Figure 1
Figure 1. FTY720 treatment blocks autophagy in MCL cells. (A) Four MCL cell lines were treated with FTY720 (Jeko-1, 12.5μM; Mino, 7.5μM; UPN-1, 12.5μM; and Z-138, 7.5μM), chloroquine (40μM), rapamycin (10μM), or combinations, harvested at 24 hours, and immunoblotted for the microtubule-associated protein light-chain 3 (LC3-I and LC3-II). Actin was used as a loading control. Representative histograms summarizing 3 independent experiments are also shown. Histograms were obtained using densitometry data for LC3-II levels in treated samples relative to untreated samples and normalized to the actin control. (B) The amount of total cellular LC3 was determined by confocal microscopy. Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY at the indicated doses, chloroquine (40μM), rapamycin (10μM), or the combination of FTY720 and chloroquine for 4, 8, and 24 hours. LC3 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 LC3 fluorescence intensity are shown. P values were calculated comparing FTY720, chloroquine, and rapamycin treatment with the untreated control. (C) Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY720 at the doses indicated in panel A, chloroquine (40μM), rapamycin (10μM), or combinations, harvested at 24 hours, and immunoblotted for p62. Actin was used as loading control. Levels of p62 normalized with actin are also shown in panel C. Representative histograms summarizing 3 independent experiments are also shown. Histograms were obtained using densitometry data for p62 levels in treated samples relative to untreated samples and normalized to the actin control. (D) Representative TEM images showing ultrastructural changes observed with Jeko-1 and Mino cells treated with or without FTY720 at the indicated concentrations for 8 hours. The arrows indicate accumulation of autophagic vacuoles containing cytoplasmic material after exposure to FTY720 or chloroquine.

FTY720 treatment blocks autophagy in MCL cells. (A) Four MCL cell lines were treated with FTY720 (Jeko-1, 12.5μM; Mino, 7.5μM; UPN-1, 12.5μM; and Z-138, 7.5μM), chloroquine (40μM), rapamycin (10μM), or combinations, harvested at 24 hours, and immunoblotted for the microtubule-associated protein light-chain 3 (LC3-I and LC3-II). Actin was used as a loading control. Representative histograms summarizing 3 independent experiments are also shown. Histograms were obtained using densitometry data for LC3-II levels in treated samples relative to untreated samples and normalized to the actin control. (B) The amount of total cellular LC3 was determined by confocal microscopy. Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY at the indicated doses, chloroquine (40μM), rapamycin (10μM), or the combination of FTY720 and chloroquine for 4, 8, and 24 hours. LC3 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 LC3 fluorescence intensity are shown. P values were calculated comparing FTY720, chloroquine, and rapamycin treatment with the untreated control. (C) Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY720 at the doses indicated in panel A, chloroquine (40μM), rapamycin (10μM), or combinations, harvested at 24 hours, and immunoblotted for p62. Actin was used as loading control. Levels of p62 normalized with actin are also shown in panel C. Representative histograms summarizing 3 independent experiments are also shown. Histograms were obtained using densitometry data for p62 levels in treated samples relative to untreated samples and normalized to the actin control. (D) Representative TEM images showing ultrastructural changes observed with Jeko-1 and Mino cells treated with or without FTY720 at the indicated concentrations for 8 hours. The arrows indicate accumulation of autophagic vacuoles containing cytoplasmic material after exposure to FTY720 or chloroquine.

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