Figure 4
Figure 4. Cathepsin B activity is required for arsenic-induced autophagic degradation of BCR-ABL. (A) K562 cells were treated with AS2O3 (2μM) for 24 hours. The cells were then stained with a pan-cathepsin probe (Prosense 680; red), and aftercollection stained with either anti-ABL (blue) or anti-p62/SQSTM1 (green), and signals were detected by confocal microscopy. Merged panels indicate overlapping images of the 3 fluorescing signals, and magnified sections indicate colocalization of p62/SQSTM1, BCR-ABL, and cathepsins. (B) K562 cells were treated with or without As2O3 (2μM) or CA-074 (5μM) for 24 hours. The cells were then stained with quenched probe DQ-BSA (red), and after collection stained with either anti-ABL (blue), or anti-p62/SQSTM1 (red), and signals were detected by confocal microscopy. Merged panels indicate overlapping images of the 3 fluorescing signals, and arrows show the colocalization of p62/SQSTM1, BCR-ABL, and lysosomal probe DQ-BSA. (C left panel) Lysates from K562 cells stably expressing control (Ctrl)–shRNA or cathepsin B-shRNA where incubated with the biotinylated probe DCG-04 and, after resolution by SDS-PAGE, were immunoblotted with anti-streptavidin or anti-Hsp90 antibodies, as indicated. (Right panel) K562 cells stably expressing Ctrl-shRNA or Cathepsin B shRNA were treated with As2O3 (2μM) for 24 hours. Total lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D left panel) K562 cells were incubated in the presence or absence of As2O3 (2μM) and/or CA-074 (5μM) for 24 hours. Cell lysates where incubated with the biotinylated probe DCG-04, resolved by SDS-PAGE and immunoblotted with anti-streptavidin or anti-Hsp90, as indicated. (Right panel) K562 cells were incubated in the presence or absence of As2O3 (2μM) and/or CA-074 (5μM) for 24 hours. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (E) K562 cells were plated in a methylcellulose assay system in the presence of either As2O3 (0.5μM) and/or CA-074 (5μM) as indicated. Data are expressed as percent control of CFU-L colony numbers for control untreated cells and represent means ± SE of 4 independent experiments. Paired t test analysis comparing the effects As2O3 in the absence or presence of CA-074 combination showed a paired P value = .0013. (F) K562 cells stably expressing control (CTRL)–shRNA or cathepsin B (CTSB)–shRNA were plated in a methylcellulose assay system in the presence of either AS2O3 (0.5μM) as indicated. Data are expressed as percent control of CFU-L colony numbers for control untreated cells and represent means ± SE of 4 independent experiments. Paired t test analysis comparing the effects AS2O3 showed a paired P value = .0249.

Cathepsin B activity is required for arsenic-induced autophagic degradation of BCR-ABL. (A) K562 cells were treated with AS2O3 (2μM) for 24 hours. The cells were then stained with a pan-cathepsin probe (Prosense 680; red), and aftercollection stained with either anti-ABL (blue) or anti-p62/SQSTM1 (green), and signals were detected by confocal microscopy. Merged panels indicate overlapping images of the 3 fluorescing signals, and magnified sections indicate colocalization of p62/SQSTM1, BCR-ABL, and cathepsins. (B) K562 cells were treated with or without As2O3 (2μM) or CA-074 (5μM) for 24 hours. The cells were then stained with quenched probe DQ-BSA (red), and after collection stained with either anti-ABL (blue), or anti-p62/SQSTM1 (red), and signals were detected by confocal microscopy. Merged panels indicate overlapping images of the 3 fluorescing signals, and arrows show the colocalization of p62/SQSTM1, BCR-ABL, and lysosomal probe DQ-BSA. (C left panel) Lysates from K562 cells stably expressing control (Ctrl)–shRNA or cathepsin B-shRNA where incubated with the biotinylated probe DCG-04 and, after resolution by SDS-PAGE, were immunoblotted with anti-streptavidin or anti-Hsp90 antibodies, as indicated. (Right panel) K562 cells stably expressing Ctrl-shRNA or Cathepsin B shRNA were treated with As2O3 (2μM) for 24 hours. Total lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D left panel) K562 cells were incubated in the presence or absence of As2O3 (2μM) and/or CA-074 (5μM) for 24 hours. Cell lysates where incubated with the biotinylated probe DCG-04, resolved by SDS-PAGE and immunoblotted with anti-streptavidin or anti-Hsp90, as indicated. (Right panel) K562 cells were incubated in the presence or absence of As2O3 (2μM) and/or CA-074 (5μM) for 24 hours. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (E) K562 cells were plated in a methylcellulose assay system in the presence of either As2O3 (0.5μM) and/or CA-074 (5μM) as indicated. Data are expressed as percent control of CFU-L colony numbers for control untreated cells and represent means ± SE of 4 independent experiments. Paired t test analysis comparing the effects As2O3 in the absence or presence of CA-074 combination showed a paired P value = .0013. (F) K562 cells stably expressing control (CTRL)–shRNA or cathepsin B (CTSB)–shRNA were plated in a methylcellulose assay system in the presence of either AS2O3 (0.5μM) as indicated. Data are expressed as percent control of CFU-L colony numbers for control untreated cells and represent means ± SE of 4 independent experiments. Paired t test analysis comparing the effects AS2O3 showed a paired P value = .0249.

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