Figure 4

Siva represses endogenous IL-2 gene expression. A. The flow plot gating scheme used in B and C is shown. An FSC high gate was drawn to include 7AAD^pos cells, but exclude debris events that would falsely appear as viable, 7AAD^neg cells in subsequent analyses. 7AAD^neg events included in the FSC high gate were used to calculate viable cell number. GFP is being detected in the YFP channel, which was not used in any of these experiments. In B-D, IL-2 expression levels were divided by the mean viable cell number for each indicated sample. B. Flow plots indicating viability and transduction efficiency based on GFP expression for Jurkat T cells transduced with pHSPG retrovirus (RV) or RV expressing an EGFP/Siva-1 fusion protein (pHSP-EGFP/Siva-1). Transduced Jurkat T cells were subsequently tested for IL-2 production in response to PMA and Ionomycin. C. Similarly to data shown in B, the flow plots show Jurkat T cells transduced with RV from pHSPG or pHSPG encoding untagged Siva-1. IL-2 protein expression in response to PMA and Ionomycin for the same cells is shown. D. Knockdown (KD) of endogenous Siva with shRNA enhanced endogenous IL-2 expression. Siva-1 KD efficiency was evaluated by standard RT-PCR and DNA bands are shown. In B-D, error bars represent standard deviations for n = 3; * indicates p < 0.05 for two-tailed Student's t-test between vector transduced cells and the respective EGFP/Siva-1, Siva-1 or shSIVA expressing cells. Data shown in B-D is from one experiment representative of 3-5 replicates.