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Fig. 6 | Molecular Cancer

Fig. 6

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. 6

PKP3 stabilizes PD-L1 via OTUB1-mediated PD-L1 deubiquitination. A IF staining of PD-L1 in A549 cells treated with shControl or shPKP3. PD-L1 (green), nuclear DAPI staining (blue). Scale bars, 20 μm. B IF staining to detect PD-1 binding ability. PD-L1 in A549 cells treated with shControl or shPKP3. PD-1 (red), nuclear DAPI staining (blue). Scale bars, 20 μm. C Western blotting analysis of PD-L1 stability in A549 cells expressing PKP3 shRNA and in SK-MES-1 cells transfected with PKP3-overexpressing plasmid. D The protein level of PD-L1 in PKP3-silenced or PKP3-overexpressing NSCLC cells was detected by western blotting. E Western blotting analysis of PD-L1 stability in NSCLC cells transfected with PKP3 shRNA after treatment with MG132 (100 μM). F Western blotting analysis of PD-L1 abundance in CTRL or PKP3-overexpressing H1650 cells treated with or without OTUB1 siRNA. G OTUB1 overexpression increased PD-L1 protein levels in NSCLC cells, whereas OTUB1 inhibition reduced these levels, as determined by western blotting. H IP analysis of PD-L1 ubiquitination in A549 cells transfected with the indicated constructs and/or PKP3 shRNA. I IP analysis of PD-L1 ubiquitination in A549 cells transfected with the indicated constructs and/or OTUB1 siRNA. J Correlation between PKP3 and OTUB1 mRNA levels in LUAD (upper) and LUSC (lower) from the TCGA data set, as analyzed by TIMER 2.0. K Representative FISH-IF images of PKP3 protein and OTUB1 mRNA in NSCLC samples from Tongji Hospital. Scale bars, 100 μm. L K-M OS analysis of NSCLC patients in the TCGA data set according to the OTUB1 expression level. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined by the log-rank test (L). Correlations were determined by the Spearman correlation test (J)

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