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A: Immunoblot analyses of ER stress markers ATF4 <t>and</t> <t>BIP</t> in ARCaP(M) and PC3 cells (C) grown alone or in TW co-culture with adipocytes, treated with 1μM CAY10566. ARCaP(M) (B) and PC3 (D) cells were cells grown alone or TW co-culture with adipocytes and treated with vehicle control (0.1% DMSO) or 1μM CAY10566 and scrambled siRNA or siRNA targeting SCD for 48 hours and subjected to immunoblot analysis of total and phosphorylated downstream mTOR proteins: <t>AKT,</t> P70S6K, PRAS40 and NDRG1. E: PC3 cells were grown in 3-dimensional cultures on reconstituted basement membrane (rBM) with a 2% Cultrex overlay. Live/Dead assay on 3D cultures grown alone (top panels) or with adipocytes (bottom panels) and treated with scrambled siRNA and vehicle control (0.1% DMSO) (Control), siRNA targeting SCD and 50nM Everolimus (EVO). Live cells (green fluorescence; calcein AM); dead cells (red fluorescence; ethidium homodimer. F: Quantification of live (green) spheroid volume compared to control for PC3 cells grown in monoculture (top panel) or with adipocytes (bottom panel). G: PC3 cells were grown in alone conditions or in TW co-culture with adipocytes and treated with vehicle control (0.1% DMSO) or 10nM EVO for 48 hours and subjected to immunoblot analysis of phosphorylated downstream mTOR proteins: AKT, P70S6K, NDRG1, and 4EBP1. Data represent at least 3 experiments; ** p < 0.01; *** p < 0.001 and ns = not significant.
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A: Immunoblot analyses of ER stress markers ATF4 and BIP in ARCaP(M) and PC3 cells (C) grown alone or in TW co-culture with adipocytes, treated with 1μM CAY10566. ARCaP(M) (B) and PC3 (D) cells were cells grown alone or TW co-culture with adipocytes and treated with vehicle control (0.1% DMSO) or 1μM CAY10566 and scrambled siRNA or siRNA targeting SCD for 48 hours and subjected to immunoblot analysis of total and phosphorylated downstream mTOR proteins: AKT, P70S6K, PRAS40 and NDRG1. E: PC3 cells were grown in 3-dimensional cultures on reconstituted basement membrane (rBM) with a 2% Cultrex overlay. Live/Dead assay on 3D cultures grown alone (top panels) or with adipocytes (bottom panels) and treated with scrambled siRNA and vehicle control (0.1% DMSO) (Control), siRNA targeting SCD and 50nM Everolimus (EVO). Live cells (green fluorescence; calcein AM); dead cells (red fluorescence; ethidium homodimer. F: Quantification of live (green) spheroid volume compared to control for PC3 cells grown in monoculture (top panel) or with adipocytes (bottom panel). G: PC3 cells were grown in alone conditions or in TW co-culture with adipocytes and treated with vehicle control (0.1% DMSO) or 10nM EVO for 48 hours and subjected to immunoblot analysis of phosphorylated downstream mTOR proteins: AKT, P70S6K, NDRG1, and 4EBP1. Data represent at least 3 experiments; ** p < 0.01; *** p < 0.001 and ns = not significant.

Journal: bioRxiv

Article Title: Targeting SCD in SCD-amplified prostate cancer inhibits growth in bone by modulating cellular stress, mTOR, and DNA damage pathways

doi: 10.1101/2025.06.09.658523

Figure Lengend Snippet: A: Immunoblot analyses of ER stress markers ATF4 and BIP in ARCaP(M) and PC3 cells (C) grown alone or in TW co-culture with adipocytes, treated with 1μM CAY10566. ARCaP(M) (B) and PC3 (D) cells were cells grown alone or TW co-culture with adipocytes and treated with vehicle control (0.1% DMSO) or 1μM CAY10566 and scrambled siRNA or siRNA targeting SCD for 48 hours and subjected to immunoblot analysis of total and phosphorylated downstream mTOR proteins: AKT, P70S6K, PRAS40 and NDRG1. E: PC3 cells were grown in 3-dimensional cultures on reconstituted basement membrane (rBM) with a 2% Cultrex overlay. Live/Dead assay on 3D cultures grown alone (top panels) or with adipocytes (bottom panels) and treated with scrambled siRNA and vehicle control (0.1% DMSO) (Control), siRNA targeting SCD and 50nM Everolimus (EVO). Live cells (green fluorescence; calcein AM); dead cells (red fluorescence; ethidium homodimer. F: Quantification of live (green) spheroid volume compared to control for PC3 cells grown in monoculture (top panel) or with adipocytes (bottom panel). G: PC3 cells were grown in alone conditions or in TW co-culture with adipocytes and treated with vehicle control (0.1% DMSO) or 10nM EVO for 48 hours and subjected to immunoblot analysis of phosphorylated downstream mTOR proteins: AKT, P70S6K, NDRG1, and 4EBP1. Data represent at least 3 experiments; ** p < 0.01; *** p < 0.001 and ns = not significant.

Article Snippet: Antibodies to HSPA5 (BIP; #3177), ATF4 (#11815), γ-H2AX (#9718), p-AKT (#4060), Total AKT (#9272), p-p70S6K (#9205), Total p70S6K (#34475), p-PRAS40 (#13175), Total PRAS40 (#2691), p-NDRG1 (#5482), Total NDRG1 (#9485), and p-4EBP1 (#9459) were from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Co-Culture Assay, Control, Membrane, Live Dead Assay, Fluorescence