [MBP pellet/(supernatant + pellet)] was subtracted from the (101–210) pellet percentage. Such normalized (101–210) pellet percentages are plotted as Partition Percentage as mean ± SD of n = 3 independent experiments. It is most likely that all supernatants may also have substantial portion of condensates. Moreover, the size cutoff of condensates is also arbitrary, as protein assemblies may take a continuum of size distribution71. e, Immunoblotting by α-AKAP95 (top) or GAPDH (bottom) of total lysates from Flp-In T-Rex 293 cell lines induced to express full-length AKAP95 WT or indicated mutants fused to GFP. Repeated 3 times. f, g, Indicated full-length AKAP95 WT or mutants fused to GFP were induced by various concentrations of doxycycline in Flp-In T-Rex 293 cell lines. Immunoblotting of total cell lysates with indicated antibodies (f). The doxycycline concentrations in red font activated the transgene at the near endogenous level, and were selected for treating cells and fluorescence microscopy assays of fixed cells in (g). Scale bar, 5 µm. Repeated 2 times. h, 293T cells transiently expressing indicated constructs with FLAG-HA-tag were used for α-FLAG immunoprecipitation and immunoblotting with indicated antibodies and Ponceau S staining. Repeated 2 times. i, Immunoblotting of 293T cells transfected with empty vector or indicated FLAG-HA-tagged AKAP95 chimeras fused to GFP. Bottom, by anti-GAPDH. Top, by anti-AKAP95 (Bethyl Laboratories, A301–062A, recognizes an epitope in a region between residue 575 and 625 of human AKAP95). Repeated 2 times. P values by two-sided Student’s t-test for d. Uncropped blots and statistical source data are provided as in Source Data Extended Data Fig. 6.

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Extended Data Fig. 7 YF mutation alters material properties of AKAP95 condensates.

a, OD450 at different time after TEV protease treatment of 50 µM MBP-AKAP95 (101–210) WT, YS, and YF in 150 mM NaCl, as mean ± SD of readings after subtracting that of MBP at each time (constant at 0.07–0.08) from n = 3 independent assays. Dashed lines show half of the maximum turbidity and time (τ1/2) to reach it. b, Ratio of protein concentration inside the droplets over sum of inside and outside for (101–210) WT and YF at increasing protein concentrations and in 30 mM NaCl, as mean ± SD from n = 6 randomly picked droplets each, in one representative assay from 3 repeats based on Fig. 7a. c, Confocal microscopy images of GFP-AKAP95 (101–210) WT and YF at increasing protein concentrations, all in 150 mM NaCl and 10% of PEG 6000 after TEV protease treatment for 20 min. Repeated 2 times with similar results. d, DIC and fluorescence microscopy images for 20 µM MBP-AKAP95 (101–210) YF spiked with Oregon-green-labelled same protein (molar ratio 10:1), after TEV protease treatment for 30 min. Changes in NaCl concentration is indicated. Images were taken 5 min after salt adjustment. Repeated 2 times with similar results. e, Different extent of droplet fusion (arrows) by AKAP95 (101–210) WT and YF, both at 50 µM and in 30 mM NaCl and 10% of PEG 6000 after TEV protease treatment for 30 min. A similar trend was observed in 5 fusion events (or attempted fusion for YF) for each. Also see Supplementary Videos 46. f, Equation for Line Raster Scan Image Correlation fitting autocorrelation G(Ψ), which depends on G(0)=γ/N (γ: beam profile, N = number of mobile particles), diffusion coefficient D (µm2/s), line scan time tl, pixel size Ψ, radial beam waist w0 and axial beam waist wz. The radial waist w0 (0.218 µm) was calibrated with sub-diffraction beads (0.1 µm) diluted solution as reported before72. The axial waist wz was considered equal to 3*w0. g, Fluorescence confocal microscope image of HeLa cell expressing full-length AKAP95 WT or YF fused to GFP. Arrow indicates the scanned region for in vivo Line RICS. h, Fluorescence confocal microscope image of GFP-AKAP95 (101–210) WT and YF in 150 mM NaCl and 10% of PEG 6000 after TEV protease treatment for 20 min, for in vitro Line RICS experiments. i, Line Fluorescence carpet formed by ~104 lines (each line is composed by 128 pixels, 50 nm/pixel) acquired with 0.101 s line scan time and 32.8 µs/pixel. Line RICS autocorrelation curves were computed on 64 sections of 128 lines followed by averaging all the curves. The sectioning of the line carpet permitted to avoid the effect of the movement of the condensates on the measurement of the autocorrelation curves. (λex=488 nm). j, Line RICS autocorrelation curves for experimental data and fitted. k, Residuals of the fitting. Experiments in gi were repeated 3 times with similar results. Scale bar, 2 µm for c and h, 5 µm for d, e, and g.

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Extended Data Fig. 8 Regulation of tumorigenesis and gene expression by AKAP95 requires its condensation with appropriate material properties.

a,b, MDA-MB-231 cells were virally infected to stably express scramble (control) or AKAP95 shRNA #1 (KD) and the indicated constructs including empty vector (vec) and FLAG-HA-tagged full-length AKAP95 WT or mutants. a, Relative CCNA2 mRNA level as determined by RT-qPCR and normalized to GAPDH, and plotted for each of the 2 biological repeats individually. b, RT-PCR for ratios for exon-included over -skipped PPM1K transcript, as mean ± SD from n = 3 biological repeats. c-f, MYC-transduced Akap95 KO MEFs were transduced with vector or constructs expressing HA-tagged full-length AKAP95 WT or mutants. c, Heatmap showing relative expression levels of genes changed in MYC-transduced KO MEFs (from 2 embryos each) stably expressing indicated rescue constructs. Also see Supplementary Table 2d. d, Relative mRNA levels of indicated SASP genes as determined by RNA-seq reads from 2 biological repeats (KO1 and KO2). e,f, Sashimi plots showing example genes for which the alternative exon inclusion was promoted (e) or suppressed (f) by introduction of AKAP95 WT, but not as effectively by YS or YF, and RT-PCR for the inclusion of the alternative exon, as mean from 2 embryos each. g, A model for how AKAP95 condensates may regulate gene expression for tumorigenesis. P values by one-way ANOVA followed by Tukey’s post hoc test. Uncropped blots and statistical source data are provided as in Source Data Extended Data Fig. 8.

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Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1 Changes of gene expression and alternative splicing by AKAP95 KD and rescue in TNBC cells. This table contains two tabs as explained below: a, Gene expression changes by AKAP95 KD and rescue in TNBC cells. By expression analyses of the RNA-seq results from MDA-MB-231 cells that were virally infected to stably express scramble control shRNA or AKAP95 shRNA (KD) as well as vector or AKAP95 construct. Shown are steps to transform the expression values to generate heatmap as described in Materials and Methods. b, Changes of gene alternative splicing by AKAP95 KD and rescue in TNBC cells. By splicing analyses of the RNA-seq results from MDA-MB-231 cells that were virally infected to stably express scramble control shRNA or AKAP95 shRNA (KD) as well as vector or AKAP95 construct. Supplementary Table 2 Changes of gene expression and alternative splicing by Akap95 KO and rescue in MYC-transduced MEFs. This table contains seven tabs as explained below: a, Gene expression changes by Akap95 KO in MYC-transduced MEFs. By expression analyses of the RNA-seq results from MYC-transduced Akap95 Het and KO MEFs derived from n = 2 embryos for each. Shown are steps to transform the expression values to generate heatmap as described in Materials and Methods. b, Gene expression changes by Akap95 KO and rescue in MYC-transduced MEFs. By expression analyses of the RNA-seq results from MYC-transduced Akap95 Het, and KO MEFs, and KO MEFs that were introduced with vector or AKAP95 WT. c, Changes of gene alternative splicing by Akap95 KO in MYC-transduced MEFs. By splicing analyses of the RNA-seq results from MYC-transduced Akap95 Het and KO MEFs derived from n = 2 embryos for each. d, Gene expression changes by AKAP95 expression in MYC-transduced Akap95 KO MEFs. By expression analyses of the RNA-seq results from MYC-transduced Akap95 KO MEFs (from n = 2 embryos) that were introduced with vector, AKAP95 WT, YS or YF mutant. e, Changes of gene alternative splicing by AKAP95 expression in MYC-transduced Akap95 KO MEFs. By splicing analyses of the RNA-seq results from MYC-transduced Akap95 KO MEFs (from n = 2 embryos) that were introduced with vector, AKAP95 WT, YS or YF mutant. f, Gene expression changes by AKAP95 expression in Akap95 KO MEFs. By expression analyses of the RNA-seq results from un-transduced Akap95 Het and KO MEFs derived from n = 2 embryos for each. g, Changes of gene alternative splicing by AKAP95 expression in Akap95 KO MEFs. By splicing analyses of the RNA-seq results from un-transduced Akap95 Het and KO MEFs derived from n = 2 embryos for each. Supplementary Table 3 AKAP95 chimeras information Information of the IDRs from four different proteins that were used to replace the 101–210 region of AKAP95 to make AKAP95 chimeras. Supplementary Table 4 Information for Primers, shRNAs and siRNAs.

Supplementary Video 1

AKAP95 droplets in vitro are highly dynamic and can fuse. Purified MBP-AKAP95 (101–210) formed droplets at 50 µM, in 30 mM NaCl and 10% of PEG 6000 after treatment with TEV protease for 30 min. The yellow triangle points to a fusion event of two droplets. Played at 134x speed. Repeated three times.

Supplementary Video 2

Rapid fusion of AKAP95 foci in the cell nucleus. Full-length AKAP95 (ZFC-S)-GFP foci in a HeLa cell nucleus following transfection. The arrows point to two different fusion events. Played at 20x speed. Repeated three times.

Supplementary Video 3

FRAP assays for intracellular AKAP95. Representative FRAP assays for AKAP95 fused to GFP and transfected into HeLa cells. Played at 20x speed. Repeated three times.

Supplementary Video 4

Different extent of droplet fusion by AKAP95 WT and YF. Purified AKAP95 (101–210) WT and YF both at 50 µM in 30 mM NaCl and 10% of PEG 6000 after treatment with TEV protease for 30 min. Yellow square and green circle highlight the fusion event for YF and WT, respectively. These Supplementary Videos are the first 100-s excerpts from Supplementary Supplementary Videos 5 and 6. Repeated three times.

Supplementary Video 5

AKAP95 WT droplets in vitro. Purified AKAP95 (101–210) WT at 50 µM in 30 mM NaCl and 10% of PEG 6000 after treatment with TEV protease for 30 min, and were videoed under microscope for 60 min in total, with one image every 4 s. Played at 20x speed. Repeated three times.

Supplementary Video 6

AKAP95 YF droplets in vitro. Same as Supplementary Video 5 except using purified AKAP95 (101–210) YF.

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Li, W., Hu, J., Shi, B. et al. Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis. Nat Cell Biol 22, 960–972 (2020). https://doi.org/10.1038/s41556-020-0550-8

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