Extended Data Fig. 3: Role of phosphorylation residue, catalytic domain, and nuclear export sequence (NES) regions in modulating HDAC6 phase separation.
Related to Fig. 2. a. Immunoblotting analysis of HDAC6 in WT and HDAC6 knockout (KO) MDA-MB231 and BT-549 cells. b. Immunofluorescence images of phosphorylated form of HDAC6 (or HDAC6 truncates) when overexpressing HDAC6 deletion segments(ΔIDR1/2) in the cell nuclei of HDAC6 KO MDA-MB231 cells. N = 15 cells per group. Scale bar, 5 μm. c. Analysis of droplet formation in the indicated protein mixture (160 μM each) before and after 5% 1,6-hexanediol treatment. WT refers to the wide-type HDAC6 IDR1 fused with mEGFP. S22F, HDAC6 IDR1 Ser 22 to Phe; S22E, HDAC6 IDR1 Ser 22 to Glu. N = 3 per group. Scale bar, 5 μm. Each point in the statistical chart represents the average diameter of all droplets in each experiment. d. Cell viability assays were performed using CellTiter-Glo in BT-549 and MDA-MB321 cells transduced with HDAC6 wild-type (WT) or mutations (S22E or S22F). N = 3 per group, statistical analysis is conducted after a 72-hour period. e. Sphere formation assay was conducted in BT-549 and MDA-MB321 cells transduced with HDAC6 WT or mutations, and the results were compared with the empty vector control. N = 3 per group, the number of spheres formed was counted on day 7. f. Invasion assays were performed in BT-549 and MDA-MB321 cells transduced with HDAC6 WT or mutations, respectively, n = 3 per group. g. Representative images of orthotopic tumor tissue (day 14) derived from MDA-MB231 cells transfected with HDAC6 WT, S22E, or S22F mutations (6 mice in each group). The tumor volume and weight data (day 14) are also presented. h. Immunofluorescence images of phosphorylated form of HDAC6 (or HDAC6 truncates) when overexpressing HDAC6 deletion segments (ΔBUZ, ΔDD1, ΔDD2, ΔDD) in the cell nuclei of HDAC6 KO MDA-MB231 cells. N = 15 cells per group. Scale bar, 5 μm. i. Immunofluorescence images of phosphorylated form of HDAC6 (or HDAC6 truncates) when overexpressing HDAC6 deletion segments (ΔNES1, ΔSE14, ΔNES2, ΔNES) in the cell nuclei of HDAC6 KO MDA-MB231 cells. N = 15 cells per group. Scale bar, 5 μm. j. Sphere formation assay of BT-549 cells overexpressing the empty vector (EV), full-length (FL), or NES-truncated HDAC6 variants. k. Extreme limiting dilution assay of BT-549 cells overexpressing the empty vector (EV), full-length (FL), or NES-truncated HDAC6 variants (ΔNES). (Left) An extreme limiting dilution algorithm was used to calculate the frequency of cancer stem cells, showing significant differences between EV and ΔNES (P = 0.0492). (Right) The table displays the estimated stem cell frequency and 95% confidence interval (CI). In b, h-i the boxplots represent the median values and quartiles, and the whiskers represent the maximum and minimum values. Data in c-g, j are shown as the mean ± s.d.; P values in b, e-j were calculated by one-way ANOVA with multiple comparisons. P values in c-d were calculated by two-way ANOVA with multiple comparisons. P values in k were calculated by chi-square test. Experiments were repeated three times independently with similar results; representative images are shown in a-c and g-j.
