Journal: Oncotarget

Article Title: KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models

doi:

Figure Lengend Snippet: A. Increased GFP-LC3 puncta formation in KMS-11/Cfz versus parental KMS-11 cells treated overnight with carfilzomib (50 nM). B. Increased stabilization of LC3B-II in KMS-11/Cfz versus parental KMS-11 cells upon autophagy inhibition. Representative western blot of LC3B levels of four independent experiments is shown ( P < 0.0001). Cells were treated overnight with carfilzomib (50 nM) in the presence or absence of the lysosomal protease inhibitors E64d (10 μg/ml), pepstatin A (PA; 10 μg/ml), and leupeptin (Leu; 1 μg/ml) or chloroquine (10 μM). Densitometry was used to calculate the LC3B-II/LC3B-I ratios and the relative increases in the ratios upon autophagy inhibition with respect to carfilzomib only-treated controls are indicated in the graph on the right. C. Cells were treated with the indicated concentrations of carfilzomib for 72 hours in the presence or absence of chloroquine (10 μM). Cell viability was determined by alamarBlue assay. The data are presented as the mean ± S.D of three experiments. IC 50 values were calculated by linear regression of the plots in the absence or presence of chloroquine: KMS-11/Cfz, 35 nM versus 22 nM; KMS-11, 12 nM versus 12 nM; KMS-34/Cfz, 35 nM versus 6 nM; KMS-34, 6 nM versus 6 nM.

Article Snippet: Carfilzomib was obtained from Active Biochem (Cat. No. A-1098), chloroquine was purchased from Selleck Chemicals (Cat. No. S4157), and the lysosomal inhibitors E-64d (Cat. No. E8640), leupeptin (Cat. No. 103476-89-7) and pepstatin A (Cat. No. 77170) were from Sigma-Aldrich.

Techniques: Inhibition, Western Blot, Alamar Blue Assay

Journal: Oncotarget

Article Title: KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models

doi:

Figure Lengend Snippet: Gene expression changes associated with acquisition of carfilzomib resistance (KMS-11/Cfz and KMS-34/Cfz) and KLF4 overexpression (KMS-11/KLF4) in MM cells

Article Snippet: Carfilzomib was obtained from Active Biochem (Cat. No. A-1098), chloroquine was purchased from Selleck Chemicals (Cat. No. S4157), and the lysosomal inhibitors E-64d (Cat. No. E8640), leupeptin (Cat. No. 103476-89-7) and pepstatin A (Cat. No. 77170) were from Sigma-Aldrich.

Techniques: Gene Expression, Over Expression

Journal: Oncotarget

Article Title: KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models

doi:

Figure Lengend Snippet: A. Gene set: GSE13411_IGM_MEMORY_BCELL_VS_PLASMA_CELL_UP (M3249). B. Gene set: GSE13411_NAIVE_BCELL_VS_PLASMA_CELL_UP (M3243). C. Gene set: GSE13411_SWITCHED_MEMORY_BCELL_VS_PLASMA_CELL_UP (M3251). FDR, false discovery rate; NES, normalized enrichment score; CfzR, carfilzomib-resistant derivatives; KLF4 is indicated.

Article Snippet: Carfilzomib was obtained from Active Biochem (Cat. No. A-1098), chloroquine was purchased from Selleck Chemicals (Cat. No. S4157), and the lysosomal inhibitors E-64d (Cat. No. E8640), leupeptin (Cat. No. 103476-89-7) and pepstatin A (Cat. No. 77170) were from Sigma-Aldrich.

Techniques: Clinical Proteomics

Journal: Oncotarget

Article Title: KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models

doi:

Figure Lengend Snippet: A. Fluorescence histograms of cells stained with the Cyto-ID Green autophagy detection reagent. Representative examples of three independent experiments are shown ( P < 0.05, paired Student's t test). B. , C. To assess complete autophagic flux, cells were treated overnight with or without the lysosomal protease inhibitors E64d (10 μg/ml), pepstatin A (PA; 10 μg/ml), and leupeptin (Leu; 1 μg/ml) . Cell lysates were probed with anti-LC3B B. or anti-SQSTM1 C. . D. Cells were labeled with anti-LC3B (Alexa Fluor 568, red) or anti-SQSTM1 (Alexa Fluor 488, green) antibodies, and immunofluorescence staining was analyzed by confocal laser scanning microscopy. Increased colocalization of the two factors in KMS-11/Cfz versus parental KMS-11 cells (yellow signals) correlated with acquisition of carfilzomib resistance ( P < 0.003).

Article Snippet: Carfilzomib was obtained from Active Biochem (Cat. No. A-1098), chloroquine was purchased from Selleck Chemicals (Cat. No. S4157), and the lysosomal inhibitors E-64d (Cat. No. E8640), leupeptin (Cat. No. 103476-89-7) and pepstatin A (Cat. No. 77170) were from Sigma-Aldrich.

Techniques: Fluorescence, Staining, Labeling, Immunofluorescence, Confocal Laser Scanning Microscopy

Journal: Oncotarget

Article Title: KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models

doi:

Figure Lengend Snippet: A. Increased GFP-LC3 puncta formation in KMS-11/Cfz versus parental KMS-11 cells treated overnight with carfilzomib (50 nM). B. Increased stabilization of LC3B-II in KMS-11/Cfz versus parental KMS-11 cells upon autophagy inhibition. Representative western blot of LC3B levels of four independent experiments is shown ( P < 0.0001). Cells were treated overnight with carfilzomib (50 nM) in the presence or absence of the lysosomal protease inhibitors E64d (10 μg/ml), pepstatin A (PA; 10 μg/ml), and leupeptin (Leu; 1 μg/ml) or chloroquine (10 μM). Densitometry was used to calculate the LC3B-II/LC3B-I ratios and the relative increases in the ratios upon autophagy inhibition with respect to carfilzomib only-treated controls are indicated in the graph on the right. C. Cells were treated with the indicated concentrations of carfilzomib for 72 hours in the presence or absence of chloroquine (10 μM). Cell viability was determined by alamarBlue assay. The data are presented as the mean ± S.D of three experiments. IC 50 values were calculated by linear regression of the plots in the absence or presence of chloroquine: KMS-11/Cfz, 35 nM versus 22 nM; KMS-11, 12 nM versus 12 nM; KMS-34/Cfz, 35 nM versus 6 nM; KMS-34, 6 nM versus 6 nM.

Article Snippet: Carfilzomib was obtained from Active Biochem (Cat. No. A-1098), chloroquine was purchased from Selleck Chemicals (Cat. No. S4157), and the lysosomal inhibitors E-64d (Cat. No. E8640), leupeptin (Cat. No. 103476-89-7) and pepstatin A (Cat. No. 77170) were from Sigma-Aldrich.

Techniques: Inhibition, Western Blot, Alamar Blue Assay