Fig 2: Knockdown of ZAP70 does not affect survival of T/NK Lymphoma cell lines.(A) KHYG cell line was treated with si Non Targeting Control (NT) or siZAP70 and immunoblot analysis of the protein expression of phosphorylated ZAP-70, total Zap-70 phosphorylated LAT, total LAT, phosphorylated SLP-76, total SLP-76, phosphorylated p38 MAPk, total p38MAPK were evaluated with Actin as the loading control. (B) KHYG cells which have been either treated with the siNT or si-ZAP-70 was evaluated for apoptosis by Annexin V PI staining. Obatoclax is a positive control demonstrating induction of apoptosis. (C) ZAP70 siRNA knockdown was performed in a wider range of cell lines. Protein expression in WT and ZAP70 knockdown TCL cells (JURKAT and KARPAS-299) and NKTL cells (KHYG1, HANK-1, NK-YS, SNK-1 and SNK-6) cell lines were analysed by immunoblotting. ß- ACTIN was used as a loading control. (D) ZAP70 mRNA was measured in control and ZAP70 siRNA conditions by RT-PCR in TCL cells (JURKAT and KARPAS-299) and NKTL cells (KHYG1, HANK-1, NK-YS, SNK-1 and SNK-6) (E) TCL cells (JURKAT and KARPAS-299) and NKTL cells (KHYG1, HANK-1, NK-YS, SNK-1 and SNK-6) were stained with Annexin V-APC and PI and analyzed by flow cytometry. Obatoclax 5µM treatment is a positive control demonstrating induction of apoptosis. (F) KHYG1cells which have been either treated with the siNT or si-ZAP-70 was evaluated by Cell-Titer Glo (Promega) after 48h.

Fig 3: RNAseq highlights that there are not many genes differentially regulated with the depletion of ZAP-70 protein.(A) GSEA plots showing the analysis plot of genes sets significantly enriched in the NKTL cell lines (HANK-1, NK-YS, SNK-1) after ZAP-70 knockdown via siRNA utilising the (A) C2 (curated gene sets) (B) Hallmark gene set parameters for gene set analysis. (C) Differential gene expression analysis is represented by individual volcano plots for indicated NKTL cell lines shows that there are very few genes that are differentially expressed between the si-non targeting and si-ZAP70 in the three cell lines. (D) KHYG cells were treated with siNT or siZAP70 and then the effect on the mRNA expression levels of ZAP70, PMEPA1, AHNAK2 and KLHD7CB measured via qRT-PCR.

Fig 4: DLG1 knockout inhibits TCR-MC formation and T cell signaling (A). Microclusters of phosphorylated Y493 ZAP-70 (green) and phosphorylated TCR-? (red) in immunostained wild-type or DLG1 KO Jurkat cells were visualized 5 min after the cells were dropped onto coverslips coated with anti-CD3 (stimulated) or anti-CD45 (unstimulated). Scale bars, 10 µm. Quantitative data for specific signals’ intensities (using Image J; collected from eight fields) are graphically represented. (B). Immunoblot analysis of lysates from wild-type and DLG1 KO Jurkat cells activated with soluble anti-CD3 (monoclonal antibody OKT3) and antibody to mouse immunoglobulin G (IgG). Blots were probed for the indicated phosphorylated and total proteins. Data are representative of three experiments. Student’s unpaired t tests were used for (A). ** p < 0.01, **** p < 0.0001.

Fig 5: DLG1 is critical for ZAP70 recruitment to the TCR complex. (A). Immunoblot analysis (IB) of the indicated co-immunoprecipitated partners after immunoprecipitation (IP) of DLG1 from activated Jurkat cells. (B). Immunoblot analysis (IB) of the indicated co-immunoprecipitated partners after immunoprecipitation (IP) of TCR-? from activated wild-type and DLG1 KO Jurkat cells. Data are representative of three experiments.