Figure 2
Figure 2. Resistance to romidepsin in HuT78, DpVp50, and DpP75 cells does not involve P-glycoprotein but does involve the MAPK pathway. (A) Whole-cell lysates were prepared from HuT78 or DpP75 cells after a 24-hour incubation in the noted concentrations of romidepsin (Dp), subjected to polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose. Blots were probed with acetylated histone H3 (AcH3), histone H3 (H3), or GAPDH antibodies. Results from 1 of 3 independent experiments are shown; densitometry of AcH3, with normalization to histone H3 levels, is shown on the bottom row. (B) Pgp-expressing Dp500 cells were incubated with the noted concentrations of romidepsin in the presence or absence of 3 µg/mL of the Pgp inhibitor valspodar (PSC). Whole-cell lysates were prepared, subjected to PAGE, transferred to nitrocellulose, and probed with antibodies to AcH3 and GAPDH as in (A). Results from 1 of 3 independent experiments are shown. (C) HuT78 parental, DpVp50, and DpP75 cells were incubated with either an isotype control antibody (red histogram) or with the anti-Pgp antibody MRK-16 (Kamiya Biomedical, Seattle, WA) to detect cell surface expression (blue histogram) for 30 minutes, after which cells were washed and incubated with a phycoerythrin-labeled secondary antibody (Vector Laboratories, Burlingame, CA) and subsequently analyzed by flow cytometry. (D) HuT78, DpVp50, DpP75, and Dp500 cells were treated with 1 or 10 ng/mL of romidepsin (Dp) for 48 hours in the presence or absence of 200 nM of the Pgp inhibitor tariquidar (Tar.) or with tariquidar alone for 48 hours. Cells were harvested and incubated with anti-annexin V antibody and propidium iodide as outlined in Figure 1. Graphed results are from at least 3 independent experiments. Whole-cell lysates were prepared from HuT78 parental, DpVp35, DpVp50, and DpP75 cells, subjected to PAGE, and transferred to nitrocellulose membranes. The membranes were incubated with antibodies to (E) insulin receptor beta or GAPDH; (F) MEK, phosphorylated MEK (pMEK), ERK, or phosphorylated ERK (pERK); (G) Akt, phosphorylated Akt (pAKT), mTOR, phosphorylated mTOR (p-mTOR), IRS2, or GAPDH; and (H) STAT3, phosphorylated STAT3 (pSTAT3), or GAPDH. (I) HuT78 cells were treated with the noted concentrations of romidepsin for 24 hours. Expression of IR beta and pMEK was then examined by immunoblot analysis of whole-cell lysates. GAPDH served as a loading control. All experiments were performed at least 3 times. C, control.

Resistance to romidepsin in HuT78, DpVp50, and DpP75 cells does not involve P-glycoprotein but does involve the MAPK pathway. (A) Whole-cell lysates were prepared from HuT78 or DpP75 cells after a 24-hour incubation in the noted concentrations of romidepsin (Dp), subjected to polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose. Blots were probed with acetylated histone H3 (AcH3), histone H3 (H3), or GAPDH antibodies. Results from 1 of 3 independent experiments are shown; densitometry of AcH3, with normalization to histone H3 levels, is shown on the bottom row. (B) Pgp-expressing Dp500 cells were incubated with the noted concentrations of romidepsin in the presence or absence of 3 µg/mL of the Pgp inhibitor valspodar (PSC). Whole-cell lysates were prepared, subjected to PAGE, transferred to nitrocellulose, and probed with antibodies to AcH3 and GAPDH as in (A). Results from 1 of 3 independent experiments are shown. (C) HuT78 parental, DpVp50, and DpP75 cells were incubated with either an isotype control antibody (red histogram) or with the anti-Pgp antibody MRK-16 (Kamiya Biomedical, Seattle, WA) to detect cell surface expression (blue histogram) for 30 minutes, after which cells were washed and incubated with a phycoerythrin-labeled secondary antibody (Vector Laboratories, Burlingame, CA) and subsequently analyzed by flow cytometry. (D) HuT78, DpVp50, DpP75, and Dp500 cells were treated with 1 or 10 ng/mL of romidepsin (Dp) for 48 hours in the presence or absence of 200 nM of the Pgp inhibitor tariquidar (Tar.) or with tariquidar alone for 48 hours. Cells were harvested and incubated with anti-annexin V antibody and propidium iodide as outlined in Figure 1. Graphed results are from at least 3 independent experiments. Whole-cell lysates were prepared from HuT78 parental, DpVp35, DpVp50, and DpP75 cells, subjected to PAGE, and transferred to nitrocellulose membranes. The membranes were incubated with antibodies to (E) insulin receptor beta or GAPDH; (F) MEK, phosphorylated MEK (pMEK), ERK, or phosphorylated ERK (pERK); (G) Akt, phosphorylated Akt (pAKT), mTOR, phosphorylated mTOR (p-mTOR), IRS2, or GAPDH; and (H) STAT3, phosphorylated STAT3 (pSTAT3), or GAPDH. (I) HuT78 cells were treated with the noted concentrations of romidepsin for 24 hours. Expression of IR beta and pMEK was then examined by immunoblot analysis of whole-cell lysates. GAPDH served as a loading control. All experiments were performed at least 3 times. C, control.

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