American Society of Hematology

Figure 2

$Figure 2. Involvement of TWIST-1 in CML response to TKIs, KCL22, or Lama-84 cell lines was analyzed for TWIST-1 expression. (A) Comparative expression by quantitative PCR of TWIST-1 in Lama-84 and KCL-22 imatinib-sensitive (□) and imatinib-resistant (■) Ph1+ cell lines. Results are expressed as a ratio to normal (healthy donors) CD34− cell values. (B) TWIST-1, BCR-ABL, and KU80 (used as loading control) protein analysis by Western blot in HEL expressing BCR-ABL (BA) or not (WT), KCL-22 and Lama 84 imatinib-sensitive (S) and -resistant (R) cell extracts. (C) Viability and viable cell numbers in untreated and imatinib-treated (1.5μM) Lama84-resistant Ph1+ cell line, submitted to TWIST-1 siRNA or negative scramble as control. Cells were incubated in 96-well plates in serum-free medium with stem cell factor (100 ng/mL), in the presence of 50nM of fluorescent siRNA. Results are presented as a percentage of viable cells or as total viable cell numbers ± SEM. After 2 days of culture, cell proliferation and viability were determined by trypan blue counting. Fold proliferation was determined by reference to the input number of viable cells. Results represent the mean ± SEM of 7 independent experiments. *Significant value (P < .05). (D) TWIST-1 expression in KCL22-resistant cells (KCL22R) transduced with a pSR vector empty or containing sh sequence against TWIST-1 or in KCl22-sensitive cells (KCL22S) transduced with a pMIG vector empty or containing TWIST-1 expressing sequence. After 3 days in the presence of puromycin selection for KCL22R-shTWIST-1 or after 48 hours for KCL22S-TWIST-1, cells were incubated in the presence of imatinib at the indicated dose. After 3 days of culture, cell proliferation and viability were determined by trypan blue counting. The percentage of proliferation was determined by reference to the nontreated number of viable cells ± SEM from 5 experiments. (E) Follow-up analysis for TWIST-1 expression by quantitative PCR in the CD34− fraction. Results are expressed as a ratio to normal (healthy donors) CD34− cell values. The values obtained for the BCR-ABL/ABL ratio are indicated as a percentage (IS) at each data point for both patients. BC indicates blast crisis; and CCyR, complete cytogenetic remission. †Deceased.$

Involvement of TWIST-1 in CML response to TKIs, KCL22, or Lama-84 cell lines was analyzed for TWIST-1 expression. (A) Comparative expression by quantitative PCR of TWIST-1 in Lama-84 and KCL-22 imatinib-sensitive (□) and imatinib-resistant (■) Ph1⁺ cell lines. Results are expressed as a ratio to normal (healthy donors) CD34⁻ cell values. (B) TWIST-1, BCR-ABL, and KU80 (used as loading control) protein analysis by Western blot in HEL expressing BCR-ABL (BA) or not (WT), KCL-22 and Lama 84 imatinib-sensitive (S) and -resistant (R) cell extracts. (C) Viability and viable cell numbers in untreated and imatinib-treated (1.5μM) Lama84-resistant Ph1⁺ cell line, submitted to TWIST-1 siRNA or negative scramble as control. Cells were incubated in 96-well plates in serum-free medium with stem cell factor (100 ng/mL), in the presence of 50nM of fluorescent siRNA. Results are presented as a percentage of viable cells or as total viable cell numbers ± SEM. After 2 days of culture, cell proliferation and viability were determined by trypan blue counting. Fold proliferation was determined by reference to the input number of viable cells. Results represent the mean ± SEM of 7 independent experiments. *Significant value (P < .05). (D) TWIST-1 expression in KCL22-resistant cells (KCL22R) transduced with a pSR vector empty or containing sh sequence against TWIST-1 or in KCl22-sensitive cells (KCL22S) transduced with a pMIG vector empty or containing TWIST-1 expressing sequence. After 3 days in the presence of puromycin selection for KCL22R-shTWIST-1 or after 48 hours for KCL22S-TWIST-1, cells were incubated in the presence of imatinib at the indicated dose. After 3 days of culture, cell proliferation and viability were determined by trypan blue counting. The percentage of proliferation was determined by reference to the nontreated number of viable cells ± SEM from 5 experiments. (E) Follow-up analysis for TWIST-1 expression by quantitative PCR in the CD34⁻ fraction. Results are expressed as a ratio to normal (healthy donors) CD34⁻ cell values. The values obtained for the BCR-ABL/ABL ratio are indicated as a percentage (IS) at each data point for both patients. BC indicates blast crisis; and CCyR, complete cytogenetic remission. †Deceased.

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