Extended Data Fig. 4: Inhibition of HDAC6 LLPS condensates by Nexturastat A suppresses TNBC growth in vitro and in vivo.
Related to Fig. 3. a. MDA-MB231 cells were transduced with HA tagged HDAC6 WT or S22F. Co-immunoprecipitation (Co-IP) analysis showing the interaction between HDAC6 and PPP1CA in MDA-MB231 cells. b. Immunoblotting analysis of HDAC6 and phosphorylated HDAC6 levels in MDA-MB231 cells transduced with shPPP1CA and control shRNA. c. Immunoblotting analysis of HDAC6 and phosphorylated HDAC6 in MDA-MB231 cells transduced with shPPP1CA or control shRNA, followed by treatment with 5 μM Nexturastat A or 5 mM HPOB (a reference compound) for 24 h. d. Immunoblotting analysis of HDAC6 and phosphorylated HDAC6 in MDA-MB231 cells treated with or without 5 mM Nexturastat A or 1.5% 1,6-hexanediol. e. Cell viability assays were performed in chondrocytes (as a normal control), MCF-7 cells treated with Nexturastat A for 48 h, n = 3 per group. f. Transwell assay was conducted in BT-549 cells treated with DMSO, HPOB, or Nexturastat A for 24 h, respectively. g. Flow cytometry analysis of apoptotic cells in BT-549 and MDA-MB231 cells treated with DMSO, HPOB, or Nexturastat A. h. Flow cytometry analysis of apoptotic cells in MDA-MB231, BT-549, and MCF-7 cells treated with Nexturastat A at the indicated concentrations. i. Flow cytometry analysis of apoptotic cells in MDA-MB231 cells stably transduced with HDAC6 WT or S22F mutation, with or without treatment of Nexturastat A (5 μM). j. Cell cycle analysis of BT-549 cells treated with Nexturastat A at the indicated concentrations for 48 h, n = 3 per group. k. Representative images of IHC staining and quantification of Ki67-positive cells in MDA-MB231-derived subcutaneous tumors treated with Nexturastat A at the indicated concentration, n = 3 per group. l. Representative immunohistochemistry (IHC) staining for ER, PR, and HER2 in TNBC patient-derived xenografts. m. Weight curve of mice bearing MDA-MB231-derived subcutaneous tumors after treatment with Nexturastat A at the indicated concentration, n = 5 per group. n. Cell viability assays were conducted in MDA-MB231 and BT-549 cells stably transduced with non-targeting scrambled control shRNA (shCtrl) or two HDAC6 shRNAs (shHDAC6-1 and shHDAC6-2), respectively. Related cell viability values are normalized to the control (day 0), n = 3 per group. o. Sphere formation assays were conducted in MDA-MB231 and BT-549 cells stably transduced with shCtrl, shHDAC6-1, or shHDAC6-2, respectively, with or without Nexturastat A (5 μM). N = 3 per group, the number of spheroids was counted on day 7. p. Invasion assays were conducted in MDA-MB231 cells stably transduced with shCtrl, shHDAC6-1, or shHDAC6-2, respectively, with treatment of DMSO or Nexturastat A (5 μM), n = 3 per group. q. Flow cytometry analysis of apoptotic cells in MDA-MB231 and BT-549 cells stably transduced with shCtrl, shHDAC6-1, or shHDAC6-2, respectively, with or without Nexturastat A (5 μM), n = 3 per group. Data in e-k and m-q are shown as the mean ± s.d.; P values in f-h, k were calculated by one-way ANOVA with multiple comparisons. P values in i, n-q were calculated by two-way ANOVA with multiple comparisons. Experiments were repeated three times independently with similar results; representative images are shown in a-d and k-l.
