GATA6 acts as an oncogenic gene in CTCL tumor cells through directly regulating CD137L expression. (A) GATA6 expression in lesional skin of CTCL was determined by immunohistochemistry. Diaminobenzidine was used for visualizing the staining and counterstaining with Mayer hematoxylin was performed. Representative results are shown (original magnification ×100). Insets, High-magnification images (original magnification ×400). (B) GATA6⁺ cells were counted per high-power field (×400). (C) Quantitative RT-PCR was performed to measure expression levels of GATA6 using mRNA extracted from CTCL lesional skin (n = 57; 51 MF cases and 6 SS cases) and healthy skin (n = 20). (D) A correlation between GATA6 and CD137L mRNA levels in CTCL lesional skin. (E) Western blotting analysis for GATA6 protein expression was conducted on the lysates of PBMCs from 1 SS patient as well as healthy controls and CTCL cell lines. (F) A correlation between GATA6 and CD137L mRNA levels in CTCL cell lines. (G) ChIP assay in Hut78 and MyLa cells was performed with anti-GATA6 antibody and the primer specific for the designed area of the CD137L gene promoter. (H) Luciferase reporter assay using CD137L promoter constructs. HEK293 cells were cotransfected with CD137L promoter constructs and GATA6 expression vector. Forty-eight hours after transfection, luciferase activity was measured. (I-P) Hut78 and MyLa cells were transfected with either GATA6-targeting shRNA (shGATA6) or control scrambled shRNA (shSCR). (I-J) GATA6 expression levels were assayed by western blotting analysis (I) and quantitative RT-PCR (J). (K-L) CD137L (K) and CXCR4 (L) expression levels were determined by flow cytometric analysis and quantitative RT-PCR. Representative results are shown. (M) Western blotting analysis was conducted on the lysates of shGATA6 and shSCR cells. Expression levels of p-AKT and p-ERK1/2 were measured. (N) shGATA6 or shSCR transduced cells (4.0 × 10⁴ per well) were cultured for 72 hours. Viable cells were counted at indicated time points by trypan blue exclusion. (O) Apoptosis (Annexin V⁺ and 7-AAD⁻) in shGATA6 and shSCR cells was evaluated with Annexin V and 7-AAD staining for flow cytometric analysis. Data are presented as mean plus or minus SD. *P < .05, **P < .01. (P) Hut78-shGATA6 or Hut78-shSCR cells (5.0 × 10⁶) were injected into NSG mice. The tumor volume was calculated using the equation: V = π (L1 × L22)/6, where V = volume (mm³), L1 = longest diameter (mm), and L2 = shortest diameter (mm). Values are means plus or minus SEM (n = 12). *P < .05, **P < .01 by the Mann-Whitney U test compared with control group.