Additional file 2 of MKL-1 is a coactivator for STAT5b, the regulator of Treg cell development and function

Additional file 1: Figure S1. Over-expression MKL-1 and STAT5b increase the number of Treg in CD3+ T cells and enhance the Treg markers expression. A. Western blot analysis of MKL-1 and STAT5b protein level in CD3+T cells transfected with myc-MKL-1 or flag-STAT5b for 48 h. B. The number of Treg in CD3+T cells transfected with myc-MKL-1 or flag-STAT5b for 48 h by flow cytometry. C. QPCR analysis of Foxp3 and CD25 mRNA level in CD3+T cells transfected with myc-MKL-1 or flag-STAT5b for 48 h. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. D and E. Western blot analysis of Foxp3 and CD25 protein level in CD3+T cells transfected with myc-MKL-1 or flag-STAT5b for 48 h. Data were quantified using Quantity One software. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. Figure S2. Inhibited or knock-down MKL-1 and STAT5b weaken the Treg markers expression. A. QPCR analysis of Foxp3 and CD25 mRNA level in CD3+T cells treated with AG490 or Y27632 for 48 h. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. B. QPCR analysis of Foxp3 and CD25 mRNA level in CD3+T cells transfected with MKL-1 and STAT5b siRNA for 48 h. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. C and E. Western blot analysis of Foxp3 and CD25 mRNA level in CD3+T cells treated with AG490 or Y27632 for 48 h. Data were quantified using Quantity One software. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. D and F. Western blot analysis of Foxp3 and CD25 protein level in CD3+T cells transfected with MKL-1 and STAT5b siRNA for 48 h. Data were quantified using Quantity One software. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. Figure S3. IL2 affects the effect MKL-1 and STAT5b on the Treg marker expression. A. QPCR analysis of Foxp3 protein level in CD3+T cells transfected with MKL-1 and STAT5b and treated with IL2 for 48 h. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. B and C. Western blot analysis of Foxp3 protein level in CD3+T cells transfected with MKL-1 and STAT5b and treated with IL2 for 48 h. Data were quantified using Quantity One software. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. D. The luciferase reporter assays were used to test the transactivity of Foxp3 in CD3+T cells transfected with MKL-1 and STAT5b and treated with IL2 for 48 h. **, P < 0.01, *, P < 0.05, n = 6. Figure S4. Ag490 and Y27632 affect the phosphorylation of Foxp3 and nuclear accumulation of Foxp3. A and B. Western blot analysis to detect phosphorylated Foxp3 in CD3+T cells treated with AG490 or Y27632 for 48 h. Data were quantified using Quantity One software. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3. C and D. Western blot analysis to detect nuclear or membrane Foxp3 in CD3 + T cells treated with AG490 or Y27632 for 48 h. Data were quantified using Quantity One software. GAPDH is the loading control. **, P < 0.01, *, P < 0.05. n = 3.

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