Fig 2: MEF2B mutations alter the abundance of protein from MEF2B target genes and decrease cell migration.(a–f) CARD11, MYC, NDRG1, MEF2C, vimentin, fibronectin and SNAI2 protein abundance in cells expressing WT or mutant MEF2B-V5. The MEF2B-V5 bands shown in a were detected on the same western blot as was used for MYC and CARD11 detection. (b,c) The same lysates were used for NDRG1 and MEF2C western blots as for the MEF2B-V5 western blots shown in b. (d–f) The same lysates were used for the SNAI2, vimentin and fibronectin western blots as for the MEF2B-V5 western blots shown below them. Mean relative abundance was calculated compared with untransfected cells using densitometry on three biological replicates of western blots. Representative western blots are shown. MEF2B-V5 was detected using V5 antibody. (g) Movement into the scratched area of a confluent monolayer is increased more by WT than mutant MEF2B-V5 expression. Scratches were assessed in at least two biological replicates (K4E: two biological replicates; Y69H and D83V: three biological replicates; WT, untransfected and empty vector: four biological replicates). The total number of scratches assessed for each sample is shown in parentheses for 12 and 20 h time points, respectively. For all panels, *P<0.05 in comparison with WT MEF2B-V5 expressing cells (Student's two-tailed t-test, unpaired). Error bars represent the s.e.m.

Fig 3: Synergistic activity of Card11 L232 LI and Bcl6. (a) Venn diagrams showing the percentages of cells expressing the surrogate markers for exogenously transduced Card11 L232 LI (GFP, green circles) and Bcl6 (hCD4, blue circles). Fractions of cells coexpressing GFP and hCD4 are shown in red. The remaining four mice, out of 11 mice used in the experiment (Exp.), were found dead. Thus, viable cells were unavailable for flow cytometric analysis. (b) Expression of CARD11 in the developed lymphoma. (c) BCL6 expression in the developed lymphoma. BCL6 protein levels are normalized to the levels of actin (protein loading control) and BCL6/actin ratios are presented and compared to that of OCI-Ly8, a human lymphoma cell line, being set to 1.0.

Fig 4: Tumorigenicity of Card11 L232 LI and Bcl6 with/without Bcl2-transduced germinal center B cells. (a) Kaplan–Meier analysis of the probability of event-free survival of mice transplanted with induced germinal center B (iGCB) cells transduced with Card11 L232 LI, Bcl6, and Bcl2 (red line). An event is defined as the presence of lymphoma on dissection or death. The probability of event-free survival of the mice transplanted with iGCB cells transduced with Card11 L232 LI and Bcl6 is shown by the blue line. The difference was not statistically significant (P = 0.56), as determined by the log–rank test. (b) Flow cytometric analysis of gene-transduced iGCB cells before transplantation (upper panels; experiment [Exp.] 1) and cells in lymphoma that developed after transplantation in a mouse (lower panels). Percentages of cells expressing GFP (representing Card11 L232 LI), hCD4 (Bcl6), and hCD8 (Bcl2) are depicted and truncated after the decimal point. (c) Venn diagrams showing the percentages of cells expressing exogenously transduced Card11 L232 LI (GFP, green circles), Bcl6 (hCD4, blue circles), and Bcl2 (hCD8, yellow circles) before (upper panel) and after transplantation, on lymphoma development (lower panel). One (#1) and two (#2 and #3) mice developed lymphoma in two independent experiments. Percentages of cells co-expressing Card11 and Bcl6 are shown in red. The remaining three out six mice used in the experiment were found dead. Thus, viable cells were unavailable for flow cytometric analysis.

Fig 5: Tumorigenicity of induced germinal center B cells singly transduced with Card11 L232 LI or Bcl6. Kaplan–Meier analysis of the probability of event-free survival of the mice receiving induced germinal center B cells transduced with Card11 L232 LI (red line) or Bcl6 alone (black line). An event is defined as the presence of lymphoma on dissection or death. The probability of event-free survival was compared with that of mice transplanted with cells transduced with Card11 L232 LI and Bcl6 (dashed line). The difference in the probability of event-free survival was statistically significant (P = 1.17 × 10-4), as determined by the log–rank test. Post-hoc pairwise comparison was undertaken using Bonferroni correction. All P-values <0.05 are shown in the figure.