Fig. 6
IFN induces expression of LNK as a negative feedback. a MTT assay of A375 melanoma cells with either force expressing or silencing of LNK either in the presence or absence of IFN-γ (2000 U/ml). Upper panel, silenced LNK using shRNA (shLNK16). Lower panel, silenced LNK using CRISPR-Cas9 sgRNA (sgLNK). Ctrl, control A375 cells; LNK, LNK overexpressing cells. Mean ± SD; ***P < 0.001; ****P < 0.0001; Two way ANOVA. b Foci formation assays of melanoma cell lines with either force expression of LNK (LNK OE) or silencing of LNK (sgLNK), either with or without IFN-γ exposure. IFN-γ (2000 U/ml) was added 2 h after seeding the cells. Ctrl, control. c Higher levels of LNK expression occurred in pretreatment biopsy samples of melanoma patients who did not respond to PD-1 antibody therapy. Data were retrieved from GEO dataset GSE78220, unpaired two tailed t-test. d, e Silencing Lnk enhanced tumor suppressive effect of Pd-1 antibody. Control (control CRISPR-Cas9 vector) and Lnk silenced (CRISPR-Cas9 with sgLNK guide RNA) B16/F10 (left panel, n = 8) or D4M.3 A cells (right panel, n = 6. Two mice did not growth any tumor at day 10 were excluded before separation of the cohorts) were subcutaneously injected into both flanks of the same mice (8–12-weeks-old syngeneic C57BL/6 mice). Mean + SEM; *P < 0.05; **P < 0.01; unpaired two tailed t-test. f Western blot analysis showed Lnk silencing in either B16/F10 or D4M.3 A cells. g Exposure to IFN-γ induced LNK expression in melanoma cells. Melanoma cells were treated with IFN-γ (2000 U/ml, 24 h) and expression of LNK was examined by western blot. Ctrl, control. h ChIP-seq binding pattern of STAT1 and IRF-1 at transcription start site of LNK. Data were retrieved from ENCODE project database. Number of each panel reflects scale of ChIP enrichment. i Diagram shows the IFN-STAT1-LNK signaling axis in melanoma cells. Error bars represent SEM (d) or SD (a)
