Journal: Autophagy

Article Title: Type I interferons induce autophagy in certain human cancer cell lines

doi: 10.4161/auto.23921

Figure Lengend Snippet: Figure 10. Role of the MTORC1 activity in IFN-induced autophagy. ( A ) siRNA-mediated RNA silencing of MTOR . T98G cells were transfected for 48 h with 100 nM SignalSilence R MTOR siRNA or SignalSilence R control siRNA follow by IFNA2c (3.6 ng/mL) treatment for 48 h. The effect of MTOR -siRNA on MAP1LC3-II level was examined by western blot. Lanes: (1) molecular weight marker; (2) negative siRNA control; (3) negative siRNA control plus IFNA2c (3.6 ng/mL); (4) specific siRNA for MTOR ; (5) specific siRNA for MTOR plus IFNA2c (3.6 ng/mL). Data are representative of three individual experiments. Ratios of MAP1LC3 were calculated as the division of the ratio of induced MAP1LC3-I to induced MAP1LC3-II by the ratio of basal MAP1LC3-I to basal MAP1LC3-II, and the numbers are shown below the MAP1LC3 lanes. ( B ) Detection of MAP1LC3-I, MAP1LC3-II, and p-RPS6 upon treatment with inhibitors rapamycin, LY294002 and IFNA2c. Lanes: (1) molecular weight marker; (2) negative control, untreated cells; (3) IFNA2c (3.6 ng/mL); (4) rapamycin (2.7 nM); (5) IFNA2c (3.6 ng/mL) + rapamycin (2.7 nM); (6) LY294002 (10 µM); (7) IFNA2c (3.6 ng/mL) + LY294002 (10 µM) Data are representative of two individual experiments. Ratios of MAP1LC3 were calculated as the division of the ratio of induced MAP1LC3-I to induced MAP1LC3-II by the ratio of basal MAP1LC3-I to basal MAP1LC3-II, and the numbers are shown below the MAP1LC3 lanes.

Article Snippet: HeLa S3 cells were seeded at 1 × 10 5 cells/well in 6-well plates and immediately transfected for 48 h with 100 nM SignalSilence R MTOR siRNA (Cell Signaling 6381), or SignalSilence R control siRNA (Cell Signaling 6568), using 5 μg Lipofectamine TM 2000 (Invitrogen, 11668019) diluted in Opti-MEM (Gibco, 31985070).

Techniques: Activity Assay, Transfection, Control, Western Blot, Molecular Weight, Marker, Negative Control

Journal: Autophagy

Article Title: Type I interferons induce autophagy in certain human cancer cell lines

doi: 10.4161/auto.23921

Figure Lengend Snippet: Table 2. MTOR siRNA and IFNA2c inhibit cell growth

Article Snippet: HeLa S3 cells were seeded at 1 × 10 5 cells/well in 6-well plates and immediately transfected for 48 h with 100 nM SignalSilence R MTOR siRNA (Cell Signaling 6381), or SignalSilence R control siRNA (Cell Signaling 6568), using 5 μg Lipofectamine TM 2000 (Invitrogen, 11668019) diluted in Opti-MEM (Gibco, 31985070).

Techniques: Inhibition, Negative Control

Journal: International Journal of Oncology

Article Title: Curcumin inhibits cancer-associated fibroblast-driven prostate cancer invasion through MAOA/mTOR/HIF-1α signaling

doi: 10.3892/ijo.2015.3202

Figure Lengend Snippet: CAFs promote MAOA, mTOR, and HIF-1α expression in PC3 cells. CM, conditioned media of CAFs, St Med, starved standard media of PC3 cells. CAFs were isolated from surgical explants from cancer regions of patients with PCa (Gleason 4+5). (A) (a–c) Cytokeratin 18 expression in CAFs was determined by immuofluorescence microscopy analysis. (d–f) α-SMA expression in CAFs was determined by immuofluorescence microscopy analysis. (B) PC3 cells were incubated with or without the indicated CM and the levels of MAOA, mTOR, HIF-1α, and β-actin were assessed by immunoblotting and qRT-PCR. (C) MAOA, mTOR, or HIF-1α were transiently silenced with shRNA in PC3 cells. The interfering efficiency was determined by immunoblotting and qRT-PCR. * P<0.05.

Article Snippet: shRNA against MAOA (Santa Cruz Biotechnology, sc-35847-SH), shRNA against mTOR (Santa Cruz Biotechnology, sc-35409-SH), shRNA against HIF-1α (Santa Cruz Biotechnology, sc-35561-SH), and a negative control shRNA (Santa Cruz Biotechnology, sc-108060) were obtained from GenePharm (Shanghai, China).

Techniques: Expressing, Isolation, Microscopy, Incubation, Western Blot, Quantitative RT-PCR, shRNA

Journal: International Journal of Oncology

Article Title: Curcumin inhibits cancer-associated fibroblast-driven prostate cancer invasion through MAOA/mTOR/HIF-1α signaling

doi: 10.3892/ijo.2015.3202

Figure Lengend Snippet: CAFs promote EMT by triggering a MAOA/mTOR/HIF-1α signaling pathway. CM, conditioned media of CAFs, St Med, starved standard media of PC3 cells. MAOA, mTOR, or HIF-1α were transiently silenced with shRNA in PC3 cells. These cells were incubated with or without the indicated CM after silencing, and (A) the levels of MAOA, mTOR, HIF-1α, and β-actin were assessed by immunoblotting. (B) PC3 cells were treated as above, and the levels of E-cadherin, vimentin, and β-actin were assessed by immunoblotting. (C and D) PC3 cells were treated as above (B), and their invasion was evaluated. Invading cells were counted and a bar graph, representative of six randomly chosen fields, is shown. * P<0.05 versus sh-control group treated with St Med group; # P<0.05 versus sh-control group treated with CM.

Article Snippet: shRNA against MAOA (Santa Cruz Biotechnology, sc-35847-SH), shRNA against mTOR (Santa Cruz Biotechnology, sc-35409-SH), shRNA against HIF-1α (Santa Cruz Biotechnology, sc-35561-SH), and a negative control shRNA (Santa Cruz Biotechnology, sc-108060) were obtained from GenePharm (Shanghai, China).

Techniques: shRNA, Incubation, Western Blot, Control

Journal: International Journal of Oncology

Article Title: Curcumin inhibits cancer-associated fibroblast-driven prostate cancer invasion through MAOA/mTOR/HIF-1α signaling

doi: 10.3892/ijo.2015.3202

Figure Lengend Snippet: CAFs promote a MAOA/mTOR/HIF-1α-dependent oxidative response and CXCR4 and IL-6 receptor expression. CM, conditioned media of CAFs, St Med, starved standard media of PC3 cells. MAOA, mTOR, or HIF-1α were transiently silenced with shRNA in PC3 cells, and these cells were incubated with or without the indicated CM, and (A) hydrogen peroxide production was evaluated with DCF-DA and normalized based on total protein content. CXCR4 (B) and IL-6 receptor (C) mRNA expression levels were evaluated by qRT-PCR. * P<0.05 versus sh-control group treated with St Med group; # P<0.05 versus sh-control group treated with CM.

Article Snippet: shRNA against MAOA (Santa Cruz Biotechnology, sc-35847-SH), shRNA against mTOR (Santa Cruz Biotechnology, sc-35409-SH), shRNA against HIF-1α (Santa Cruz Biotechnology, sc-35561-SH), and a negative control shRNA (Santa Cruz Biotechnology, sc-108060) were obtained from GenePharm (Shanghai, China).

Techniques: Expressing, shRNA, Incubation, Quantitative RT-PCR, Control

Journal: International Journal of Oncology

Article Title: Curcumin inhibits cancer-associated fibroblast-driven prostate cancer invasion through MAOA/mTOR/HIF-1α signaling

doi: 10.3892/ijo.2015.3202

Figure Lengend Snippet: Curcumin suppresses CAF-induced EMT in PC3 cells via MAOA/mTOR/HIF-1α signaling. CM, conditioned media of CAFs, St Med, starved standard media of PC3 cells. (A) PC3 cells were incubated with or without the indicated CM in the presence or absence of 25 μM curcumin, and the levels of E-cadherin, vimentin, and β-actin were assessed by immunoblotting. (B) PC3 cells in which MAOA, mTOR, or HIF-1α was transiently silenced with shRNA, and then were incubated with the indicated CM in the presence or absence of 25 μM curcumin, and the levels of E-cadherin, vimentin, and β-actin were assessed by immunoblotting. (C and D) PC3 cells were treated as in (A), and their invasion was evaluated. Invading cells were counted and a bar graph, representative of six randomly chosen fields, is shown. * P<0.05 versus control group treated with St Med; # P<0.05 versus control group treated with CM. (E and F) PC3 cells were treated as in (B), and their invasion was evaluated. Invading cells were counted and a bar graph, representative of six randomly chosen fields, is shown. * P<0.05 versus sh-control group treated with CM; # P<0.05 versus sh-control group treated with CM+curcumin.

Article Snippet: shRNA against MAOA (Santa Cruz Biotechnology, sc-35847-SH), shRNA against mTOR (Santa Cruz Biotechnology, sc-35409-SH), shRNA against HIF-1α (Santa Cruz Biotechnology, sc-35561-SH), and a negative control shRNA (Santa Cruz Biotechnology, sc-108060) were obtained from GenePharm (Shanghai, China).

Techniques: Incubation, Western Blot, shRNA, Control

Journal: International Journal of Oncology

Article Title: Curcumin inhibits cancer-associated fibroblast-driven prostate cancer invasion through MAOA/mTOR/HIF-1α signaling

doi: 10.3892/ijo.2015.3202

Figure Lengend Snippet: Curcumin abolishes CAF-induced ROS production and CXCR4 and IL-6 receptor expression in PC3 cells through the MAOA/mTOR/HIF-1α signaling. CM, conditioned media of CAFs. PC3 cells in which MAOA, mTOR, or HIF-1α was transiently silenced with shRNA were incubated with the indicated CM in the presence or absence of 25 μM curcumin (A), hydrogen peroxide production was evaluated with DCF-DA and normalized based on total protein content. CXCR4 (B) and IL-6 receptor (C) mRNA expression levels were evaluated by qRT-PCR. * P<0.05 versus sh-control group treated with CM.

Article Snippet: shRNA against MAOA (Santa Cruz Biotechnology, sc-35847-SH), shRNA against mTOR (Santa Cruz Biotechnology, sc-35409-SH), shRNA against HIF-1α (Santa Cruz Biotechnology, sc-35561-SH), and a negative control shRNA (Santa Cruz Biotechnology, sc-108060) were obtained from GenePharm (Shanghai, China).

Techniques: Expressing, shRNA, Incubation, Quantitative RT-PCR, Control

Journal:

Article Title: Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival

doi: 10.1038/onc.2008.318

Figure Lengend Snippet: (A)Effect of increasing doses of rapamycin on LNCaP and C4-2 cells treated for 48 hours. Raptor expression was not affected by rapamycin (upper panel), but its activation was impaired as indicated by the de-phosphorylation of the p70S6K substrate, the ribosomal protein S6 (rpS6) (2nd panel). On the other hand, rictor expression was severely impeded (3rd panel). (B) Effect of inhibition of mTORC1 and mTORC2 individually on apoptosis. C4-2 cells were transfected with a control siRNA or with raptor or rictor siRNA, and then treated with DMSO (vehicle) or 10 μM bicalutamide. Cells were collected after 48 hours and analyzed for percent of apoptotic cells by flow cytometry. (C) Effect of overexpression of raptor or rictor on apoptosis induced by rapamycin + bicalutamide. C4-2 cells were transfected with vector (pCMV) or with myc-raptor or myc-rictor, then treated with DMSO, 10 μM bicalutamide, 1 nM rapamycin or both. Apoptosis was analyzed as described above.

Article Snippet: Cells were plated in 60 mm dishes and transfected with 50 pmoles of one of the following: AR siRNA, mTOR siRNA, Akt1 siRNA, raptor siRNA (Santa Cruz Biotechnlogy, Santa Cruz, CA) and rictor siRNA, consisting of a pool of 4 siRNA duplexes (Dharmacon Research Inc., Lafayette, CO).

Techniques: Expressing, Activation Assay, De-Phosphorylation Assay, Inhibition, Transfection, Control, Flow Cytometry, Over Expression, Plasmid Preparation

Journal:

Article Title: Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival

doi: 10.1038/onc.2008.318

Figure Lengend Snippet: (A) Effect of raptor on Akt phosphorylation. LNAI cells were treated with vehicle or 10 μM bicalutamide and 1 nM rapamycin for 48 hours in the presence of control or raptor siRNA. (B) Effect of the same doses of rapamycin and bicalutamide on Akt phosphorylation in the presence of an empty vector (pCMV) or a plasmid overexpressing raptor (myr-raptor) in LNAI cells. (C) Effect of a control siRNA or siRNAs against raptor or (D) a vector control or myr-raptor on AR transcriptional activity of LNCaP cells in the presence of vehicle or bicalutamide as determined by luciferase assay. Data shows hPSA-luc activity normalized to β-gal and represent mean ± S.D. fold change over vehicle control (n=3).

Article Snippet: Cells were plated in 60 mm dishes and transfected with 50 pmoles of one of the following: AR siRNA, mTOR siRNA, Akt1 siRNA, raptor siRNA (Santa Cruz Biotechnlogy, Santa Cruz, CA) and rictor siRNA, consisting of a pool of 4 siRNA duplexes (Dharmacon Research Inc., Lafayette, CO).

Techniques: Phospho-proteomics, Control, Plasmid Preparation, Activity Assay, Luciferase

Journal:

Article Title: Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival

doi: 10.1038/onc.2008.318

Figure Lengend Snippet: (A) Effect of rictor on Akt phosphorylation. LNAI cells were treated with vehicle or 10 μM bicalutamide and 1 nM rapamycin for 48 hours in the presence of control or rictor siRNA. (B) Effect of the same doses of rapamycin and bicalutamide on Akt phosphorylation in the presence of an empty vector (pCMV) or a plasmid overexpressing rictor (myr-rictor) in LNAI cells. (C) Effect of a control siRNA or siRNAs against rictor or (D) a vector control or myr-rictor on AR transcriptional activity of LNCaP cells in the presence of vehicle or bicalutamide as determined by luciferase assay. Data shows hPSA-luc activity normalized to β-gal and represent mean ± S.D. fold change over vehicle control (n=3).

Article Snippet: Cells were plated in 60 mm dishes and transfected with 50 pmoles of one of the following: AR siRNA, mTOR siRNA, Akt1 siRNA, raptor siRNA (Santa Cruz Biotechnlogy, Santa Cruz, CA) and rictor siRNA, consisting of a pool of 4 siRNA duplexes (Dharmacon Research Inc., Lafayette, CO).

Techniques: Phospho-proteomics, Control, Plasmid Preparation, Activity Assay, Luciferase

Journal:

Article Title: Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival

doi: 10.1038/onc.2008.318

Figure Lengend Snippet: (A) AR transcriptional activity in C4-2 cells transfected with control or Akt1 siRNA and treated with 1 nM rapamycin and 10 μM bicalutamide. Cells were co-transfected with hPSA-luc and β-gal and luciferase activity was estimated in the transfected cells after 48 hours of treatment. Data represents mean ± S.D. of three independent readings normalized to β-gal activity. Shown is the fold change over control (cells transfected with control siRNA and treated with vehicle only). (inset) PSA levels were estimated by Western blotting. Loading control was demonstrated by the levels of tubulin. (B) AR transcriptional activity in cells transfected with vector only or myr-Akt, together with hPSA-luc and β-gal, and treated with 1 nM rapamycin and 10 μM bicalutamide for 48 hours. In all cases, data represents mean ± S.D. (n=3) normalized to β-gal activity. Shown is the fold change over vector controls treated with vehicle only. (C) Transfection of constitutively active Akt (myr-Akt) inhibited rapamycin + bicalutamide induced apoptosis. C4-2 cells were transfected with vector control (pCMV) or myr-Akt, and treated with vehicle (DMSO), 1 nM rapamycin, 10 μM bicalutamide, or both, for 48 hours. % cells undergoing apoptosis was measured by flow cytometry.

Article Snippet: Cells were plated in 60 mm dishes and transfected with 50 pmoles of one of the following: AR siRNA, mTOR siRNA, Akt1 siRNA, raptor siRNA (Santa Cruz Biotechnlogy, Santa Cruz, CA) and rictor siRNA, consisting of a pool of 4 siRNA duplexes (Dharmacon Research Inc., Lafayette, CO).

Techniques: Activity Assay, Transfection, Control, Luciferase, Western Blot, Plasmid Preparation, Flow Cytometry

Journal:

Article Title: Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival

doi: 10.1038/onc.2008.318

Figure Lengend Snippet: mTOR forms two complexes mTORC1 (with raptor) and mTORC2 (with rictor), both of which are inhibited by rapamycin in prostate cancer cells. Inhibition of mTORC2 inhibits AR transcriptional activity and cell survival in an Akt-dependent manner, as mTORC2 is a kinase for Akt phosphorylation. On the other hand, inhibition of mTORC1 induces apoptosis in an Akt- and AR-independent manner, but also stimulates Akt-phosphorylation and AR transcriptional activity. Stimulation of AR transcriptional activity and Akt phosphorylation by rapamycin treatment stimulated cell survival. Thus, the mTORC1 and mTORC2/Akt/AR pathways regulate cell survival in parallel in prostate cancer cells. This indicates that both pathways have to be simultaneously inhibited in order to induce apoptosis in these cells.

Article Snippet: Cells were plated in 60 mm dishes and transfected with 50 pmoles of one of the following: AR siRNA, mTOR siRNA, Akt1 siRNA, raptor siRNA (Santa Cruz Biotechnlogy, Santa Cruz, CA) and rictor siRNA, consisting of a pool of 4 siRNA duplexes (Dharmacon Research Inc., Lafayette, CO).

Techniques: Inhibition, Activity Assay, Phospho-proteomics