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

Figure 3. VGLL4 interacts with IRF2BP2 independent of TDU domains.

Figure 3

  • A
    The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti‐VGLL4 antibody and visualized by anti‐IRF2BP2 antibody in A549 cells.
  • B
    The endogenous IRF2BP2 was immunoprecipitated by VGLL4‐FLAG proteins in HEK293T cells.
  • C
    Interaction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co‐immunoprecipitation of differentially tagged proteins in HEK293T cells.
  • D
    C‐terminus of IRF2BP2 interacted with VGLL4 by co‐immunoprecipitation assay in HEK293T cells.
  • E
    Interaction of IRF2BP2 with VGLL1‐4 was revealed by co‐immunoprecipitation of differentially tagged proteins in HEK293T cells.
  • F
    TDU domains in VGLL4 are required for the interaction with TEADs by co‐immunoprecipitation assay in HEK293T cells.
  • G, H
    TDU‐deleted or HF4A VGLL4 mutations interacted with IRF2BP2 (G) and IRF2BP1 (H) by co‐immunoprecipitation analysis in HEK293T cells.
  • I
    Expression of VGLL4‐WT or VGLL4‐HF4A enhances PD‐L1 expression in A549 cells by immunoblot analysis. A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated antibodies.
  • J
    VGLL4‐HF4A rescues PD‐L1 expression in VGLL4‐knockdown A549 cells. A549 cells were transfected with siRNA targeting to the 3′UTR of VGLL4 mRNA and followed by transduction with lenti‐VGLL4‐HF4A virus. Cell lysates were analyzed by Western blot with indicated antibodies.
  • L
    Expression of VGLL4‐HF4A rescues tumor growth of VGLL4‐knockdown LLC cells in C57BL/6 mice. Control, Vgll4 knockdown, or Vgll4 knockdown together with VGLL4‐HF4A‐overexpressing LLC cells were transplanted into nude mice or C57BL/6 mice. Tumor volumes were measured 15 days for nude mice and 21 days for C57BL/6 mice after tumor cell inoculation. n = 8 tumors for each group. ***P < 0.001, two‐tailed Student's t‐test. The solid line represents the average volume ± SEM.
  • M
    Expression of VGLL4‐HF4A attenuates the T cell‐mediated tumor cell killing. Activated T cells and A549 cells were co‐cultured in 24‐well plates for 4 days, and relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal violet. n = 3, mean ± SEM.

Source data are available online for this figure.