Figure 4.
Sec61 inhibitors increase cell surface expression of BCMA in multiple myeloma cells. (A-C) KMS11 cells, (D-F) AMO1 cells. (A,D) KMS11 and AMO1 cells were treated with increasing concentrations of the Sec61 inhibitors CT8 and PS3061 (100, 200, 400, and 800 nM) or DMSO as a control for 24 hours. Cells were stained for cell surface expression of BCMA and CD38 and analyzed by using flow cytometry. Data are means of 3 biological replicates, and error bars denote standard deviations. (B,E) Total protein lysates from cells treated with increasing concentration of CT8 and PS3061 (200, 400, and 800 nM) for 24 hours were processed for western blotting. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used to normalize for differences in loading amounts. Data are represented as fold change relative to the normal protein expression level after normalization with GAPDH. Data points are means of 2 technical replicates, and error bars denote standard deviations. (C,F) Cells treated with increasing concentrations of PS3061 and CT8 (200, 400, and 800 nM) for 24 hours were processed for quantitative polymerase chain reaction to determine transcript levels of BCMA and CD38. Fold change in transcript levels were determined after normalizing to β-actin. Data are means of 2 biological replicates, and error bars denote standard deviations. (G) AMO1 cells were treated for 24 hours with the Sec61 inhibitor PS3061 (100 and 200 nM) and the γ-secretase inhibitor RO4929097 (5 μM) as single agents or in combination using DMSO as a control. Cells were stained for cell surface expression of BCMA and CD38 and analyzed by using flow cytometry. Data are means of 3 biological replicates, and error bars denote standard deviations. (H-I) KMS11 cells were treated with 800 nM of PS3061, 5 µM of RO4929097, and DMSO for 24 hours. (H) Drug-treated cells were analyzed by flow cytometry for cell surface expression of BCMA. Histograms indicate distribution of PE/CY7 BCMA in the drug-treated cells. Data are a representation of 2 biological replicates. (I) Concentration of sBCMA in the cell culture supernatant after drug treatment was measured by using enzyme-linked immunosorbent assay. Data are means of 2 biological replicates, and error bars denote standard deviations. **P < .005. n.s., not significant, 2-tailed, unpaired Student t test.

Sec61 inhibitors increase cell surface expression of BCMA in multiple myeloma cells. (A-C) KMS11 cells, (D-F) AMO1 cells. (A,D) KMS11 and AMO1 cells were treated with increasing concentrations of the Sec61 inhibitors CT8 and PS3061 (100, 200, 400, and 800 nM) or DMSO as a control for 24 hours. Cells were stained for cell surface expression of BCMA and CD38 and analyzed by using flow cytometry. Data are means of 3 biological replicates, and error bars denote standard deviations. (B,E) Total protein lysates from cells treated with increasing concentration of CT8 and PS3061 (200, 400, and 800 nM) for 24 hours were processed for western blotting. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used to normalize for differences in loading amounts. Data are represented as fold change relative to the normal protein expression level after normalization with GAPDH. Data points are means of 2 technical replicates, and error bars denote standard deviations. (C,F) Cells treated with increasing concentrations of PS3061 and CT8 (200, 400, and 800 nM) for 24 hours were processed for quantitative polymerase chain reaction to determine transcript levels of BCMA and CD38. Fold change in transcript levels were determined after normalizing to β-actin. Data are means of 2 biological replicates, and error bars denote standard deviations. (G) AMO1 cells were treated for 24 hours with the Sec61 inhibitor PS3061 (100 and 200 nM) and the γ-secretase inhibitor RO4929097 (5 μM) as single agents or in combination using DMSO as a control. Cells were stained for cell surface expression of BCMA and CD38 and analyzed by using flow cytometry. Data are means of 3 biological replicates, and error bars denote standard deviations. (H-I) KMS11 cells were treated with 800 nM of PS3061, 5 µM of RO4929097, and DMSO for 24 hours. (H) Drug-treated cells were analyzed by flow cytometry for cell surface expression of BCMA. Histograms indicate distribution of PE/CY7 BCMA in the drug-treated cells. Data are a representation of 2 biological replicates. (I) Concentration of sBCMA in the cell culture supernatant after drug treatment was measured by using enzyme-linked immunosorbent assay. Data are means of 2 biological replicates, and error bars denote standard deviations. **P < .005. n.s., not significant, 2-tailed, unpaired Student t test.

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