Figure 7. H3K9me³ repression of SATB1 in malignant Sézary patient cells.

(A) CD4⁺CD26^– isolated T cells from peripheral blood apheresis of Sézary patients (n = 4) were cultured in R10 media with 100 U/mL human recombinant IL-2 and treated with increasing doses of SUV39H1/2 inhibitors chaetocin and F5446 (72 hours) as well as romidepsin (72 hours) and GSK126 (48 hours) versus the vehicle control (DMSO) prior to MTT analysis. IC₅₀ values (nM) were calculated for each patient sample for the respective treatments. (B) Summary of IC₅₀ values for chaetocin, F5446, romidepsin, and GSK126 for each malignant sample (n = 4). One-way ANOVA with Tukey’s multiple-comparison test: *P < 0.05; **P ≤ 0.01. (C) RNA was extracted from primary CD4⁺CD26^– Sézary patient cells that were treated with chaetocin, F5446, romidepsin, or vehicle control (DMSO) for 24–36 hours, and SATB1 mRNA expression was quantified by q-PCR normalized to human TBP mRNA. Data pooled from 3 patient samples with 2 independent experiments shown (n = 6). Two-tailed Student’s t test: ****P ≤ 0.0001. (D) Chromatin immunoprecipitation quantified by real-time q-PCR with anti-H3K27me³ (clone C36B11) or control IgG isotype immunoprecipitation from isolated CD4⁺ T cells from peripheral blood of Sézary patients (n = 2) calculated against 2.5% input values. Regions amplified at the predicted occupied region (approximately 4.8 kb)of the SATB1 promoter. Representative of 2 independent experiments. Two-tailed Student’s t test: *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001. (E) Similar to D except with anti-H3K9me³ (Abcam, ab8898) at the approximately 5.6-kb region versus the control region (n = 2). Representative of 2 independent experiments. Two-tailed Student’s t test: *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001. (F) Primary Sézary CD4⁺CD26^– cells (n = 2) were transduced with retrovirus containing human SATB1 and sorted for GFP⁺ and GFP^– cells. Western blot was performed with antibodies against human p-STAT5 and STAT5 protein in cells endogenously expressing SATB1 versus cells with ectopic expression of SATB1 (n = 2). (G) Primary Sézary cells were labeled with CellTrace Violet and were serum starved for 24 hours prior to stimulation with anti-CD3/anti-CD28 beads and cultured in complete medium with 100 U/mL rhIL-2 for 5 days. Proliferation was assessed by CellTrace Violet dilution using FACS for cells ectopically (n = 2) or endogenously (n = 2) expressing SATB1. Experiment was performed on 2 replicates per patient (n = 4). Two-tailed Student’s t test: **P ≤ 0.01.