Supplementary figure S1 A B C D Figure S1. EGF in combination with LY294002 induces survival and NE differentiation of LNCaP cells. LNCaP cells were cultured.

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Supplementary figure S1 A B C D Figure S1. EGF in combination with LY induces survival and NE differentiation of LNCaP cells. LNCaP cells were cultured in RPMI with 7% FBS for 2 days and then placed in serum-free medium for 24 hours. The cells were then incubated with LY29 (20 µM), EGF (10 -8 M) or LY29 plus EGF. After 24 hours: (A) the cells were treated with TMRM (200 nM) and the signal of TMRM-stained cells was determined by flow cytometry. The graph shows the percentage of TMRM-negative cells. Error bars represent the standard error of the mean (SEM). Significance of t-test **p < 0,01; (B) cell lysates were isolated and NSE levels were analyzed by western blot; error bars represent the standard error (SE). Significance of t-test *p < 0,1; (C) the relative amount of chromogranin B (CrgB) mRNA was quantified by real-time RT-PCR; error bars represent the standard error (SE); (D) cells were fixed and immunostained with anti-chromogranin A (red) and DAPI (blue). The figure is representative of three different experiments, all of which exhibited similar trends.

Supplementary figure S2 A B C Figure S2. The increase of ErbB2 levels and MAPK-Erk activity induced by EGF in combination with LY were maintained over time. LNCaP cells were cultured in RPMI with 7% FBS for 2 days and then placed in serum-free medium for 24 hours. The cells were then incubated with EGF (10 -8 M) or LY29 (20 µM) plus EGF LNCaP cells for the indicated times. The ErbB2 levels (A) or MAPK-ERK activity (B) were analyzed by western blot. Each figure is representative of three different experiments, all of which exhibited similar trends.

Supplementary figure S3 Figure S3. The increase of ErbB2 levels and MAPK-Erk activity induced by EGF in combination with LY were dose-dependent. LNCaP cells were cultured in RPMI with 7% FBS for 2 days and then placed in serum-free medium for 24 hours. The cells were then incubated with (A) indicated dose of LY in the presence of EGF (10 -8 M) or (B) indicated dose of EGF in the presence of LY (20 µM). After 24 hours, the ErbB2 levels were analyzed by western blot. The figure is representative of three different experiments, all of which exhibited similar trends. A B

Supplementary figure S4 PC3 DU145 Figure S4. EGF in combination with LY did not increase ErbB2 levels in PC3 and DU145 cells. PC3 cells (up) and DU145 (Bottom) were cultured in RPMI with 10% FBS for 2 days and then placed in serum-free medium for 24 hours. The cells were then incubated with EGF (10 -8 M) or LY29 (20 µM) plus EGF for the indicated times. The ErbB2 levels were analyzed by western blot. Each figure is representative of three different experiments, all of which exhibited similar trends.

Supplementary figure S5 EGF LY P-p70S6k (T389) P-Akt (S473) β-tubulin Figure S5. mTORC1 is constitutively active in LNCaP cells. LNCaP cells were cultured in RPMI with 7% FBS for 2 days and then placed in serum-free medium for 24 hours. The cells were then incubated with EGF (10 -8 M) or LY29 (20 µM) plus EGF LNCaP cells for 24 hours. Cellular lysates were analysed by immunoblotting using the indicated specific antibodies. Each figure is representative of three different experiments, all of which exhibited similar trends.