Figure 7.

Lentivirus-mediated correction of SASH3 deficiency in Jurkat cells and in patient-derived T cells. (A) Top: transduction efficiency as measured by mCherry expression in wild-type (WT) and SASH3^−/− Jurkat cells upon transduction with mock and SASH3 lentivirus (LV) vectors. Bottom: western blot showing reconstitution of SASH3 protein expression in SASH3^−/− Jurkat cells upon transduction with the SASH3 LV vector. (B) Correction of the proliferative defect of SASH3^−/− Jurkat cells upon transduction with the SASH3 LV vector. WT, wild-type Jurkat; mock, SASH3^−/− Jurkat cells transduced with mock LV vector; SASH3, SASH3^−/− Jurkat cells transduced with the SASH3 LV vector. Statistical significance was assessed with 2-way analysis of variance for multiple comparisons. (C) Dot-plot showing transduction efficiency (as measured by mCherry expression) in control- and P2-derived T-cell blasts upon transduction with mock and SASH3 LV vectors. (D) Top: western blot showing reconstitution of PLCγ1 phosphorylation in SASH3-transduced P2 T cells upon in vitro stimulation with anti-CD3 and anti-CD28. Bottom: densitometric quantification of phosphorylated PLCγ1 (pPLCγ1) protein expression in mock- and SASH3-transduced P2 T cells in 2 distinct experiments (identified by different symbols). Results are expressed as pPLCγ1:glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio and compared with what was detected in mock-transduced control cells, which were given a value of 1.0.