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. Author manuscript; available in PMC: 2020 Jul 27.
Published in final edited form as: Cell Rep. 2020 Feb 25;30(8):2776–2790.e6. doi: 10.1016/j.celrep.2020.01.093

Figure 6. TIFAB Is Critical for the Function of a Mouse Model of MLL-AF9 AML.

Figure 6.

(A) Generation of Tifab+/+ and Tifab−/− MLL-AF9 leukemia model and schematic overview of the experimental setup.

(B) Colony formation in methylcellulose of Tifab+/+;MLL-AF9 or Tifab−/−;MLL-AF9. Two biological experiments were performed in triplicate. Representative images are shown. ***p < 0.0005.

(C) Kaplan-Meier plot for overall survival of recipient mice transplanted with Tifab+/+;MLL-AF9 or Tifab−/−;MLL-AF9 cells.

(D) Relative mRNA expression of the indicated genes in Tifab+/+;MA9 or Tifab−/−;MA9 cells. Three independent experiments were performed in triplicate. Error bars represent SD. *p < 0.05, ***p < 0.0005.

(E) Cell growth analysis in liquid culture of Tifab+/+;MLL-AF9 or Tifab−/−;MLL-AF9 cells expressed as live cell number per day, determined by trypan blue exclusion. *p < 0.05.

(F) Relative cell growth of Tifab−/−;MLL-AF9 (GFP+) cells transduced with lentivirus encoding empty vector (Vector) or USP15 and mCherry was determined by flow cytometry (GFP+mCherry+) at the indicated time points (n = 6 replicates). *p < 0.05.

(G) Relative colony formation in methylcellulose of Tifab+/+;MLL-AF9 or Tifab−/−;MLL-AF9 cells treated with vehicle (DMSO) or doxorubicin (Dox; 5 nM). The number of colonies was normalized to DMSO controls for each group (n = 2 biological experiments, performed in triplicate). ***p < 0.0005.

(H) Experimental setup for generation of Tifab−/−;MLL-AF9 knockin (KI) mice.

(I) Colony formation in methylcellulose of cKit+ MLL-AF9 KI cells expressing FLAG-TIFAB (MSCV-IRES-GFP) or empty vector. Representative images are shown to the right as image sections of the entire well (n = 3). *p < 0.005.

(J) Immunoblotting of p53 and FLAG-TIFAB on lysate isolated from MLL-AF9 KI cKit+ BM cells transduced with FLAG-TIFAB (MSCV-IRES-GFP) or empty vector, with actin as a loading control.