Fig. 2: Deletion of Tfap4 removes the selection pressure to acquire defects in the TRP53 pathway during c-MYC-driven lymphoma development.

A Western blot analysis of sgTfap4/Eµ-MYC/Cas9 and sgControl/Eµ-MYC/Cas9 primary lymphomas for TRP53, p19/ARF, TFAP4 and HSP70 (protein loading control). The blot includes positive and negative control cell lysates for mutant (Mut) and wild-type (WT) TRP53. Molecular weight markers are indicated in kDa. B Summary graph representing percentages of sgTfap4/Eµ-MYC/Cas9 (n = 9) and sgControl/Eµ-MYC/Cas9 (n = 9) lymphomas that have defects in the TRP53 pathway as assessed by Western blotting for TRP53 and p19/ARF. TRP53 wt determined as lack of detectable protein expression of both TRP53 and p19/ARF. TRP53 knockout (ko) is determined as no detectable TRP53 protein expression with high expression of p19/ARF. Mutant TRP53 is identified by high level expression of both TRP53 and p19/ARF proteins. Cell viability response curves and corresponding IC50 graphs in sgTfap4/Eµ-MYC/Cas9 and sgControl/Eµ-MYC/Cas9 lymphoma cell lines 24 h after treatment with the indicated doses of nutlin-3A (C) or etoposide (D). Cell viability was determined by flow cytometry; live cells were identified as the Annexin V/PI double negative population. Data represent percentage mean survival of lymphoma cell lines at each dose (sgControl n = 3, sgTfap4 n = 4–9). Data were log transformed and fitted to non-linear regression mean ± SEM. IC50 values were calculated using Prism Graphpad software. Each dot represents an independent lymphoma cell line; error bars represent mean ± SEM. Two-tailed Student’s t test, *p < 0.05.