Study the effects of serum starvation for 24hrs on 4 cell types - TERV, TERV-ST, TERV-ST110, TERV-ASB56.

Transcription profiling of human serum starvation cell lines for 24hrs, 4 cell types - TERV, TERV-ST, TERV-ST110, TERV-ASB56

Signaling and transcriptional changes critical for transformation of human cells by simian virus 40 small tumor antigen or protein phosphatase 2A B56gamma knockdown

Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Affymetrix GeneChip Human Genome HG-U133B [HG-U133B]

Cross-platform expression profiling demonstrates that SV40 small tumor antigen activates Notch, Hedgehog, and Wnt signaling in human cells

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Study the effects of serum starvation for 24hrs on 4 cell types - TERV, TERV-ST, TERV-ST110, TERV-ASB56.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-3647

Stable human embryonic kidney (HEK) cell lines HEK-TERST, HEK-TERST110, and HEK-TERV have been previously described (1).  The HEK-TERASB56 cell line is described elsewhere (2).  Briefly, HEK TER cells were generated from human embryonic kidney (HEK) cells as described. HEK TERAS56Gamma3 (B56Gamma3 antisense construct) and corresponding control retroviral vectors were introduced into HEK TER cells and selected for green fluorescent protein (GFP)-expressing cells using a fluorescent-activated cell sorter (FACS). After two rounds of infection, 70 to 90% of the cells expressed GFP and 50% of these GFP-expressing cells were retained by FACS.  Thus, each line used in this study is actually a pool of hundreds of individual clones.  Cells were grown in alpha-MEM, 10% FBS, and serum-starved in alpha-MEM, 0.1% FBS for 24 hrs.    HekTERST:  Human epithelial kidney, Human telomerase catalytic subunit hTERT, SV40 small T, SV40 large T, activated Ras-V12 mutant.  HekTERST110: Human epithelial kidney, Human telomerase catalytic subunit hTERT, SV40 small T with mutation, SV40 large T, activated Ras mutant.  HekTERV: Human epithelial kidney, Human telomerase catalytic subunit hTERT, SV40 large T, activated Ras mutant, vector.  HekASB56Gamma3: Human epithelial kidney, Human telomerase catalytic subunit hTERT, SV40 large T, activated Ras mutant, PP2A B56gamma3 antisense.   Serum starve protocol: aspirate off media, wash 2x with PBS then add 10mls (per P100) alpha MEM + 0.1% fetal calf serum. Grow for 24 hours at 37deg C, 10% CO2.  1.        Hahn, W. C., Dessain, S. K., Brooks, M. W., King, J. E., Elenbaas, B.,Sabatini, D. M., DeCaprio, J. A., and Weinberg, R. A. Enumeration of the simian virus 40 early region elements necessary for human cell transformation. Mol Cell Biol, 22: 2111-2123., 2002. 2.        Chen, W., Possemato, R., Campbell, K. T., Plattner, C. A., Pallas, D. C., and Hahn, W. C. Identification of specific PP2A complexes involved in human cell transformation. Cancer Cell, 5: 127-136, 2004.

P-MEXP-3648

RNA was prepared using QIAGEN RNEasy Kit according to Manufacturer's protocol.  cDNA synthesis: First strand cDNA synthesis: 1. 25ug of RNA + 100picoM dTT7 primer. 2. Heat 70deg C for 10min. 3. Add first strand mix(4ul 5X first strand buffer, 2ul 0.1mM DTT, 1ul 10mM dNTP), mix, heat 37deg C for 2 min. 4. Add 2ul SuperScript II, mix, incubate at 42deg C for 1hr. Second strand cDNA synthesis 1. Add second strand buffer (91.33ul ddH2O, 30ul 5x second strand buffer, 4ul DNA pol I, 3ul 10mM dNTP, 1ul DNA ligase, 0.67ul RNaseH), mix, incubate at 16deg C for 2 hrs. 2. Clean up DNA using Zymo Clean and Concentrator according to manufacturers instructions.  

P-MEXP-4686

Serum starve protocol: aspirate off media, wash 2x with PBS then add 10mls (per P100) alpha MEM + 0.1% fetal calf serum. Grow for 24 hours. 

P-MEXP-4688

Label cDNA according to ENZO labeling kit instructions. Following ENZO protocol, purify RNA using Qiagen RNeasy kit and following manufacturers instructions.

Signaling and transcriptional changes critical for transformation of human cells by simian virus 40 small tumor antigen or protein phosphatase 2A B56gamma knockdown.

Transcription profiling of human serum starvation cell lines for 24hrs, 4 cell types - TERV, TERV-ST, TERV-ST110, TERV-ASB56

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