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Blood, Vol. 113, Issue 8, 1689-1698, February 19, 2009
Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases
Blood Joshi et al.
113: 1689
Supplemental materials for: Joshi et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 64.9 KB)
- Table S1. Calculation of relative G1 rescue index for rescue from GSI-induced G1 arrest with ectopic expression of Cyclin D3 (PDF, 225 KB)
- Table S2. Calculation of relative G1 rescue index for rescue from GSI-induced G1 arrest with ectopic expression of Cyclin D3 and cdk4 or cdk6 (PDF, 301 KB)
- Figure S1. Purified CD4+ T cells from bulk splenocytes were incubated with varying concentrations of ILCHO (left panel) for 30 minutes prior to stimulating in vitro with plate-bound anti-CD3 plus anti-CD28 for 48 hours (JPG, 22.1 KB)
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Whole cell lysates were generated, resolved by SDS page and immunoblotted with anti-cleaved Notch1. Blots were reprobed with PKC-θ to demonstrate equal protein loading.
- Figure S2. Mice were fed GSI in rodent chow formulated to deliver 5 mg/kg/day for the indicated number of days (JPG, 21.5 KB)
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Splenocytes were harvested, CD4+ T cells isolated, and stimulated in vitro with plate-bound anti-CD3 plus anti-CD28 for 48 hours. Whole cell lysates were generated, resolved by SDS page, and immunoblotted with an antibody that recognizes both the transmembrane (NTM) and the cleaved (NIC) isoforms of Notch1, as indicated. Absence of the lower, NIC, band indicates reduction in cleaved Notch1. Blot was reprobed with PKC-θ to demonstrate equal protein loading.
- Figure S3 (JPG, 72.8 KB)
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(A) Cell cycle analysis was performed on human T-ALL cell lines DND-41, HPB-ALL, and T-ALL1 that were treated with 0.1% DMSO (top panels) or GSI, 1 µm DAPT (bottom panels) for 7 days. (B) Whole cell extracts were prepared from human T-ALL cell lines after 7 days of DAPT or DMSO-treatment to analyze differences in Cyclin D3, Notch1, CDK4, CDK6, pRb780, or pRb795.
- Figure S4. Human T-ALL cell lines were transduced with Cyclin D3 expression construct (JPG, 23 KB)
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Whole cell lysates were generated, resolved by SDS page, and immunoblotted with Cyclin D3. Blots were reprobed with GAPDH to demonstrate equal protein loading.
- Figure S5. Partial rescue of Human T-ALL cell lines by ectopic expression of Cyclin D3 from GSI induced G1 arrest (JPG, 28.6 KB)
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Scatter plot represents the percentage of cells in G1 after 7 days of DMSO (solid symbols) and GSI treatment (open symbols). Uninfected cells/ non-transduced (■, □), MSCV-IRES-YFP (▲, △), and MSCV-Cyclin D3-IRES-YFP (●, ○).
- Figure S6. Partial rescue of Human T-ALL cell lines by ectopic expression of CDK4 or CDK6 and Cyclin D3 from GSI induced G1 arrest (JPG, 26.1 KB)
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Scatter plot represent the percentage of cells in G1 after 7 days of DMSO (■) and GSI treatment (▲). Uninfected cells (uninfected), MSCV-IRES-DsRed II and MSCV-IRES-YFP (vector), MSCV-CDK4-IRES-DsRed II and MSCV-Cyclin D3-IRES-YFP (CDK4+D3), MSCV-CDK6-IRES-DsRed II and MSCV-Cyclin D3-IRES-YFP (CDK6+D3).
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