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Blood, Vol. 113, Issue 17, 4063-4073, April 23, 2009
A novel molecular mechanism of primary resistance to FLT3-kinase inhibitors in AML
Blood Breitenbuecher et al.
113: 4063
Supplemental materials for: Breitenbuecher et al
Files in this Data Supplement:
- Figure S1. Amino acid sequences of FLT3_ITD, FLT3_ITD627E, and FLT3_ITD627A (JPG, 43 KB)
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- Figure S2. MCL-1 expression and PKC412-resistant phenotype of clonal 32D_ITD627E cell lines and new 32D_ITD627E transfectands (JPG, 68.4 KB)
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(A) Protein expression of MCL-1 in two independent clonal cell lines established from early passages of 32D_ITD627E cells was determined by immunoblot using anti–MCL-1 antibody and anti-actin antibody as loading control (left panel). The percentage of apoptotic cells was assessed by flow cytometric measurement of subG1 DNA content in 32D_ITD, 32D_ITD627Ec1, and 32D_ITD627Ec2 cells after incubation with PKC412 for 24h (right panel). The means of three independent experiments are shown, and error bars represent mean +/− standard deviation. (B) Protein expression of MCL-1 in the newly transfected cell line 32D_ITD627Ep4 was determined by immunoblot using anti–MCL-1 antibody and anti-actin antibody as loading control (left panel). The percentage of apoptotic cells was assessed by flow cytometric measurement of subG1 DNA content in 32D_ITD and 32D_ITD627Ep4 cells after incubation with PKC412 for 24h (right panel). The means of three independent experiments are shown and error bars represent mean +/− standard deviation.
- Figure S3. MCL-1 regulation and GRB2/FLT3_ITD627E interaction (JPG, 84.2 KB)
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(A) Suppression of STAT3 or MCL-1 expression by transient siRNA transfection sensitizes 32D_ITD cells to PKC412 and downregulation of STAT3 leads to decreased MCL-1 protein expression in 32D_ITD cells. Induction of apoptosis was determined by flow cytometry 48h after introduction of STAT3- or MCL-1–specific siRNA in response to incubation with and without PKC412 (5nM and 10 nM) for 48h (right panel). Protein expression of STAT3 and MCL-1 in 32D_ITD cells was assessed by immunoblotting at 24h (left panel). Results of one representative experiment out of a total of two are shown. (B) Primary AML blasts isolated at the time of primary resistance harbouring the FLT3_ITD627E allele were treated ex vivo with different concentrations of PKC412 for 1h. Tyrosine phosphorylation of FLT3 and protein expression of MCL-1 was analyzed by immunoblotting of cellular lysates. (C) MCL-1 and FLT3 protein expression was analyzed by immunoblot 24h after transient transfection of 32D_ITD cells with FLT3-specific siRNA. An anti-GAPDH antibody was used to control equal loading. (D) Analysis of GRB-2/FLT3_ITD627E interaction upon incubation with PKC412. FLT3 was immunoprecipitated from protein lysates of 32D_ITD and 32D_ITD627E cells after treatment with 50nM PKC412 for 2h and the amount of co-immunoprecipitated GRB-2 was assessed by immunoblot analysis using anti–GRB-2 and anti-FLT3 antibody.
- Figure S4. Phosphorylation of STAT3 and protein expression of BCL-2 and BCL-XL (JPG, 51.4 KB)
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(A) Phosphorylation of STAT3 is downregulated in 32D_ITD and 32D_ITD627E cells upon treatment with PKC412. Phosphorylation of STAT3 at S727 was determined in 32D_ITD and 32D_ITD cells after treatment with PKC412 for 3h by immunoblot using a specific phospho-STAT3S727 antibody. An anti-GAPDH antibody was used to control equal loading. (B) Phosphorylation of STAT3 at Y705 was determined in 32D_ITD and 32D_ITD cells after treatment with PKC412 for 2h by immunoblot using a specific phospho-STAT3Y705 antibody. An anti-STAT3 antibody was used to control equal loading. (C) Protein expression of BCL-2 and BCL-XL in 32D_ITD and 32D_ITDmMCL-1 cells was determined by immunoblot using specific antibodies. Equal loading was determined by hybridization with anti-tubulin antibody.
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