Date of download: 9/17/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Differential Responses of Human Papillary Thyroid Cancer Cell Lines Carrying the RET/PTC1 Rearrangement or a BRAF Mutation to MEK1/2 Inhibitors Arch Otolaryngol Head Neck Surg. 2007;133(8): doi: /archotol Expression of phosphorylated extracellular signal-related kinase 1/2 (p-ERK1/2) in papillary thyroid carcinoma (PTC) cells after treatment with PD98059 or U0126. A, Cells with a BRAF mutation (BHP5-16) were treated with 50μM, 75μM, 100μM, or 150μM PD98059 or 1μM, 5μM, 10μM, or 50μM U0126 for 24 hours. The expression of p-ERK1/2 was detected using Western blot analysis. Total ERK1/2 was used as a loading control and cells treated with dimethyl sulfoxide (DMSO) only were used as positive controls. The concentrations used in the experiments conducted hereafter (B and C) are indicated by asterisks. B, Cells with a BRAF mutation (BHP5-16) were treated for 1, 3, 6, 24, 48, or 72 hours. Expression of p-ERK1/2 and total ERK1/2 was detected using Western blot analysis. One group of cells (last lane) was treated with PD98059 or U0126 for 24 hours first and then incubated in regular medium without the inhibitors for 24 hours (24 hours + 24 hours). Cells treated with DMSO only were used as positive controls. C, Cells with RET/PTC1 rearrangement (BHP 2-7) or a BRAF mutation (BHP5-16) were treated (+) for 1 hour. Cells treated with DMSO only (−) were used as positive controls. Figure Legend:

Date of download: 9/17/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Differential Responses of Human Papillary Thyroid Cancer Cell Lines Carrying the RET/PTC1 Rearrangement or a BRAF Mutation to MEK1/2 Inhibitors Arch Otolaryngol Head Neck Surg. 2007;133(8): doi: /archotol Inhibition of the growth of papillary thyroid carcinoma (PTC) cells treated with PD98059 or U0126. Cells with the RET/PTC1 rearrangement (BHP2-7) (A) or a BRAF mutation (BHP5-16) (B) were treated with 75μM PD98059 or 10μM U0126 starting on day 0 for up to 4 days. Each day, the cells were counted according to absorption at 570 nm (Abs 570) after staining with 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) using a microplate reader. Cells treated with dimethyl sulfoxide (DMSO) only were used as positive controls. Figure Legend:

Date of download: 9/17/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Differential Responses of Human Papillary Thyroid Cancer Cell Lines Carrying the RET/PTC1 Rearrangement or a BRAF Mutation to MEK1/2 Inhibitors Arch Otolaryngol Head Neck Surg. 2007;133(8): doi: /archotol Detection of apoptosis only in cells with a BRAF mutation. A, Cells with the RET/PTC1 rearrangement (BHP2-7) or a BRAF mutation (BHP5-16) were treated with 75μM PD98059 (+) for 4 days and 1 to 4 days, respectively. B, Cells with the RET/PTC1 rearrangement (BHP2-7) or a BRAF mutation (BHP5-16) were treated with 10μM U0126 (+) for 4 days and up to 2 days, respectively. The cleaved poly(adenosine diphosphate–ribose) polymerase (PARP) protein was detected in cells with a BRAF mutation when treated with PD98059 for 1 to 4 days or with U0126 for 1 to 2 days using Western blot analysis. The PARP protein was detected in cells with either the RET/PTC1 rearrangement or a BRAF mutation. Actin was used as a loading control and cells treated with dimethyl sulfoxide only were used as positive controls (−). Figure Legend: