10.6084/m9.figshare.12638067.v1 Yuan Xiang Yuan Xiang Jun Wang Jun Wang Jia Peng Li Jia Peng Li Wei Guo Wei Guo Feng Huang Feng Huang Hui Min Zhang Hui Min Zhang Han Han Li Han Han Li Zhou Tong Dai Zhou Tong Dai Zi Jian Zhang Zi Jian Zhang Hui Li Hui Li Le Yuan Bao Le Yuan Bao Chao Jiang Gu Chao Jiang Gu Kun Chen Kun Chen Tong Cun Zhang Tong Cun Zhang Xing Hua Liao Xing Hua Liao Additional file 2 of MKL-1 is a coactivator for STAT5b, the regulator of Treg cell development and function Springer Nature 2020 MKL-1 STAT5b Treg ITP 2020-07-10 04:26:49 Journal contribution https://springernature.figshare.com/articles/journal_contribution/Additional_file_2_of_MKL-1_is_a_coactivator_for_STAT5b_the_regulator_of_Treg_cell_development_and_function/12638067 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.