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[Preprint]. 2023 Oct 9:2023.04.10.536234. Originally published 2023 Apr 10. [Version 3] doi: 10.1101/2023.04.10.536234

Fig. 2. Loss of TES1 but not TES2 signaling triggers LCL apoptosis.

Fig. 2.

(A) Schematic diagram of LMP1 WT with TES1 and TES2 domains highlighted. Wildtype (WT) or point mutants abrogated for signaling from TES1 (TES1m), TES2 (TES2m) or double TES1/TES2 mutant (DM) are shown.

(B) Immunoblot analysis of WCL from GM12878 LCLs that expressed control or LMP1 sgRNAs and puromycin selected for 3 days, then induced for expression with the indicated LMP1 rescue cDNA construct for 6 days. Blots are representative of n = 3 experiments.

(C) Growth curve analysis of GM12878 LCLs at the indicated day post expression of control or LMP1 sgRNAs and the indicated LMP1 WT, TES1m, TES2m or DM rescue cDNA. Shown are mean ± SD from n=3 independent experiments. **P<0.01.

(D) FACS analysis of 7-AAD vital dye uptake in GM12878 on day 7 post- expression of LMP1 sgRNAs and the indicated LMP1 rescue cDNA. Shown are percentages of 7-AAD+ cells within the indicated gates. Representative of n=3 experiments.

(E) Mean ± SD of fold change 7-AAD values from n=3 independent experiments of GM12878 with the indicated control or LMP1 sgRNA and rescue cDNA expression, as in (D). Values in GM12878 with control sgRNA and no LMP1 rescue cDNA were set to 1.

(F) FACS analysis of plasma membrane annexin V abundance in GM12878 on day 7 post- expression of control or LMP1 sgRNAs and the indicated LMP1 rescue cDNA. Shown are percentages of 7-AAD+ cells within the indicated gates. Representative of n=3 experiments.

(G) Mean ± SD of fold-change caspase 3/7 activity levels, as determined by caspase 3/7 Glo assay, from n=3 independent experiments of GM12878 with the indicated control or LMP1 sgRNA and rescue cDNA expression. Values in GM12878 with control sgRNA and no LMP1 rescue cDNA were set to 1.