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. Author manuscript; available in PMC: 2022 Feb 2.
Published in final edited form as: Nat Cancer. 2021 Aug 2;2:978–993. doi: 10.1038/s43018-021-00237-1

Extended Data Fig. 6. Atg5 deletion blocks ESK981-induced vacuolization and CXCL10-mediated immune response.

Extended Data Fig. 6.

(a) Myc-CaP wild-type (WT) and Atg5 knockout (Atg5 KO) cells were treated with increasing concentrations of ESK981 for 24 hours. Atg5 and LC3 levels were assessed by western blot from three independent experiments. GAPDH served as a loading control.

(b) Representative morphology of vacuolization in Myc-CaP wild-type (WT) and Atg5 knockout (Atg5 KO) cells after treatment with control or 100 nM ESK981 for 24 hours from three independent experiments.

(c) Autophagosome content of Myc-CaP WT and Atg5 KO cells were measured by CYTO-ID® assay after being treated with increasing concentrations of ESK981 for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.

(d) Mouse cytokine array using Myc-CaP WT and Atg5 KO cell supernatant after treatment with 10 ng/ml mouse interferon gamma (mIFNγ) or mIFNγ + 100 nM ESK981 for 24 hours. Differential expression candidate dots are highlighted by boxes.

(e) Mouse CXCL10 protein levels were measured by ELISA in Myc-CaP WT and Atg5 KO conditioned medium with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.

(f) mRNA levels of Cxcl10 and Cxcl9 were measured by qPCR in Myc-CaP WT and Atg5 KO cells with 50 nM or 100 nM ESK981 and 10 ng/ml mIFNγ treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.