Fig 1: Six1 promotes cell proliferation/migration and suppresses monocytes/macrophages (Mophs) infiltration in vivo. (A) mRNA expression of cyclin genes (Ccna 1, Ccnd 1 and C-myc), glycolytic genes (Glut1, Pgk and Ldha) and Mmp9 in adeno-associated viral vector serotype 9 (AAV9)-Six1 treated (AAV-Six1), and AAV empty-vector treated (AAV-Control) kidneys at 1, 2, and 3 days after ischemia/reperfusion injury (IRI) assessed by qRT-PCR. (B) Representative F4/80 stained kidney sections from AAV-Six1 and AAV-Control mice at indicated time after IRI and quantification of the number of F4/80+ Mophs. F4/80 (green) and 4',6-diamidino-2-phenylindole (DAPI, blue). Bar = 50 µm. (C) qRT-PCR analysis of inflammatory factors (Mcp-1, Tnfa, Il-1ß), and cofactor genes of NF?B subunit RELA (Aes and Fus) in AAV-Six1 and AAV-Control IRI kidneys. (D, E) Protein expression of Six1, Cyclin D1 and Aes in AAV-Six1 and AAV-Control kidneys at 1, 2, and 3 days after IRI were assessed by Western blotting. Data are mean ± SD for groups of three mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus AAV-Control (t-test). IRI 1d, 1 day after IRI; IRI 2d, 2 days after IRI; IRI 3d, 3 days after IRI; GAPDH, glyceraldehydes-3-phosphate dehydrogenase.
Fig 2: Mouse Six1 suppresses monocyte chemotactic protein-1 (MCP-1) expression by inhibiting NF-?B activation. (A) pNF?B-TA-luc reporter containing four NF?B binding sites (top). pNF?B-TA-luc co-transfected with pRL-SV40-N (internal control reporter plasmid) in mouse Six1 overexpression HK2 cells (HK2-Six1 6#) and the control 1#. Cells were harvested 24 h after transfection or treatment with 25 ng/ml TNFa for another 24 h, and luciferase activity was measured (bottom). (B) Plasmids (pNF?B-TA-luc and pRL-SV40-N) were co-transfected into the mouse renal tubular epithelial cells Six1 knockout (TCMK1-Six1-/- 5#) and the Control 1#. Luciferase activity was measured. The relative luciferase activity was quantified by adjusting it to the renilla luciferase activity and untreated control samples were normalized to 1.0. (C) mRNA expression of mouse Six1, human NF?B subunit RELA, human cofactors genes of RELA (AES and FUS) were assessed by qRT-PCR in the HK2-Six1 6# and the Control 1# cell lines. (D, E) Western blotting was applied to SIX1, RELA, p-RELA, AES, and FUS in HK2-Six1 cell lines (4# and 6#) and the Control cell lines (1# and 2#). (F)SIX1 DNA binding compatible motif (ACCTGA) in AES and FUS gene promoter regions and chromatin immunoprecipitation (ChIP) analysis of SIX1 occupancy of the AES and FUS genes from HK2-Six1 6# and the control 1# cell lines. The location of each primer set compared to the transcription start site (TSS) is shown (left). IgG control samples were normalized to 1.0. (G, H) After treatment by 25 ng/ml TNFa for 24 h or no treatment, mRNA expression of human MCP-1 and mouse Mcp-1 assessed by qRT-PCR in HK2-Six1 6# and Control 1#, TCMK1-Six1-/- 5# and Control 1#, respectively. Data are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus the Control group (t-test). GAPDH, glyceraldehydes-3-phosphate dehydrogenase.
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