Fig 2: p53 inhibition is essential for the TNFAIP8 knockdown-mediated inhibition of NSCLC proliferation and cisplatin chemoresistance. a, b qRT-PCR and western blotting analyses showed that p53 shRNA (p53-sh) increased cyclin D1 expression levels in TNFAIP8 shRNA2 (TNFAIP8-sh2)-transfected NCI-H460 and A549 cells. *P < 0.05 and n.s., not significant (Student’s t-test.) c p53 inhibition restored NSCLC cell viability after TNFAIP8-sh2 transfection. d, e qRT-PCR and western blotting analyses showed that p53 shRNA (p53-sh) increased RAD51 expression levels in TNFAIP8 shRNA2 (TNFAIP8-sh2)-transfected A549/cDDP cells. *P < 0.05 and n.s., not significant (Student’s t-test.) f A549/cDDP cells were transfected with shRNA (Ctrl), TNFAIP8 shRNA2 (TNFAIP8-sh2) or p53 shRNA (p53-sh), and CCK-8 assay was used to measure cisplatin sensitivity. *P < 0.05 (Student’s t-test)

Fig 3: The pro-tumorigenic effect of DTX3 is dependent on mutant p53. (A, B) Depletion of mtp53 impairs DTX3-induced cell growth. OVCA420 cells were transfected with combinations of plasmids and siRNAs as indicated followed by the CCK-8 cell viability assay at 72 h post-transfection (A). SKOV3 cells stably-expressing the empty vector or mtp53-R273H were transfected with plasmids as indicated followed by the CCK-8 cell viability assay at 72 h post-transfection (B). (C, D) Depletion of mtp53 impairs DTX3-induced cell invasion. ES-2 (C) and OVCA420 (D) cells stably expressing the empty vector or DTX3 were transfected with siRNAs as indicated followed by the transwell cell invasion assay. Quantification of the migrating cells is shown in the right panels. All the experiments were performed in biological triplicate.

Fig 4: DTX3 stabilizes mutant p53 by reducing MDM2-p53 binding. (A) DTX3 induces ubiquitination of mtp53. HCT116p53-/- cells were transfected with combinations of plasmids as indicated, and treated with MG132 (20 µM) for 6 h before harvested for in vivo ubiquitination assay. (B, C) The lysines K11, K27, K29, K33, K48, and K63 of the ubiquitin are important for DTX3-induced mtp53 ubiquitination. The same experiment was performed as that in the panel A, except that the lysine-mutant ubiquitin plasmids were used. (D) Overexpression of DTX3 increases mtp53 protein level. ES-2 cells infected with control or DTX3-containing lentivirus were subjected to IB analysis using antibodies as indicated. (E) Knockdown of DTX3 reduces mtp53 protein level. ES-2 cells were transfected with siRNAs against DTX3 followed by IB analysis using antibodies as indicated. (F) DTX3 siRNA-mediated reduction of mtp53 level is restored by the proteasome inhibitor MG132. OVCA420 cells were transfected with siRNAs against DTX3 and treated with MG132 for 6 h before harvest for IB analysis using antibodies as indicated. (G) Ectopic DTX3 interferes with the MDM2-mtp53 interaction. ES-2 cells stably expressing the empty vector or DTX3 were subjected to co-IP-IB analysis using antibodies as indicated. The arrows indicate non-specific bands.

Fig 5: UVB-induced melanocyte migration elicited by suppression of miR-211 and the upregulation of MMP9. (A) Quantitative PCR was performed to measure the miR-211 levels of melanocytes treated with repeated UVB. Data represent the mean ± standard deviation of three independent experiments. **P<0.01 vs. contl. (B) Melanocytes were immunostained with antibodies against MMP9 (red) and then counter-stained with DAPI to identify nuclei (blue). Representative fluorescence images of MMP9 merged with DAPI staining are shown. Scale bars: 50 µm. (C) Transwell cell culture chambers were used for cell migration assays. A total of 48 h later, migrating cells on the bottom surface of the inserts were stained with crystal violet and counted in five microscopic fields using a microscope. Representative images of migrating cells are shown. Scale bars: 50 µm. (D) Histogram showing the number of migrating cells in each Transwell as the mean ± standard deviation of three independent experiments. **P<0.01 vs. contl or 8xUVB. UVB, ultraviolet B; ACTB, ß-actin; MMP, matrix metalloproteinase; TRPM1, p53-transient receptor potential cation channel subfamily M member 1; miR, microRNA; DAPI, 4'6'-diamidino-2-phenylindole; contl, control.