Figure 2

MCL1 is transcriptionally regulated by ganetespib. (a) MSTO-211H, H460 and H23 have been treated with ganetespib 200 nm for 48 h. PARP cleavage and the expression of the pro-survival BCL-2 family members have been assessed by western blot. (b) MCL1 mRNA expression was evaluated by qRT-PCR on RNA extracted from MSTO-211H, H460 and H23 cells treated for 48 h with ganetespib 200 nm. Data were normalized to untreated control (MSTO-211H: **P=0.0033; H460 *P= 0.0150; H23: ***P=0.0006). (c) The MCL1 promoter activity was measured by a luciferase reporter assay in MSTO-211H, H460 and H23 cells transfected with pGL2 basic (EV) or pGL2-MCL1 (FL) and then treated with ganetespib 200 nm for 24 h. Data were normalized to untreated full-length (FL) control (MSTO-211H: *P=0.0119; H460 ****P<0.0001; H23: ***P=0.0010). (d) MSTO-211H cells were transfected with three fragments of the promoter and treated with ganetespib 200 nm for 24 h. The MCL1 promoter activity was measured by a reporter assay. Data were normalized to untreated FL control (Fragment A: **P=0.0031; Fragment B **P=0.0077; Fragment C: ****P<0.0001). (e) The effect of siSTAT5A and sip53 on MCL1 mRNA expression and MCL1 promoter activity was measured by qRT-PCR on RNA extracted from MSTO-211H and NCI-H28 cells transfected for 24 h or luciferase reporter assay. Cells treated with ganetespib 200 nm were used as a positive control. Data were normalized to untreated control (qRT-PCR: MSTO-211H, siSTAT5A: *P=0.03977, sip53: n.s.; NCI-H28, siSTAT5A: *P=0.0439, sip53: n.s. Luciferase assay: MSTO-211H, siSTAT5A: ***P<0.0001, sip53: n.s.; NCI-H28, siSTAT5A: ***P=0.0003, sip53: n.s.). (f) The effect of ganetespib on STAT5 was measured by western blot.