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. 2016 Jun 15;7(29):46127–46141. doi: 10.18632/oncotarget.10060

Figure 4. HIC1-interaction domain/Gln domain of ARID1A protein is essential for the modulation of its downstream transcriptional targets.

Figure 4

A. The domain structure of ARID1A protein and the different constructions for ectopic expression were depicted. The positions of domains and motifs were depicted according to the annotation of ARID1A protein (accession O14497, last modified: March 31, 2015) in UniProt database. The amino acid (aa) positions were indicated above the illustration. ARID1A-fl, full-length ARID1A protein. B. The full-length ARID1A protein was overexpressed in 293FT cells and cell proliferation was measured by cell counting. C. AGS cells were transfected with pCMV or pCMV-ARID1A-fl and the major components of PI3K-AKT signaling pathway were analyzed. The p-GSK3S9 was recognized using an antibody against p-GSK3α/βS21/S9, and only the band of p-GSK3βS9 was shown. D. The proliferation of Hela cell transfected with pBABE-ARID1A-C1 was measured using 5-Bromo-2-deoxyuridine (BrdU) incorporation method. E. AGS cells were transfected with pPB-CAG-ARID1A-C1 and the proliferation was measured using MTT method. The p values at each time point were shown along the curve. F. ARID1A-C1 was expressed in AGS cells and the molecules of PI3K-AKT pathway were analyzed. The p-GSK3 was recognized using an antibody against p-GSK3α/βS21/S9, and both the p-GSK3αS21 and p-GSK3βS9 were indicated. G. ARID1A was first stably silenced in HGC-27 cells and its expression was then rescued by transfecting pPB-CAG-ARID1A-C1 which expressed the C-terminal fragment of ARID1A. fl, full-length protein of ARID1A. C1, C-terminal fragment of ARID1A. H. The other three C-terminal fragments of ARID1A were overexpressed in AGS cells and PI3K and p-AKTS473 remained unchanged. I. The proliferation of AGS cells were measured after the overexpressions of ARID1A-C2, C3 and C4 fragments. J. Luciferase reporter assay of the PIK3CA promoter (PIK3CA-p1) activity was performed in AGS and SGC-7901 cells transfected with the ARID1A-C1 expression vector. K. Luciferase reporter assay was also performed to analyze the PDK1 promoter (PDK-1-p1) activity in AGS cells and SGC-7901 cells which were transfected with ARID1A-C1 expression vector.