Figure 5.
Treatment with autophagy inducer restores CRT translocation after BTZ and in vivo drug efficacy. (A) Flow cytometry analysis of CRT exposure of AMO1 WT or GABARAPKO untreated or treated with BTZ (4 nM; 16 hours), rapamycin (100 nM; 24 hours) or a combination of both drugs. Fold increase as compared with untreated cells is shown. (B) Fold increase of CRT levels on surface of KMS11 cells untreated or treated with BTZ (6 nM; 16 hours), rapamycin (500 nM; 24 hours) or a combination of both drugs. (C) Phagocytosis assay of AMO1 WT or GABARAPKO untreated or pretreated with BTZ (4 nM; 16 hours), rapamycin (100 nM; 24 hours), or a combination of both drugs cocultured with far-red DCs for 4 hours. Shown is the fold increase of the percentage of double-positive DCs in treated conditions compared with untreated cells. (D) Phagocytosis assay of CFSE-stained KMS11 untreated or pretreated with BTZ (6 nM; 16 hours), rapamycin (500 nM; 24 hours), or a combination of both drugs cocultured with far-red DCs for 4 hours. Shown is the fold increase of the percentage of double-positive DCs in treated conditions compared with untreated cells. (E) 5TGM1 WT or GabarapKO were subcutaneously injected in immunocompetent C57BL/KaLwRijHsd mice. When tumors became palpable, mice bearing WT tumors were randomized to receive either BTZ (1 mg/kg) or phosphate-buffered saline (PBS); whereas mice bearing GabarapKO tumors were randomized to receive: PBS, BTZ (1 mg/kg), rapamycin (4 mg/kg), or a combination of both drugs. Tumors were retrieved 48 hours after BTZ treatment or in the combination group, 48 hours after BTZ and 24 hours after rapamycin. CRT expression was detected by immunofluorescence. Representative images of tumors retrieved from the different groups (left) stained with CRT antibody (red). DAPI was used to label nuclei (blue); scale bars, 100 μm (63× magnification). Average of cell intensity of CRT signal is shown (right), as analyzed by the Halo software. The numbers of observations reported are as follow: WT - BTZ (21 sections from 7 tumors); WT+BTZ (18 sections from 6 tumors); GabarapKO – BTZ (15 sections from 5 tumors); GabarapKO + BTZ (18 sections from 6 tumors); GabarapKO + RAPA (9 sections from 3 tumors); and GabarapKO + RAPA + BTZ (8 sections from 2 tumors); the signal from each section is represented as a dot in the graph. (F) Fold increase of tumor growth from day 1 (start of treatment) of subcutaneous 5TGM1 GabarapKO xenografts in C57BL/KaLwRijHsd mice treated with PBS (n = 5), BTZ (1 mg/kg twice per week for 2 weeks; n = 4), rapamycin (4 mg/kg per day for 5 days; n = 5), or a combination of both drugs (n = 6) ± standard error of the mean (SEM) for each group is reported. For panels A-F, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (unpaired Student t test). RAPA, rapamycin.

Treatment with autophagy inducer restores CRT translocation after BTZ and in vivo drug efficacy. (A) Flow cytometry analysis of CRT exposure of AMO1 WT or GABARAPKO untreated or treated with BTZ (4 nM; 16 hours), rapamycin (100 nM; 24 hours) or a combination of both drugs. Fold increase as compared with untreated cells is shown. (B) Fold increase of CRT levels on surface of KMS11 cells untreated or treated with BTZ (6 nM; 16 hours), rapamycin (500 nM; 24 hours) or a combination of both drugs. (C) Phagocytosis assay of AMO1 WT or GABARAPKO untreated or pretreated with BTZ (4 nM; 16 hours), rapamycin (100 nM; 24 hours), or a combination of both drugs cocultured with far-red DCs for 4 hours. Shown is the fold increase of the percentage of double-positive DCs in treated conditions compared with untreated cells. (D) Phagocytosis assay of CFSE-stained KMS11 untreated or pretreated with BTZ (6 nM; 16 hours), rapamycin (500 nM; 24 hours), or a combination of both drugs cocultured with far-red DCs for 4 hours. Shown is the fold increase of the percentage of double-positive DCs in treated conditions compared with untreated cells. (E) 5TGM1 WT or GabarapKO were subcutaneously injected in immunocompetent C57BL/KaLwRijHsd mice. When tumors became palpable, mice bearing WT tumors were randomized to receive either BTZ (1 mg/kg) or phosphate-buffered saline (PBS); whereas mice bearing GabarapKO tumors were randomized to receive: PBS, BTZ (1 mg/kg), rapamycin (4 mg/kg), or a combination of both drugs. Tumors were retrieved 48 hours after BTZ treatment or in the combination group, 48 hours after BTZ and 24 hours after rapamycin. CRT expression was detected by immunofluorescence. Representative images of tumors retrieved from the different groups (left) stained with CRT antibody (red). DAPI was used to label nuclei (blue); scale bars, 100 μm (63× magnification). Average of cell intensity of CRT signal is shown (right), as analyzed by the Halo software. The numbers of observations reported are as follow: WT - BTZ (21 sections from 7 tumors); WT+BTZ (18 sections from 6 tumors); GabarapKO – BTZ (15 sections from 5 tumors); GabarapKO + BTZ (18 sections from 6 tumors); GabarapKO + RAPA (9 sections from 3 tumors); and GabarapKO + RAPA + BTZ (8 sections from 2 tumors); the signal from each section is represented as a dot in the graph. (F) Fold increase of tumor growth from day 1 (start of treatment) of subcutaneous 5TGM1 GabarapKO xenografts in C57BL/KaLwRijHsd mice treated with PBS (n = 5), BTZ (1 mg/kg twice per week for 2 weeks; n = 4), rapamycin (4 mg/kg per day for 5 days; n = 5), or a combination of both drugs (n = 6) ± standard error of the mean (SEM) for each group is reported. For panels A-F, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (unpaired Student t test). RAPA, rapamycin.

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