Journal: British journal of cancer

Article Title: Inhibition of neurotensin receptor 1 induces intrinsic apoptosis via let-7a-3p/Bcl-w axis in glioblastoma.

doi: 10.1038/bjc.2017.126

Figure Lengend Snippet: Figure 1. SR48692 induced mitochondrial apoptosis in GBM cells. (A) Representative western blotting of NTSR1 expression in mouse GBM GL261 cells, human GBM U87, LN229, A172 and U251 cells, and a primary GBM tissue 0807T as well as a counterpart para-carcinoma tissue 0807P. GAPDH expression levels served as a loading control. (B) Cell viability assay in GL261, A172 and U251 cells after treatment of 5 or 10 mM SR48692 or DMSO at 0, 2, 4 and 6 days. (C) Image and quantification of Sub-G1 phrase analysis by flow cytometry in GL261, A172 and U251 GBM cells after treatment of 10 mM SR48692 or DMSO for 48 h. (D) The effect of Annexin V-FITC/PI dual staining apoptosis assays analysed by flow cytometry in GL261, A172 and U251 GBM cells after treatment of 10 mM SR48692 or DMSO for 48 h. (E) The effect of relative caspase 3 activities assays in GL261, A172 and U251 GBM cells after treatment of 10 mM SR48692 or DMSO for 48 h. In all, 20 mM Ac-DEVD-CHO, a caspase 3 inhibitor, was used to perform a negative control. (F) Image of cytochrome c release assay in GL261, A172 and U251 GBM cells after treatment of 10 mM SR48692 or DMSO for 48 h. Scale bars represent 200 mm. (G) Flow cytometric analysis of JC-1 staining in GL261, A172 and U251 GBM cells after treatment of 10 mM SR48692 or DMSO for 48 h. Scale bars represent 100 mm. All data were shown as mean±s.d. Student’s t-test was carried out. *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: To determine which factor of apoptosis pathway was responsible for the cell death of GBM cells after NTSR1 inhibition, we set up a RayBio Human Apoptosis Antibody Array (AAHAPO-G1-4) analysis of 43 proteins, which was tightly related to apoptosis pathway, in U251 GBM cells after NTSR1 knockdown in the absence or presence of actinomycin D. The heatmap showed that Bcl-w was downregulated after NTSR1 knockdown in the absence or presence of actinomycin D (Figure 4A).

Techniques: Western Blot, Expressing, Control, Viability Assay, Flow Cytometry, Staining, Negative Control, Release Assay

Journal: British journal of cancer

Article Title: Inhibition of neurotensin receptor 1 induces intrinsic apoptosis via let-7a-3p/Bcl-w axis in glioblastoma.

doi: 10.1038/bjc.2017.126

Figure Lengend Snippet: Figure 2. NTSR1 knockdown induced slight mitochondrial apoptosis in GBM cells. (A) The effect of Annexin V-FITC/PI dual staining apoptosis assays analysed by flow cytometry in A172 and U251 GBM cells after NTSR1 knockdown. (B) The effect of relative caspase 3 activities assays in A172 and U251 GBM cells after NTSR1 knockdown. In all, 20 mM Ac-DEVD-CHO, a caspase 3 inhibitor, was used to perform a negative control. (C) Flow cytometric analysis of JC-1 staining in A172 and U251 GBM cells after NTSR1 knockdown. All data were shown as mean±s.d. Student’s t-test was carried out. *Po0.05, **Po0.01, ***Po0.001, NS, no sense.

Article Snippet: To determine which factor of apoptosis pathway was responsible for the cell death of GBM cells after NTSR1 inhibition, we set up a RayBio Human Apoptosis Antibody Array (AAHAPO-G1-4) analysis of 43 proteins, which was tightly related to apoptosis pathway, in U251 GBM cells after NTSR1 knockdown in the absence or presence of actinomycin D. The heatmap showed that Bcl-w was downregulated after NTSR1 knockdown in the absence or presence of actinomycin D (Figure 4A).

Techniques: Knockdown, Staining, Flow Cytometry, Negative Control

Journal: British journal of cancer

Article Title: Inhibition of neurotensin receptor 1 induces intrinsic apoptosis via let-7a-3p/Bcl-w axis in glioblastoma.

doi: 10.1038/bjc.2017.126

Figure Lengend Snippet: Figure 3. NTSR1 knockdown sensitised GBM cells to drug-induced apoptosis. (A) The effect of Annexin V-FITC/PI dual staining apoptosis assays analysed by flow cytometry in GL261, A172 and U251 GBM cells after treatment of 10 mM SR48692 or DMSO for 48 h in the presence or absence of 5 nM actinomycin D in cultural medium. (B) The effect of relative caspase 3 activities assays in GL261, A172 and U251 GBM cells after treatment of 10 mM SR48692 or DMSO for 48 h in the presence or absence of 5 nM actinomycin D in cultural medium. (C) The effect of Annexin V-FITC/PI dual staining apoptosis assays analysed by flow cytometry in A172 and U251 GBM cells after NTSR1 knockdown in the presence of 5 nM actinomycin D in cultural medium for 24 h. (D) The effect of relative caspase 3 activities assays in A172 and U251 GBM cells after NTSR1 knockdown in the presence of 5 nM actinomycin D in cultural medium for 24 h. In all, 20 mM Ac-DEVD-CHO, a caspase 3 inhibitor, was used to perform a negative control. All data were shown as mean±s.d. Student’s t-test was carried out. *Po0.05, **Po0.01, ***Po0.001, NS, no sense.

Article Snippet: To determine which factor of apoptosis pathway was responsible for the cell death of GBM cells after NTSR1 inhibition, we set up a RayBio Human Apoptosis Antibody Array (AAHAPO-G1-4) analysis of 43 proteins, which was tightly related to apoptosis pathway, in U251 GBM cells after NTSR1 knockdown in the absence or presence of actinomycin D. The heatmap showed that Bcl-w was downregulated after NTSR1 knockdown in the absence or presence of actinomycin D (Figure 4A).

Techniques: Knockdown, Staining, Flow Cytometry, Negative Control

Journal: British journal of cancer

Article Title: Inhibition of neurotensin receptor 1 induces intrinsic apoptosis via let-7a-3p/Bcl-w axis in glioblastoma.

doi: 10.1038/bjc.2017.126

Figure Lengend Snippet: Figure 4. NTSR1 inhibition induced downregulation of Bcl-w and Bcl-2 in GBM cells. (A) Heatmap of apoptosis-related protein array in U251 cells after NTSR1 knockdown in the absence or presence of 5 nM actinomycin D in cultural medium for 24 h. (B) Representative western blotting of Bcl-w, Bcl-2, caspase 3 and cleaved-caspase 3 expression in GL261, A172 and U251 cells after 10 mm SR48692 treatment in the absence or presence of 5 nM actinomycin D in cultural medium for 24 h. (C) Representative western blot of NTSR1, Bcl-w, Bcl-2, caspase 3 and cleaved-caspase 3 expression in A172 and U251 cells after NTSR1 knockdown. (D) Representative western blotting of NTSR1, Bcl-w, Bcl-2, caspase 3 and cleaved- caspase 3 expression in A172 and U251 cells after NTSR1 knockdown in the absence or presence of 5 nM actinomycin D in cultural medium for 24 h. (E) Representative western blotting of Bcl-w, Bcl-2, caspase 3 and cleaved-caspase 3 expression in Bcl-w or vector transient-transfected A172 and U251 cells after 10 mm SR48692 treatment in cultural medium for 24 h. (F) Representative western blot of Bcl-w, Bcl-2, caspase 3 and cleaved- caspase 3 expression in scramble or NTSR1-silenced A172 and U251 cells after transient transfected with Bcl-w for 48 h in the presence of 5 nM actinomycin D. GAPDH expression levels served as a loading control.

Article Snippet: To determine which factor of apoptosis pathway was responsible for the cell death of GBM cells after NTSR1 inhibition, we set up a RayBio Human Apoptosis Antibody Array (AAHAPO-G1-4) analysis of 43 proteins, which was tightly related to apoptosis pathway, in U251 GBM cells after NTSR1 knockdown in the absence or presence of actinomycin D. The heatmap showed that Bcl-w was downregulated after NTSR1 knockdown in the absence or presence of actinomycin D (Figure 4A).

Techniques: Inhibition, Protein Array, Knockdown, Western Blot, Expressing, Plasmid Preparation, Transfection, Control

Journal: British journal of cancer

Article Title: Inhibition of neurotensin receptor 1 induces intrinsic apoptosis via let-7a-3p/Bcl-w axis in glioblastoma.

doi: 10.1038/bjc.2017.126

Figure Lengend Snippet: Figure 5. NTSR1 inhibition-induced apoptosis was rescued by let-7a-3p suppression in GBM cells. (A) Relative expression of let-7a-3p in A172 and U251cells after NTSR1 knockdown in the absence or presence of actinomycin D in cultural medium for 24 h. (B) Representative western blotting of NTSR1 in A172 and U251cells after let-7a-3p inhibitor treatment. GAPDH expression levels served as a loading control. (C) Schematic diagram of the binding of let-7a-3p to the 30UTR region of Bcl-w mRNA. (D and E) Relative luciferase activity (FFL/RL) was assessed by using luciferase assay in A172 and U251 cells after negative control or let-7a-3p inhibitor and wild or mutant pGL3-Bcl-w 30UTR transfection. (F) The effect of Annexin V-FITC/PI dual staining apoptosis assays analysed by flow cytometry in DMSO or 10 mm SR48692-treated A172 and U251 cells after negative control or let-7a-3p inhibitor transfection. (G) The effect of Annexin V-FITC/PI dual staining apoptosis assays analysed by flow cytometry in NTSR1-silenced A172 and U251 cells after negative control or let-7a-3p inhibitor transfection. (H) The effect of Annexin V-FITC/PI dual staining apoptosis assays analysed by flow cytometry in DMSO or 10 mm SR48692-treated A172 and U251 cells after negative control or let- 7a-3p inhibitor transfection in the presence of 5 nM actinomycin D in cultural medium for 24 h. (I) The effect of Annexin V-FITC/PI dual staining apoptosis assays analysed by flow cytometry in NTSR1-silenced A172 and U251 cells after negative control or let-7a-3p inhibitor transfection in the presence of 5 nM actinomycin D in cultural medium for 24 h. (J) Representative western blotting of NTSR1, Bcl-w and Bcl-2 in NTSR1-silenced A172 and U251 cells after negative control or let-7a-3p inhibitor transfection. (K) Relative expression of c-Myc and LIN28 in U251 and A172 cells after NTSR1 knockdown. (L) Representative western blotting of c-Myc and LIN28 in NTSR1-silenced A172 and U251 cells. GAPDH expression levels served as a loading control. All data were shown as mean±s.d. Student’s t-test was carried out. *Po0.05, **Po0.01, ***Po0.001, NS, no sense.

Article Snippet: To determine which factor of apoptosis pathway was responsible for the cell death of GBM cells after NTSR1 inhibition, we set up a RayBio Human Apoptosis Antibody Array (AAHAPO-G1-4) analysis of 43 proteins, which was tightly related to apoptosis pathway, in U251 GBM cells after NTSR1 knockdown in the absence or presence of actinomycin D. The heatmap showed that Bcl-w was downregulated after NTSR1 knockdown in the absence or presence of actinomycin D (Figure 4A).

Techniques: Inhibition, Expressing, Knockdown, Western Blot, Control, Binding Assay, Luciferase, Activity Assay, Negative Control, Mutagenesis, Transfection, Staining, Flow Cytometry