Data represents mean Cell Cycle inhibitor fluorescence normalized to DMSO treated pLKO.1-Neg cells, n = 3. (C) Viability of transformed cells following 24 hour treatment with SW43, PB282, HCQ. Data represents percent viability compared to DMSO treated cells, n = 3, * p < 0.05. Lysosomal accumulation of sigma-2 receptor ligands is required for LMP and cell death Bxpc3 cells were treated with CMA (10 nM) for 60 minutes in order to effectively inhibit the pH gradient across the lysosomal Copanlisib membrane. Subsequent accumulation of the fluorescently labeled sigma-2 receptor ligands SW120 and PB385 showed marked decrease of fluorescence intensity by flow cytometry (Figure 5A). Bxpc3 cells pretreated

with CMA were more viable following treatment with sigma-2 ligands, but the response was greater for SW43 and HCQ compared to PB282 (Figure 5B). To determine EPZ5676 order whether LMP lead to release of proteases into the cytoplasm, the cytosolic fraction was isolated from the lysosomal fraction by selective permeabilization of the plasma membrane with digitonin, and cleavage ofcathepsin B substrate Z-RR-AMC was assessed. All compounds increased Z-RR-AMC cleavage within one hour of treatment, and CMA decreased this Z-RR-AMC cleavage to baseline (Figure 5C). CA-074-Me and pepstatin A decreased cleavage for all compounds as well, with the exception that pepstatin A was not observed to inhibit cleavage following SW43 treatment.

Functional rescue of viability in the presence of CA-074-Me and pepstatin A was assessed at 24 hours following treament, and pepstatin A was observed to rescue viability across a titrated dose range for all compounds, while CA-074-Me had a lesser effect, though the observed differences did not reach statistical significance (Figure 5D). Figure 5 Sigma-2 receptor ligand-mediated cell death is dependent on lysosomal accumulation and membrane permeabilization.

(A) Accumulation of sigma-2 receptor ligands SW120 and PB385 in Bxpc3 cells following inhibition of lysosomal pH gradient with the V-ATPase inhibitor concanamycin A (CMA) (10 nM) detected by flow cytometry. (B) Cell viability following 24 treatment with sigma-2 receptor ligands Hydroxychloroquine supplier SW43 and PB282 or lysosomal detergent hydroxychlorquine (HCQ) in the presence of CMA (10 nM). Data represents percent viability compared to DMSO treated cells, n = 3, p < 0.05 (C) Cleavage of fluorescent peptidase substrate Z-RR-AMC following one hour treatment with SW43 (30 μM), PB282 (30 μM), or HCQ (60 μM), in the presence of CMA (10 nM) or peptidase inhibitors CA-074-Me (10 μM) and pepstatin A (100 μM). Data is relative to DMSO treated cells and is representative of experiments performed in triplicate. (D) Cell viability following 24 hour treatment with SW43, PB282, or HCQ in the presence of CA-074-Me or pepstatin A. Data represents percent viability compared to DMSO treated cells, n = 3.