Extended Data Fig. 5: 14-3-3θ promotes phospho-HDAC6 LLPS in TNBCs.
Related to Fig. 4. a. Schematic diagram depicting the immunoprecipitation-mass spectrometry (IP-MS) experiment performed to identify components in phospho-HDAC6 LLPS condensates. b. The HDAC6 up-regulated interacting proteins upon 8 μM Nexturastat A treatment. c. Co-IP analysis in MDA-MB231 cells transduced with HA-tagged HDAC6 and treated with or without 10 μM Nexturastat A, showing the interaction between HDAC6 and a-Tubulin. EV, empty vector. d. Mass spectrometry images showing the 14-3-3θ interacted with HDAC6. e. qPCR showing the knockdown efficiency of 14-3-3θ in BT-549 cells. f-g. In vitro droplet formation assays demonstrating that HDAC6-WT droplets but not HDAC6-S22F droplets incorporate 14-3-3θ protein. Scale bar = 50 μm. h. Statistical analysis of the diameter distribution of mixed protein droplets formed by HDAC6-WT, HDAC6-S22F, and HDAC6-S22E with 14-3-3θ protein in vitro. i. Quantitative PCR (qPCR) demonstrating the knockdown efficiency of Importin β in BT-549 cells j. Representative super-resolution microscope images showing total HDAC6 localization in BT-549 cells with or without Importin β KD (n = 15). Nuclei were counterstained with DAPI. Scale bars, 5 μm. k. qPCR demonstrating the knockdown efficiency of NUP153, NUP214, NUP88, and NUP98 in BT-549 cells. In j the boxplots represent the median values and quartiles, and the whiskers represent the maximum and minimum values. Data in e and h-k are shown as the mean ± s.d.; P values in e, i-k were calculated by one-way ANOVA with multiple comparisons. P values in h were calculated by two-way ANOVA with multiple comparisons. Experiments were repeated three times independently with similar results; representative images are shown in c, f-g and j.
