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Blood, Vol. 111, Issue 4, 2238-2245, February 15, 2008
RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia
Blood Tyner et al.
111: 2238
Supplemental materials for: Tyner et al
Files in this Data Supplement:
- Document 1. Primers for qPCR (PDF, 11.1 KB)
- Table S1. CMK cell viability after siRNA knockdown of individual members of each tyrosine kinase as well as N-RAS and K-RAS (JPG, 314 KB)
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GENE refers to siRNA target, VIABILITY values represent percent mean (normalized to non-specific siRNA control wells), S.E. refers to standard error of the mean, T-TEST 1 refers to p-value of t-test between individual gene viability and single non-specific siRNA viability, T-TEST 2 refers to p-value of t-test between individual gene viability and pooled non-specific siRNA viability, and AVG. OF T-TEST refers to mean of T-TEST 1 and T-TEST 2 values.
- Table 2. HMC1.1 cell viability after siRNA knockdown of individual members of each tyrosine kinase as well as N-RAS and K-RAS (JPG, 315 KB)
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GENE refers to siRNA target, VIABILITY values represent percent mean (normalized to non-specific siRNA control wells), S.E. refers to standard error of the mean, T-TEST 1 refers to p-value of t-test between individual gene viability and single non-specific siRNA viability, T-TEST 2 refers to p-value of t-test between individual gene viability and pooled non-specific siRNA viability, and AVG. OF T-TEST refers to mean of T-TEST 1 and T-TEST 2 values.
- Figure S1. RNAi targeting in HMC1.1 cells leads to effective knockdown (JPG, 100 KB)
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HMC1.1 cells were treated with siRNA targeting JAK1, JAK2, JAK3, PTK2, PTK2B, PTK6, PTK9, EPHA4, LTK, LYN, SRC, or c-KIT and electroporated as in Figure 5. Forty-eight hours later, cell lysates or total RNA were harvested and subjected to (A) immunoblot or (B) quantitative PCR analysis for each targeted gene as well as  -ACTIN or GAPDH as a loading control. Densitometric or qPCR values were normalized to their respective loading controls and percent knockdown was calculated. For determination of reciprocal off-target effects by SRC and LYN siRNA, HMC1.1 cells were transfected with SRC and LYN siRNA and immunoblotted with antibodies specific for both SRC and LYN.
- Figure S2. HMC1.1 cells are sensitive to inhibitors targeting JAK3 and SRC (JPG, 60.1 KB)
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(A) HMC1.1 and K562 control cells were treated with increasing concentrations of the SRC family inhibitor, SRC kinase inhibitor I. 72 hours later, cells were subjected to an MTS assay for determination of total viable cells. Values represent mean ± s.e.m. (n = 3). (B) HMC1.1 and K562 control cells were treated with increasing concentrations of the JAK3-specific inhibitor, JAK3 inhibitor III. 72 hours later, cells were subjected to an MTS assay for determination of total viable cells. Values represent mean ± s.e.m. (n = 3). (C) HMC1.1 and HEL control cells were treated with increasing concentrations of the JAK2-specific inhibitor, AG-490. 72 hours later, cells were subjected to an MTS assay for determination of total viable cells. Values represent mean ± s.e.m. (n = 6).
- Figure S3. c-KIT associates with and activates JAK2 in HMC1.1 cells (JPG, 34.6 KB)
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HMC1.1 cells were serum-starved overnight and stimulated with 100 ng/ml SCF for 5 minutes. Cell lysates were immunoprecipitated for c-KIT and JAK2. Immunoprecipitates and whole cell extract (WCE) were analyzed by immunoblot for c-KIT, total and phospho JAK2, and -actin.
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