Figure 5
Figure 5. Functional role of S727-STAT3 and of MCL-1 up-regulation for resistance to PKC412. (A) Antibody array analyzing expression and/or phosphorylation of 615 proteins in duplicates using lysates from 32D_ITD and 32D_ITD627E cells. Data obtained from the antibody array was filtered as follows: (1) Flag 0. This means spot quality is acceptable based on morphology and background; (2) change from control (CFC) > 50% or < −50% CFC. The percentage change from control is a measure of the change in normalized signal intensity averages between the experimental sample (FLT3_ITD627E) and the control sample (FLT3_ITD); (3) total error range: < 30%. The total error range is the sum of “% error range” from the experimental and control samples. (B) Phosphorylation of STAT3 at S727 in 32D_ITD and 32D_ITD627E cells was determined by immunoblotting (top panel). In FLT3_ITD627E cells, introduction of FLT3-specific siRNA results in down-regulation of P-S727-STAT3 levels while protein levels of STAT3 remain unchanged (bottom panel). FLT3 expression and phosphorylation of STAT3 at S727 was assessed in 32D_ITD627E cells by immunoblotting 24 hours after siRNA-mediated knockdown of FLT3. (C) Suppression of STAT3 expression by RNA interference increases sensitivity of FLT3_ITD627E cells to PKC412. The percentage of apoptotic 32D_ITD627E cells was determined by flow cytometry 48 hours after introducing STAT3-specific siRNA and treatment of cells with and without PKC412 (right panel). As a control, down-regulation of STAT3 expression was assessed at 24 hours by immunoblotting (left panel). Shown are the results of 1 representative experiment of a total of 2. (D) MCL-1 is up-regulated in 32D_ITD627E cells. Protein expression of MCL-1 in cellular lysates of 32D_ITD and 32D_ITD627E cells treated with and without different concentrations of PKC412 (2 hours) was determined by immunoblotting (top panel). → indicate subspecies of MCL-1 as detected by Western blot analysis. In FLT3_ITD627E cells, introduction of FLT3-specific siRNA results in down-regulation of MCL-1 levels while protein levels of STAT3 remain unchanged (bottom panel). Expression of FLT3, MCL-1, and STAT3 was assessed in 32D_ITD627E cells at 24 hours by immunoblotting. Results shown correspond to 1 representative experiment (partially depicted in Figure 3E left panel) of a total of 2. (E) Suppression of MCL-1 expression by RNA interference rescues sensitivity of FLT3_ITD627E cells to PKC412. Induction of apoptosis was determined by flow cytometry 48 hours after introduction of MCL-1–specific siRNA in response to incubation with and without PKC412 (10 nM and 20 nM) for 48 hours (right panel). As a control, protein expression of MCL-1, BCL-XL, and BCL-2 in 32D_ITD627E cells was assessed by immunoblotting at 24 hours (left panel). Results of 1 representative experiment of a total of 2 are shown. (F) Suppression of BCL-2 expression by transient siRNA transfection. Induction of apoptosis was determined by flow cytometry 48 hours after introduction of BCL-2–specific siRNA in response to incubation with and without PKC412 (5 nM and 10 nM) for 48 hours (right panel). As a control, protein expression of BCL-2 and of MCL-1 in 32D_ITD627E cells was assessed by immunoblotting at 24 hours (left panel). Results of 1 representative experiment of a total of 2 are shown. (G) Ectopic expression of FLAG-tagged murine MCL-1 (FLAG-mMCL-1) in 32D_ITD cells confers resistance to PKC412. Protein expression of FLAG-mMCL-1 in 32D_ITDmMCL-1 cells was determined by immunoblotting using an anti-FLAG antibody, and actin was used as a loading control (left panel). The percentage of apoptotic cells was assessed by flow cytometric measurement of subG1 DNA content in 32D_ITD and 32D_ITDmMCL-1 cells after incubation with PKC412 for 24 hours (right panel). The means of 3 independent experiments are shown and error bars represent mean (± SD).

Functional role of S727-STAT3 and of MCL-1 up-regulation for resistance to PKC412. (A) Antibody array analyzing expression and/or phosphorylation of 615 proteins in duplicates using lysates from 32D_ITD and 32D_ITD627E cells. Data obtained from the antibody array was filtered as follows: (1) Flag 0. This means spot quality is acceptable based on morphology and background; (2) change from control (CFC) > 50% or < −50% CFC. The percentage change from control is a measure of the change in normalized signal intensity averages between the experimental sample (FLT3_ITD627E) and the control sample (FLT3_ITD); (3) total error range: < 30%. The total error range is the sum of “% error range” from the experimental and control samples. (B) Phosphorylation of STAT3 at S727 in 32D_ITD and 32D_ITD627E cells was determined by immunoblotting (top panel). In FLT3_ITD627E cells, introduction of FLT3-specific siRNA results in down-regulation of P-S727-STAT3 levels while protein levels of STAT3 remain unchanged (bottom panel). FLT3 expression and phosphorylation of STAT3 at S727 was assessed in 32D_ITD627E cells by immunoblotting 24 hours after siRNA-mediated knockdown of FLT3. (C) Suppression of STAT3 expression by RNA interference increases sensitivity of FLT3_ITD627E cells to PKC412. The percentage of apoptotic 32D_ITD627E cells was determined by flow cytometry 48 hours after introducing STAT3-specific siRNA and treatment of cells with and without PKC412 (right panel). As a control, down-regulation of STAT3 expression was assessed at 24 hours by immunoblotting (left panel). Shown are the results of 1 representative experiment of a total of 2. (D) MCL-1 is up-regulated in 32D_ITD627E cells. Protein expression of MCL-1 in cellular lysates of 32D_ITD and 32D_ITD627E cells treated with and without different concentrations of PKC412 (2 hours) was determined by immunoblotting (top panel). → indicate subspecies of MCL-1 as detected by Western blot analysis. In FLT3_ITD627E cells, introduction of FLT3-specific siRNA results in down-regulation of MCL-1 levels while protein levels of STAT3 remain unchanged (bottom panel). Expression of FLT3, MCL-1, and STAT3 was assessed in 32D_ITD627E cells at 24 hours by immunoblotting. Results shown correspond to 1 representative experiment (partially depicted in Figure 3E left panel) of a total of 2. (E) Suppression of MCL-1 expression by RNA interference rescues sensitivity of FLT3_ITD627E cells to PKC412. Induction of apoptosis was determined by flow cytometry 48 hours after introduction of MCL-1–specific siRNA in response to incubation with and without PKC412 (10 nM and 20 nM) for 48 hours (right panel). As a control, protein expression of MCL-1, BCL-XL, and BCL-2 in 32D_ITD627E cells was assessed by immunoblotting at 24 hours (left panel). Results of 1 representative experiment of a total of 2 are shown. (F) Suppression of BCL-2 expression by transient siRNA transfection. Induction of apoptosis was determined by flow cytometry 48 hours after introduction of BCL-2–specific siRNA in response to incubation with and without PKC412 (5 nM and 10 nM) for 48 hours (right panel). As a control, protein expression of BCL-2 and of MCL-1 in 32D_ITD627E cells was assessed by immunoblotting at 24 hours (left panel). Results of 1 representative experiment of a total of 2 are shown. (G) Ectopic expression of FLAG-tagged murine MCL-1 (FLAG-mMCL-1) in 32D_ITD cells confers resistance to PKC412. Protein expression of FLAG-mMCL-1 in 32D_ITDmMCL-1 cells was determined by immunoblotting using an anti-FLAG antibody, and actin was used as a loading control (left panel). The percentage of apoptotic cells was assessed by flow cytometric measurement of subG1 DNA content in 32D_ITD and 32D_ITDmMCL-1 cells after incubation with PKC412 for 24 hours (right panel). The means of 3 independent experiments are shown and error bars represent mean (± SD).

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