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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

Fig. 7. Identification of lipid kinase PIKfyve as the target of ESK981-induced effects on autophagy and CXCL10 levels.

Fig. 7.

(a) Gene ontology analysis for top elevated genes after 300 nM ESK981 in VCaP cells for 6 and 24 hour treatments. The top three processes are listed.

(b) Heatmap representation of untargeted lipidomics analysis after 300 nM ESK981 treatment for 6 and 24 hours in VCaP cells. N=3 technical replicates per group. The PE class is highlighted in red.

(c) Percent inhibition of 1 μM ESK981 against a panel of 22 lipid kinases. Data are presented as mean with individual data points.

(d) Representative dissociation constant (Kd) curve of ESK981 against lipid kinases PIKfyve, PIP5K1C, PIP5K1A, and PIK3CA.

(e) Representative morphology of DU145-RFP cells with control siRNA or PIKFYVE siRNA with three independent experiments.

(f) Fluorescent images showing autophagosome levels measured with CYTO-ID® assay in DU145, PC3, LNCaP, and VCaP after siRNA knockdown of PIKFYVE (top). PIKFYVE mRNA levels were quantified by qPCR in indicated cells after siNC or siPIKFYVE knockdown (bottom). Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.

(g) Quantification of autophagosome induction activity measured with CYTO-ID® assay in DU145, PC3, LNCaP, and VCaP after siRNA knockdown of PIKFYVE or treatment with ESK981. Data were analyzed by two-tailed unpaired t test from three independent experiments for VCaP siRNA and four independent experiments for the remaining conditions. Data are presented as mean ± SEM. P-values indicated.

(h) Cellular thermal shift assay (CESTA) of VCaP cells treated with control, 1 μM ESK981, or 1 μM apilimod for 2 hours with three independent experiments.

(i) CXCL10 mRNA levels in VCaP or PC3 cells after siRNA knockdown of a non-targeting control (siNC) or PIKFYVE (siPIKFYVE) 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.