Figure 3
Figure 3. CD74 expression in FTY720-treated MCL cell lines. (A) Jeko-1 and Mino cell lines were treated with FTY720, chloroquine, rapamycin, or combinations at the indicated concentrations, harvested at 24 hours, and immunoblotted for CD74 (top panels). Actin was used as loading control. Representative histograms summarizing 3 independent experiments are also shown (middle panels). Histograms were obtained using densitometry data for CD74 levels in treated samples relative to untreated samples and normalized to the actin control. CD74 mRNA expression in MCL cell lines treated with FTY720 at the indicated concentrations for 8 or 24 hours was measured by real-time RT-PCR (bottom panels). The bar graph shows normalized fold expression of CD74 mRNA relative to untreated controls using GAPDH as an internal control. (B) CD74 MFI of MCL cells treated with FTY720, chloroquine, or rapamycin at the indicated concentrations after 8 and 24 hours. Cells were stained with an anti-CD74 Ab (FITC conjugated) and CD74 MFI was measured by flow cytometry. Flow cytometric data from single experiments are shown in the top panels; representative histograms summarizing the MFI of untreated and treated Jeko-1, Mino, UPN-1, and Z-138 cells are shown in the bottom panels. (C-D) Binding, internalization, and CD74 fluorescence intensity in Jeko-1 and Mino cells were examined by laser-scanning confocal microscopy. MCL cells were incubated with 5 μg/mL of rhodamine-conjugated milatuzumab (red) in the absence (C) or presence (D) of FTY720 at the indicated concentrations for 8 hours at 37°C. MCL cells were also stained with a primary Ab (anti-LC3) and a secondary Ab (Alexa Fluor 488–conjugated, green). DAPI was used for nuclear staining (blue). (E) The amount of total cellular CD74 was determined by confocal microscopy. Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY720 at the indicated doses, chloroquine (40μM), rapamycin (10μM), or the combination of FTY720 and chloroquine for 4, 8, and 24 hours. CD74 fluorescence intensity was measured in 4 microscopic fields and integrated intensity was averaged relative to the number of cells per field (approximately 180-220 cells per condition). Representative histograms summarizing CD74 fluorescence intensity are shown. P values were calculated comparing FTY720, chloroquine, and rapamycin treatment with the untreated controls.

CD74 expression in FTY720-treated MCL cell lines. (A) Jeko-1 and Mino cell lines were treated with FTY720, chloroquine, rapamycin, or combinations at the indicated concentrations, harvested at 24 hours, and immunoblotted for CD74 (top panels). Actin was used as loading control. Representative histograms summarizing 3 independent experiments are also shown (middle panels). Histograms were obtained using densitometry data for CD74 levels in treated samples relative to untreated samples and normalized to the actin control. CD74 mRNA expression in MCL cell lines treated with FTY720 at the indicated concentrations for 8 or 24 hours was measured by real-time RT-PCR (bottom panels). The bar graph shows normalized fold expression of CD74 mRNA relative to untreated controls using GAPDH as an internal control. (B) CD74 MFI of MCL cells treated with FTY720, chloroquine, or rapamycin at the indicated concentrations after 8 and 24 hours. Cells were stained with an anti-CD74 Ab (FITC conjugated) and CD74 MFI was measured by flow cytometry. Flow cytometric data from single experiments are shown in the top panels; representative histograms summarizing the MFI of untreated and treated Jeko-1, Mino, UPN-1, and Z-138 cells are shown in the bottom panels. (C-D) Binding, internalization, and CD74 fluorescence intensity in Jeko-1 and Mino cells were examined by laser-scanning confocal microscopy. MCL cells were incubated with 5 μg/mL of rhodamine-conjugated milatuzumab (red) in the absence (C) or presence (D) of FTY720 at the indicated concentrations for 8 hours at 37°C. MCL cells were also stained with a primary Ab (anti-LC3) and a secondary Ab (Alexa Fluor 488–conjugated, green). DAPI was used for nuclear staining (blue). (E) The amount of total cellular CD74 was determined by confocal microscopy. Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY720 at the indicated doses, chloroquine (40μM), rapamycin (10μM), or the combination of FTY720 and chloroquine for 4, 8, and 24 hours. CD74 fluorescence intensity was measured in 4 microscopic fields and integrated intensity was averaged relative to the number of cells per field (approximately 180-220 cells per condition). Representative histograms summarizing CD74 fluorescence intensity are shown. P values were calculated comparing FTY720, chloroquine, and rapamycin treatment with the untreated controls.

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