Transcriptomic and protein expression comparison of STAT3-mutated patients against STAT3-WT patients. (A-B) RNA-seq analysis of T-LGL leukemia patients. (A) Volcano plot of DESeq2 output of T-LGL STAT3 mutant vs STAT3 WT. Genes with positive log₂ fold changes are more highly expressed in STAT3 mutants, and genes with negative log₂ fold changes are more highly expressed in STAT3 WT. (B) Gene set enrichment analysis of the nodes from the functional modular network analysis. Positive enrichment scores indicate pathways that are positively enriched in STAT3-mutant patients. Negative enrichment scores indicate pathways enriched in STAT3-WT patients. (C) RT-qPCR comparison of mRNA levels of ZBTB46, AKT3, and PDGFB from CD8⁺-isolated normal controls (n = 3; ZBTB46 n = 2 due to signal below the limit of detection in 1 sample), WT (n = 6; ZBTB46 n = 5 due to signal below the limit of detection in 1 sample), and STAT3-mutant LGL patient samples (n = 5). Analysis of variance (ANOVA) P = .028, .014, and .018 for ZBTB46, AKT3, and PDGFB, respectively. (D) Western blotting of pS473 AKT, total AKT, phospho-p44/42 MAPK, total p44/42 MAPK, pS9 GSK3β, and total GSK3β from CD8⁺-isolated normal controls (n = 3) compared with WT and STAT3-mutant LGL patient samples (n = 5). STAT3 mutation type and LGL registry ID are indicated. (E) RT-qPCR comparison of mRNA levels of STAT3, STAT1, and PDGFRB from CD8⁺-isolated normal controls, WT, and STAT3-mutant LGL patients. ANOVA P = .049, .963, and .026 for STAT3, STAT1, and PDGFRB, respectively. (F) Western blotting of pSTAT3, total STAT3, pSTAT1, total STAT1, total PDGFRβ, and vinculin loading control from CD8⁺-isolated normal controls compared with WT and STAT3-mutant LGL patient samples. STAT3 mutation type and LGL registry ID are indicated. Welch’s or Brown-Forsythe ANOVA was used for all RT-qPCR analyses depending on the distribution of the data. Unpaired t with Welch’s correction as a post hoc test. P values are indicated as follows: *P < .05; **P < .01.