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. 2018 Nov 5;38(1):e99506. doi: 10.15252/embj.201899506

Figure 5. IFNγ stimulation elicits the release of IRF2 from PD‐L1 promoter and the dynamic interaction of IRF2 with IRF2BP2.

Figure 5

  • A
    IRF2 inhibits IRF1 transcriptional activity. A549 cells transfected with human PD‐L1 promoter luciferase reporter, and indicated plasmids were analyzed using a luciferase assay. n = 3, mean ± SEM. *P < 0.05, **P < 0.01, one‐way ANOVA followed by Dunnett's test.
  • B
    Knockdown IRF2 restores PD‐L1 expression in VGLL4‐knockdown A549 cells. Gene expression levels were analyzed by qRT–PCR in A549 cells transfected or transduced as indicated. n = 3, mean ± SEM. *P < 0.05, ***P < 0.001, one‐way ANOVA followed by Dunnett's test.
  • C
    Overexpression of IRF1 increases PD‐L1 expression in VGLL4‐knockdown A549 cells. A549 cells were transfected with VGLL4 siRNA or together with HA‐IRF1 plasmid, and total cell lysates were analyzed by Western blot.
  • D
    IRF2 is released from PD‐L1 promoter upon IFNγ stimulation. IRF2‐3 × HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP‐qPCR analysis in the PD‐L1 promoter using control and HA antibodies. Normal mouse IgG was used as control. n = 3, mean ± SEM. ***P < 0.001, one‐way ANOVA followed by Tukey's test.
  • E
    Increased association of IRF2 with PD‐L1 promoter in IRF2BP2‐knockout (KO) A549 cells revealed by ChIP‐qPCR analysis. Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower panel. n = 3, mean ± SEM. *P < 0.05, one‐way ANOVA followed by Tukey's test.
  • F
    Dynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti‐HA antibody in IRF2‐3 × HA knockin A549 cells.
  • G, H
    Loss of IRF2BP2 attenuated IFNγ‐inducible PD‐L1 protein (G) and mRNA (H) expression revealed by immunoblot and qRT–PCR analysis, respectively, in A549 control and IRF2BP2‐knockout cells. Quantification of PD‐L1 protein levels in (G) is shown in the right panel. n = 3, mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, two‐tailed Student's t‐test.

Source data are available online for this figure.