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. 2022 Nov 7;24(1):e54042. doi: 10.15252/embr.202154042

Figure 6. ZBTB2 N‐terminus‐mimetic polypeptides competitively inhibit ZBTB2 homodimerization and exhibit therapeutic effects toward p53‐deficient cancers.

Figure 6

  • A
    The same kind of luciferase assay as in Fig 5B and G was carried out by transfecting with either pEF6/ZBTB2 (ZBTB2 wt) or its empty vector (EV). To evaluate the inhibitory activity of the three truncated mutants of ZBTB2, ZBTB2 [1–23], ZBTB2 [1–91], and ZBTB2 [1–113], against wt ZBTB2, low (+) and high (++) concentrations of the corresponding expression vectors were additionally used to co‐transfect the cells.
  • B
    The same kind of split luciferase complementation assay as in Fig 5D was carried out by co‐transfecting with pcDNA4/ZBTB2‐LgBiT (ZBTB2) and either pcDNA4/ZBTB2‐SmBiT (ZBTB2) or pcDNA4/SmBiT (−). To evaluate the inhibitory activity of the two truncated mutants of ZBTB2, ZBTB2 [1–23] and ZBTB2 [1–113], against wt ZBTB2, the corresponding expression vectors were additionally used to co‐transfect the cells.
  • C
    U2OS cells transiently transfected with three concentrations (+, ++, +++) of pEF6/ZBTB2 [1–113] or pEF6/myc‐His B (EV) were cultured under < 0.1% oxygen conditions and subjected to qRT–PCR for indicated mRNA.
  • D–G
    The indicated cells were co‐transfected with either siScr or siZBTB2 and the expression vector for ZBTB2 or its empty vector (EV), cultured under < 0.1% oxygen conditions, and subjected to the luciferase assay for transactivation activity of HIF‐1α (D), that for HIF‐1 activity (E), and the q‐RT–PCR experiments for the indicated mRNA (F, G).
  • H
    HeLa cells transiently transfected with either siScr or siZBTB2 and with either ZBTB2[1–113] expression vector or its empty vector were cultured under 20% (left) or < 0.1% O2 (right) oxygen conditions for the indicated periods and subjected to the in vitro cell proliferation assay.
  • I
    Tumor growth assay using the indicated cells with s.c. transplantation.

Data information: Mean ± s.d. The number of technical replicates was 3 (A–H) and 10 (I), and reproducibility of the results was confirmed at least three times by biologically independent experiments (A–I). N.S., not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t‐test (A–I).