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. 2018 Oct 29;9:2512. doi: 10.3389/fimmu.2018.02512

Figure 3.

Figure 3

GATA3-AS1 and GATA3 form a necessary feed forward loop for TH2 polarization. (A) PBMCs were cultured under TH2 polarizing conditions for 2 days and transfected with a GATA3-AS1 specific siRNA (+) or scrambled siRNA (–). GATA3-AS1, GATA3, IL4, IL5, and IL13 transcripts were determined by qPCR on day 5 and results expressed relative to GAPDH. Statistical significance was determined using Students T-test by comparing GATA3-AS1 siRNA knockdown to scrambled control knockdown (n = 3). *P < 0.05 (B) PBMCs were cultured under TH2 or TH1 polarizing conditions and transfected with GATA3-AS1 specific siRNA (+) or scrambled siRNA (–). Protein was analyzed by ELISA (for IL-4, IL-5, and IL-13, n = 3,) or (C) Western Blot (GATA3, Densitometry). Statistics were calculated using a Paired T-test compared negative siRNA controls. *P < 0.05. (D) Similar to A, but an siRNA specific to GATA3 was transfected on day 2. Results and statistics similar to A, but (n = 4). (E) PBMCs were cultured under TH0 conditions for 2 days, and transfected with GATA3-AS1 transcripts produced from Topo-TA in vitro transcription vector. Cells were transfected with a scrambled siRNA (negative), the antisense of GATA3-AS1(antisense), GATA3-AS1 at 500 μM (AS1 High), and GATA3-AS1 at 100 μM (AS1 Low). Analysis and statistics completed similarly to A and B, (n = 5).