Fig 1.
ZAP70 is expressed in cell lines derived from TCL and NKTL cells but not in cell lines derived from B-cell malignancies.
(A) ZAP70 protein expression as assessed by Western blotting in Hela, RAJI, THP-1, T cell lines (JURKAT, KARPAS-299) and NKTL cell lines (KHYG-1, HANK-1, NK-YS, SNK-1, SNK-6). β−ACTIN was used as loading control. (B) ZAP70 protein expression as assessed by Western blotting in JURKAT, SC1, OCI-LY19, OCI-LY3, SUDHL4, GU22-650, MC116, HT, DAUDI, K231, P3HR1. GAPDH was used as loading control. (C) ZAP70 mRNA was measured by real time RT-PCR using gene-specific primers (ZAP70, GAPDH and β-ACTIN) in Hela, RAJI, THP-1 and JURKAT, KARPAS-299 and KHYG-1, HANK-1, NK-YS, SNK-1,SNK-6 cell lines. GAPDH and β−ACTIN were used as housekeeping genes.
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. β− 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.
Table 1.
Gene set enrichment analysis of siZAP-70 vs siNT in HANK-1, NKYS and SNK-1 NKTL cell lines [C2 gene set analysis].
Table 2.
Gene set enrichment analysis of siZAP-70 vs siNT in HANK-1, NKYS and SNK-1 NKTL cell lines [Hallmark gene set analysis].
Fig 4.
Overexpression of ZAP70 does not alter the sensitivity of NKT cells to etoposide or increase cell survival of NK/T lymphoma cells.
(A) NKYS cells were stably transfected with EV or ZAP-70. Activation of the TCR signalling components were analysed by Immunoblotting performed on these cell lines and the following proteins detected—phosphorylated and total ZAP70 as well as target substrates of ZAP-70, phosphorylated and total LAT, phosphorylated and total SLP76, phosphorylated and total p38 MAPK (B) The total number of live cells were determined by tryphan blue exclusion for EV and ZAP-70 overexpressing cells for the days indicated. (C) Comparison of IC50 values to etoposide treatment for 24 hours between wild type (WT) NKYS and NKYS with ZAP70 overexpression (ZAP70). Viability was assessed by Cell Titre blue.
Fig 5.
Gefitinib does not preferentially target ZAP-70 overexpressing NKTL.
(A) Gefitinib dose response curve for TCL cell lines (JURKAT, KARPAS-299), NKTL cell lines (KHYG1, HANK-1, NK-YS, SNK-1, SNK-6), monocytic leukemic cell line (THP-1) and B cell malignant cell lines (RAJI, SCI, OCI-LY19) (B) NKYS cell lines which have been stably transfected with EV or ZAP70 were treated with a range of Gefitinib concentrations and cell viability assessed by Cell–Titer Glo (Promega) (C) Jurkat a high ZAP-70 expressing T cell leukemia cell line is a positive control with growth inhibition induced by Gefitinib.