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Figure 6 | BMC Immunology

Figure 6

From: Regulation of IL-2 gene expression by Siva and FOXP3 in human T cells

Figure 6

Endogenous IL-2 in response to exogenous FOXP3 combined with Siva-1 overexpression or Siva knockdown. Jurkat T cells were transduced with two rounds of RV or LV and then activated with PMA and Ionomycin. Following stimulation, cell viability was determined using the gating method shown in Figure 4A and endogenous IL-2 expression was measured by ELISA. A. Flow cytometry for GFP expression and viability in unstimulated Jurkat T cells transduced with different combinations of pHSPG (PG), PG-Siva-1, and PG-FOXP3 RV. Plots shown have already been gated to exclude debris events using a gating scheme similar to the one shown in 4A. B. Endogenous IL-2 in response to FOXP3 and Siva-1 overexpression was determined using the cells shown in A. The IL-2 concentration was divided by the mean viable cell count for each indicated sample. C. Siva KD efficiency was determined by quantitative realtime PCR for Siva and 18S in unstimulated Jurkat T cells transduced with PG or PG-FOXP3 RV and pLKO-shEGFP or pLKO-shSIVA LV. The realtime PCR primers used in this experiment do not distinguish between the two Siva isoforms. The error bars indicate standard deviation between technical replicates. #s indicate statistically significant difference between the indicated bar and the adjacent, shEGFP-transduced control. D. Endogenous IL-2 in response to FOXP3 overexpression and Siva KD, using the transduced Jurkat T cells described in C. *s indicate statistically significant difference between the indicated bar and the PG- or shEGFP/PG-transduced control, in B and D, respectively. Statistically significant difference was based on p < 0.05 by a two-tailed Student's t-test. These transduction experiments were performed twice and representative experiments are shown.

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