Fig. 6

Knockdown of ZDHHC3 expression suppresses tumor growth and induces immune effector cell infiltration in Panc 02 tumors. A Western blot assay detecting the protein level of ZDHHC3 in Panc 02 cells that were transfected with the control-shRNA or ZDHHC3-shRNA; the stable clones were used in subsequent experiments. B Subcutaneous tumor growth curves: 5 × 10⁶ stably-expressing shZDHHC3 Panc 02 cells or shControl Panc 02 cells were injected in the right flank of C57BL6/J mice (n = 8 mice per group). C, D The tumor weight (C) and survival rate (D) of shControl and shZDHHC3 Panc 02 tumors were recorded at the end of the experiment. E–H The tumor tissues at the end point were digested into single-cell suspension and analyzed by flow cytometry. Then, cells were gated on single cells and selected on Live/Dead dye staining. Only live cells were included in the further analysis. Flow cytometry analysis of the number of NK cells (CD45⁺ CD3⁻NK1.1⁺) (E), CD8⁺ T cells (CD45⁺ CD8⁺), CD4⁺ T cells (CD45⁺ CD4⁺) (F), DCs (CD45⁺ CD11b⁺CD11c⁺) (G), type 1 macrophages (M1) (CD45⁺ CD11b⁺ F4/80⁺ CD206⁻), and type 2 macrophages (M2) (CD45⁺ CD11b⁺ F4/80⁺ CD206⁺) (H) infiltrating subcutaneous tumors, namely shControl and shZDHHC3 Panc 02 tumors at day 23. Percentages indicate different immune cell populations amongst live CD45⁺ immune cells. I Western blot assay detecting the protein level of PD-L1 in control, ZDHHC3-knockdown, and ZDHHC3-knockdown with PD-L1 overexpressed Panc 02 cells; the stable clones were used in subsequent experiments. J Subcutaneous tumor growth curves: 5 × 10⁶ stably-expressing (Control, shZDHHC3, and shZDHHC3 + PD-L1) Panc 02 cells were injected in the right flank of C57BL6/J mice (n = 6 mice per group). K, L The tumor weight (K) and survival rate (L) of (Control, shZDHHC3, and shZDHHC3 + PD-L1) Panc 02 tumors were recorded at the end of the experiment. Survival curves from the subcutaneous Panc 02 tumor model were developed until the animal’s death or the tumor volume reached > 1000 mm³. Mice were euthanized when the calculated tumor size reached ~ 1000 mm³ or when tumors became ulcerated. Survival curves were analyzed using the log-rank (Mantel-Cox) test. Results shown are presented as mean ± SEM. (ns, not significant, *p < 0.05, and **p < 0.01)