Figure 2.
Figure 2. TRRAP stabilizes mutp53 across cancer entities and p53 mutations. (A) mRNA level of TRRAP and TP53 in Namalwa and Raji cells transduced with shRNAs against TRRAP. Cells were selected with puromycin for 48 hours and harvested 3 days after transduction. Expression values (mean + SD, n = 2) were determined by qRT-PCR and normalized to GAPDH and to cells transduced with a nontargeting shRNA (NT). (B) Protein level of TRRAP and p53 in Namalwa and Raji cells transduced with shRNAs against TRRAP and p53. Cells were selected with puromycin for 48 hours and harvested 7 days after transduction. Expression was determined by western blot and normalized to GAPDH and to cells transduced with a nontargeting shRNA (NT). (C) p53 flow cytometry 7 or 8 days after shRNA-mediated knockdown of TRRAP in mutp53 cancer cell lines: BL, diffuse large B-cell lymphoma (DLBCL), and colorectal cancer (CRC). The p53 mutation is specified for each cell line. Values denote the ratio of the p53 MFI between transduced (red) and untransduced cells (gray). For cell lines with a bimodal p53 level, gates and the corresponding percentages for the p53-low and p53-high population are indicated. Dashed lines indicate isotype control stainings, and filled colored histograms indicate samples stained with anti-p53. shNT, nontargeting shRNA. (D) p53 flow cytometry 7 days after sgRNA-mediated knockout of TRRAP in Namalwa-Cas9 cells. Values denote the ratio of the p53 MFI between transduced (red) and untransduced cells (gray). sgmCherry, negative control. (E) p53 flow cytometry 72 hours after transfection with FLAG-TRRAP in Colo320 cells. Values denote the ratio of the p53 MFI between transfected (blue) and untransfected cells (gray). (F) Molecular rescue of the TRRAP knockdown phenotype by FLAG-TRRAP overexpression in Colo320 cells. Cells were transduced with either a nontargeting shRNA (shNT; gray) or with a shRNA against TRRAP (shRNA #233; red). 24 hours after transduction, part of the TRRAP-silenced cells was transfected with FLAG-TRRAP (blue). Cells were analyzed by p53 flow cytometry 4 days after transduction. Values denote p53 MFI normalized to cells transduced with the NT. (G) Rescue of the TRRAP-silencing–mediated mutp53 degradation by treatment with the proteasome inhibitor bortezomib. Namalwa cells were transduced with an IPTG-inducible shRNA against TRRAP (shRNA #233) or a nontargeting control (NT). 24 hours after induction, cells were treated with dimethyl sulfoxide (DMSO; 0.1%) or bortezomib (100 nM) for 14 hours before being subjected to p53 flow cytometry. Values denote p53 MFI normalized to DMSO-treated cells transduced with the NT.

TRRAP stabilizes mutp53 across cancer entities and p53 mutations. (A) mRNA level of TRRAP and TP53 in Namalwa and Raji cells transduced with shRNAs against TRRAP. Cells were selected with puromycin for 48 hours and harvested 3 days after transduction. Expression values (mean + SD, n = 2) were determined by qRT-PCR and normalized to GAPDH and to cells transduced with a nontargeting shRNA (NT). (B) Protein level of TRRAP and p53 in Namalwa and Raji cells transduced with shRNAs against TRRAP and p53. Cells were selected with puromycin for 48 hours and harvested 7 days after transduction. Expression was determined by western blot and normalized to GAPDH and to cells transduced with a nontargeting shRNA (NT). (C) p53 flow cytometry 7 or 8 days after shRNA-mediated knockdown of TRRAP in mutp53 cancer cell lines: BL, diffuse large B-cell lymphoma (DLBCL), and colorectal cancer (CRC). The p53 mutation is specified for each cell line. Values denote the ratio of the p53 MFI between transduced (red) and untransduced cells (gray). For cell lines with a bimodal p53 level, gates and the corresponding percentages for the p53-low and p53-high population are indicated. Dashed lines indicate isotype control stainings, and filled colored histograms indicate samples stained with anti-p53. shNT, nontargeting shRNA. (D) p53 flow cytometry 7 days after sgRNA-mediated knockout of TRRAP in Namalwa-Cas9 cells. Values denote the ratio of the p53 MFI between transduced (red) and untransduced cells (gray). sgmCherry, negative control. (E) p53 flow cytometry 72 hours after transfection with FLAG-TRRAP in Colo320 cells. Values denote the ratio of the p53 MFI between transfected (blue) and untransfected cells (gray). (F) Molecular rescue of the TRRAP knockdown phenotype by FLAG-TRRAP overexpression in Colo320 cells. Cells were transduced with either a nontargeting shRNA (shNT; gray) or with a shRNA against TRRAP (shRNA #233; red). 24 hours after transduction, part of the TRRAP-silenced cells was transfected with FLAG-TRRAP (blue). Cells were analyzed by p53 flow cytometry 4 days after transduction. Values denote p53 MFI normalized to cells transduced with the NT. (G) Rescue of the TRRAP-silencing–mediated mutp53 degradation by treatment with the proteasome inhibitor bortezomib. Namalwa cells were transduced with an IPTG-inducible shRNA against TRRAP (shRNA #233) or a nontargeting control (NT). 24 hours after induction, cells were treated with dimethyl sulfoxide (DMSO; 0.1%) or bortezomib (100 nM) for 14 hours before being subjected to p53 flow cytometry. Values denote p53 MFI normalized to DMSO-treated cells transduced with the NT.

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