Anergic signature induced by SHP2 overexpression. Hallmarks of anergy were detectable in a transduced MEC-1 cell line overexpressing wild-type SHP2 or dysfunctional C463S-mutant SHP2 in comparison with MEC-1 cells transduced with empty vector control. Groups were compared by analysis of variance using Bonferroni post hoc statistics. (A) Effect of SHP2 overexpression on proliferation. Viable cell count was measured via cell viability analyzer 120 hours after seeding (n = 9 per group). (B) Basal Ca2+ levels were measured via flow cytometry after FLUO-4 staining (n = 8 per group). (C) Basal ERK1/2 phosphorylation levels were analyzed by densitometry of western blots (n = 6 per group). (D-E) Surface IgM expression in comparison with isotype control analyzed via flow cytometry (n = 6 per group). (F) Ca2+ flux after FLUO-4 staining in response to stimulation with soluble anti-IgM-F(ab′)2 determined by flow cytometry analysis (n = 5 per group). (G) Stimulation with soluble anti-IgM-F(ab′)2–induced ERK1/2 phosphorylation was analyzed by densitometry of western blots. Cell lines were serum starved for 2 hours and stimulated for 0, 1, 5, 15, or 60 minutes. Corresponding time points were always analyzed on the same blots (n = 2 to 6 per group). (H) Antiproliferative effect of SHP2 overexpression in ibrutinib-treated cells. Cell lines were seeded in medium with and without ibrutinib (0.5 µM). Viable cell count was measured via cell viability analyzer and normalized to the respective untreated control cell counts at 120 hours (n = 9 per group). (A-D,F,H) Error bars show the standard error of the mean; *P < .05; **P < .001. FITC, fluorescein isothiocyanate.
