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.