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Fig. 4 | Molecular Cancer

Fig. 4

From: N6-methyladenosine-modified circIGF2BP3 inhibits CD8+ T-cell responses to facilitate tumor immune evasion by promoting the deubiquitination of PD-L1 in non-small cell lung cancer

Fig. 4

circIGF2BP3 attenuates antitumor immunity by upregulating PKP3 expression. A Coexpression network containing circIGF2BP3 and its 24 associated mRNAs built by Cytoscape. B The relative expression of PKP3 in 68 paired samples of NSCLC in cohort I from Tongji Hospital. GAPDH was used as the internal control. C Correlation between circIGF2BP3 and PKP3 expression in 68 paired samples of NSCLC in cohort I. D The relative expression of PKP3, OVOL1, PAX9 and FAM83H in A549 cells transfected with shControl, sh-circIGF2BP3#1 or sh-circIGF2BP3#2, as determined by qRT-PCR. E-F Representative images (left) and statistical quantitation (right) of the FACS analysis of the PBMC-mediated elimination of NSCLC cells, as determined by annexin V-FITC and propidium iodide (PI) double labeling. G The protein levels of perforin and GzmB in activated PBMCs after coculturing with the indicated NSCLC cells were detected by western blotting. H-I Ki-67 (H) and TUNEL (I) staining of PBMCs after coculturing with the indicated NSCLC cells. Scale bars, 20 μm. J The normalized expression of PKP3 in LUAD (left) and LUSC (right) from a TCGA data set. K K-M plot showing the relationship between circIGF2BP3 levels and the OS of patients with LUAD (left) and LUSC (right) in a TCGA data set. L Schematic diagram exhibiting the grouping and treatment plan of the in vivo study: C57BL/6 mice were inoculated with 106 CTRL, circIGF2BP3-OE, sh-mPKP3 or sh-mPKP3 + circIGF2BP3-OE LLC cells, and the tumor volume was measured at the indicated time points. M Time-course evaluation of the tumor volumes of different groups measured every 3 days. N Representative images and statistical quantitation of IF CD8α and PKP3 staining in the indicated groups. Scale bars, 100 μm. O Representative images and statistical quantitation of the FACS analysis of IFN-γ+, TNF-α+, GzmB+ and perforin+ CD8+ TILs from the indicated xenograft tumor samples. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined by unpaired Student’s t test (J), paired Student’s t test (B), one-way ANOVA with Tukey’s post hoc test (D, E, F, H, I, M, N and O) and the log-rank test (K). Correlations were determined by the Pearson correlation test (C)

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