Presentation on theme: "ControlNodalALK7-ca Sl Fig. 1. Identification of potential Nodal/ALK7 target genes using genearray. Total RNA was extracted from IOSE cells transfected."— Presentation transcript:
ControlNodalALK7-ca Sl Fig. 1. Identification of potential Nodal/ALK7 target genes using genearray. Total RNA was extracted from IOSE cells transfected with control vector, Nodal, or ALK7-ca and subjected to reverse transcribed. The cDNA samples were then labeled with biotin and hybridized to specific cDNA on the membrane (Human Cell Cycle Gene Array from SupperArray). Large arrow indicates cyclin G2 and small arrow indicates Skp1. Table listed genes that are up- or down- regulated by Nodal and ALK7-ca. Gene NameDescriptionGeneBank Upregulation ATMAtaxia telanlectasia mutatedNM_000051 BaxBcl-2-associated X proteinAY217036 Cyclin G2 L49506 Cyclin H U11791 Cdc16 NM_003909 Cdc7 AF015592 p21Cyclin-dependent kinase inhibitor 1AL47233 p27Cyclin-dependent kinase inhibitor 1BU10906 p57Cyclin-dependent kinase inhibitor 1CU22398 p18Cyclin-dependent kinase inhibitor 2CU17074 p19Cyclin-dependent kinase inhibitor 2DU40343 Cullin-Cul2Cullin 2U83410 Cullin-Cul5Cullin 5NM_003478 E2F1E2F transcription factor1U47677 E2F2E2F transcription factor 2NM_004191 E2F6E2F transcription factor 6AF059292 MAD2L2MAD2-like 2NM_006341 PRC1Protein regulator of cytokinesis 1NM_003981 RAD9RAD9 (S. pombe) homologU53174 RbRetinoblastoma 1 (including osteosarcoma)M15400 Rbx1Homo saplens ring-box protein1 (RBX1) mRNANM_014248 Ubiquitin CPolyubiquitinAB009010 Downregulation p55cdcp55cdc (cdc20)NM_001255 Cks1p9CDC28 protein kinase 1NM_001826 Ki67Antigen identified by monoclonal antibody Ki-67X65550 Skp1Cyclin A/CDK2-associated p19U33760 TIMP3Tissue inhibitor of metalloproteinase 3NM_000362
A pcDNA3.1-CCNG2-V5 Xba IKpn I V5His Stop Age I pcDNA4-CCNG2-V5 Kpn I V5His Stop Age I pcDNA4-CCNG2-myc-His Sac IIKpn I mycHis Stop pCCNG2-GFP Sac IIEcoR I GFP Stop p3xFLAG-CCNG2 BamH I Stop3xFlag EcoR I pcDNA3-CCNG2-V5-His Sac IIKpn I V5His Stop B CCNG2-V5 gg 12h24h48h 051015051015051015 CCNG2-V5 -actin Post-transfection 21 64 48 37 82 26 19 116 abcde 21212121 C Sl Fig. 2. Generation of cyclin G2 expression constructs and detection of cyclin G2 protein. A) Generation of human CCNG2 plasmids. These plasmids contain the fusion tags either at N- terminus or C-terminus. Arrow indicates a start site of translation. B) Detection of exogenous CG2 in a dose and time-dependent manner. IOSE397 and OV2008 cells were transiently transfected with CG2-V5 plasmid. CG2 fusion protein was detected using a V5 antibody. β-actin was used as control for the equal loading. C) Representative Western blots probed with different cyclin G2 antibodies. OV2008 cells were transiently transfected with an empty vector (1) or CCNG2-V5 (2). Proteins were extracted at 6 hours post-transfection and subjected to SDS-PAGE. Blots were probed with anti-V5 from Invitrogen (a), anti-CCNG2 from Abcam (b), anti-CCNG2 from Epitomics (c); anti-CCNG2 from Santa Cruz (d), and anti-CCNG2 from Abnova (e). Only the antibody from Santa Cruz appeared to recognize cyclin G2. OV2008 IOSE
Xu et al., Fig. Sl3 A B DAPICCNG2Merge DAPICCNG2Skp2Merge Skp2 DAPICCNG2pcDNAMerge DAPI CCNG2 Skp2 ALK7-ca Merge Sl Fig. 3. Effect of Skp2 on cyclin G2 expression as detected by immunofluorescent staining. A) Cells were co-transfected with cyclin G2 and control vector (pcDNA4, upper panel), Skp2 (middle panel), or ALK7-ca (lower panel). In cells expressing Skp2, cyclin G2 level was low whereas in cells expressing ALK7-ca, strong cyclin G2 signal was detected. B) Cells were transfected with cyclin G2 and control-siRNA (upper panel) or Skp2-siRNA (lower panel). Strong cyclin G2 signals were observed in the presence of Skp2-siRNA compared to control- siRNA. Scale bar: 100 m.
OV-GFPOV-siCCNG2 GFP Phase Sl Fig. 4. Generation of OV2008 stable cell lines expressing control vector (OV-GFP) or cyclin G2 siRNA (OV-CCNG2siRNA). GFP-positive clones were selected by fluorescent microscopy. Xu et al., Fig. Sl4
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