Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Sequences for primers used for reverse transcription-quantitative polymerase chain reaction analysis.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Notch1 and cleaved-Notch1 expression in the human prostatic carcinoma cell lines LNCaP, PC-3 and DU 145 and the immortalized human prostatic epithelial RWPE-1 cell line. (A) Western blot analysis. Total cell lysates were collected from LNCaP, PC-3, DU 145 and RWPE-1 cells, and analyzed for Notch1 and cleaved-Notch1 proteins. Anti-GAPDH antibody was included as a loading control. (B) Quantitative analysis for the changes of Notch1 and cleaved-Notch1 proteins. Intensity of the targeted protein/modification was normalized to corresponding GAPDH. Data represent the average results from three independent experiments. *P<0.05 vs. RWPE-1 group.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Control, Modification

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Knockdown of Notch1 by shRNA in LNCaP cells. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis of Notch1 mRNA and protein expression levels following transfection. (C) Quantitative analysis of Notch1 and cleaved-Notch1 protein levels. Intensity of the targeted protein and modification was normalized to corresponding GAPDH. Data represent the average results from three independent experiments. *P<0.05 vs. control group. shRNA, short hairpin RNA.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Modification, Control

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Effect of Notch1-knockdown on invasion and proliferation of LNCaP cells. (A) Results of the Matrigel invasion assay and (B) quantitative analysis. *P<0.05 vs. control group. (C) Results of the cell proliferation assay, detected by WST-1 agent. Data represent the average results from three independent experiments. shRNA, short hairpin RNA.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, Invasion Assay, Control, Proliferation Assay, shRNA

Journal: Oncology Letters

Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

doi: 10.3892/ol.2017.6761

Figure Lengend Snippet: Effect of Notch1-knockdown on the expression of genes involved in cell invasion in LNCaP cells, assessed by reverse transcription-quantitative polymerase chain reaction. Data represent the average results from three independent experiments. *P<0.05 vs. control group. MTA1, metastasis-associated 1; KISS-1, KiSS-1 metastasis-suppressor, MKK4, mitogen-activated protein kinase 4; KAI1, cluster of differentiation 82; shRNA, short hairpin RNA.

Article Snippet: In a 6-well tissue culture plate, LNCaP cells at 50–70% confluency in 800 μl of short hairpin RNA (shRNA) plasmid transfection medium (cat. no. sc-108062) was transfected with 200 μl Notch1 shRNA plasmid (cat. no. sc-36095-SH) or control shRNA plasmid-A/shRNA plasmid transfection reagent complex (cat. no. sc-108060), all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, shRNA

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: (a) Cytotoxicity analysis of the lipopeptides_Notch1 complex (MR 50 : 1) at the 72 h time point in the MDA-MB-231 breast cancer cell line compared to the HiPerFect_siRNA complex. Error bars indicate SEM from three separate replicates. (b) Flow cytometry data of time-dependent (1 h) cellular uptake studies of FAM-labelled lipopeptides (1.25 μM) in MDA-MB-231 cells and (c) flow cytometry analysis of cellular uptake studies of labelled siRNA complexes at MR 50 after 1 h of incubation in MDA-MB-231 cells. Among the designed lipopeptides, lipopeptide AB18 demonstrates the highest siRNA internalization in MDA-MB-231 cells; error bars indicate SEM from two separate replicates (*** p < 0.0001, ** p < 0.01 and n.s. – not significant compared with untreated cells). (d) Intracellular distribution of the lipopeptide_siRNA complex (MR 50) in MDA-MB-231 cells after 6 h of incubation by ApoTome microscopy. Nuclei are stained with DAPI (blue), green represents the FAM-labelled lipopeptide, and red represents DY-547 labelled siRNA and also represents the released siRNA from lipopeptide_siRNA complexes. Yellow represents the co-localization of the FAM-labelled lipopeptide and DY-547-labelled siRNA. The best cytosolic distribution of siRNA was seen in the case of the lipopeptide AB18_siRNA complex. MDA-MB-231 cells incubated with HiPerFect_siRNA has less cytoplasmic distribution of siRNA and exhibits a less healthy morphology of MDA-MB-231 cells compared to the lipopeptide AB18_siRNA complex. (e) Intracellular distribution of the lipopeptide AB36_siRNA complex (MR 50) and HiPerFect_siRNA complex after 6 h of incubation in the hard to transfect primary cell line HUVEC by ApoTome microscopy. Please note that lipopeptide AB36 has a similar peptide sequence to lipopeptide AB18, but unlike lipopeptide AB18, it does not have FAM to enable microscopic studies without interference. Nuclei are stained with DAPI (blue), and red represents DY-547 labelled siRNA. (f) Flow cytometry analysis of cellular uptake studies of labelled siRNA complexes at MR 50 after 1 h of incubation in the hard to transfect primary cell line HUVEC. Error bars indicate SEM from two separate replicates. Both (e) and (f) indicate that labelled siRNA is internalized more and shows better cytoplasmic distribution in the case of the lipopeptide AB36_siRNA complex compared to the HiPerFect_siRNA complex in HUVEC.

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: Flow Cytometry, Incubation, Microscopy, Staining, Sequencing

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: (a) Gene knockdown efficiency of the Notch1 gene by the lipopeptide_siRNA complex as compared to the HiPerFect_siRNA complex at 72 h transfection as evidenced by RT-PCR data. Lipopeptide AB18_siRNA complex shows slightly higher transfection efficacy as compared to the HiPerFect_siRNA complex at 72 h. (b) Immunofluorescence of Notch1 protein after transfection with the lipopeptide AB36_siRNA complex for 72 h compared to untreated cells, considered as a vehicle. Scale bar = 20 μM. (c) Long term gene knockdown efficiency of the lipopeptide AB36_siRNA complex compared to the HiPerFect_siRNA complex on day 3, day 7 and day 15 after the first transfection. On day 7, siRNA transporter AB36 exhibited 1.6 times more gene knockdown than HiPerFect, indicating its potential for long term gene silencing. (d) Relative expression of the epithelial marker, E-cadherin in Notch1 silenced MDA-MB-231 by the lipopeptide AB17_siRNA complex and lipopeptide AB18_siRNA complex increased and (e) relative expression of the mesenchymal marker, N-cadherin in Notch1 silenced cells by the lipopeptide AB17_siRNA complex and lipopeptide AB18_siRNA complex decreased. (f) Relative expression of the MMP-2 gene (responsible for lung metastasis) in Notch1 silenced MDA-MB-231 cells by the lipopeptide AB17_siRNA complex and lipopeptide AB18_siRNA complex. In the lipopeptide AB17_siRNA complex and lipopeptide AB18_siRNA complex treated Notch1 silenced MDA-MB-231 cells, the relative expression of (g) the CD44 gene declined and subsequently the expression of (h) the CD24 gene was increased with respect to untreated MDA-MB-231 indicating possible inhibition of stemness in the treated samples. (i and j) SEM images revealing nano-scale bridges connecting metastatic breast cancer MDA-MB-231 (epithelial) cells to HUVEC (endothelial cells) in the 3D co-culture. (i) Untreated MDA-MB-231 and (j) MDA-MB-231 cells treated with the lipopeptide AB36_Notch1 siRNA nanocomplex were co-cultured with HUVEC. Lipopeptide AB36_Notch1 silencing siRNA treated cells did not exhibit any nanobridge formation indicating the inhibition of metastasis compared to untreated cells taken as controls, which exhibited nanobridge connecting HUVEC cells (yellow arrows). (a and c–h) Data are represented as mean ± SEM of n = 3 at each data point. (a) and (c) *** p < 0.0001 and n.s. – not significant compared with untreated cells. The experiments were repeated twice. Lipopeptide AB36 (having the same peptide sequence like lipopeptide AB18) was used in (b), (c), (i) and (j) to avoid interference induced by FAM.

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Expressing, Marker, Inhibition, Co-Culture Assay, Cell Culture, Sequencing

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: Knockdown percentage of oncogenes by different siRNA transporters

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: Knockdown, Incubation

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: (a) Cytotoxicity analysis of MDA-MB-231 cells with the combination of different doses of metformin close to its IC50 and fixed dose of lipopeptide AB36_Notch1 siRNA for the determination of the combination index (CI) at 72 h. Data are represented as mean ± SEM of n = 3 at each data point (*** p < 0.0001, * p < 0.05 and n.s. – not significant compared with untreated cells). (b) CI vs. effect (Fa) at a non-fixed ratio. All of the drug combinations of drug doses are represented by blue dots (combination doses). Blue dots below CI = 1 represent synergistic interactions. (c) Relative expression of the MMP-2 gene in Notch1 silenced MDA-MB-231 cells by combination of metformin (200 μM) and the lipopeptide AB36_siRNA complex (MR 50) compared to monotherapy [ i.e. , separately by 200 μM metformin and also by lipopeptide AB36_Notch1 siRNA (MR 50)]. (d) Relative expression of the CD44 gene in Notch1 silenced MDA-MB-231 cells by combination of metformin (200 μM) and the lipopeptide AB36_siRNA complex (MR 50) compared to monotherapy. (e) Relative expression of the CD24 gene in Notch1 silenced MDA-MB-231 cells by combination of metformin (200 μM) and the lipopeptide AB36_siRNA complex (MR 50) compared to monotherapy. Combination reduces markers of metastasis (MMP-2) and stemness (CD44 and CD24) compared to monotherapy. (c–e) Data are represented as mean ± SEM of n = 3 at each data point. The experiments were repeated twice.

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: Expressing

Journal: Chemical Science

Article Title: Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer †

doi: 10.1039/d3sc01071f

Figure Lengend Snippet: In vivo zebrafish xenograft model for the evaluation of cell proliferation and micro-metastasis. (a) Day 0 images of zebrafish injected with untreated MDA-MB-231 cells at the left section, Notch1 silenced MDA-MB-231 cells with the lipopeptide AB36_Notch1 siRNA complex for 72 h at the centre and MDA-MB-231 cells treated with combination of metformin (200 μM) and the lipopeptide AB36_Notch1 siRNA complex (MR 50) for 72 h at the right section. (b) Images of zebrafish on day 5 post injection. The middle and tail portions were imaged with higher exposure for TRITC (red) for clear visualization of micro-metastasis and are inserted as an inset above each condition. Notch1 silenced MDA-MB-231 cells treated with the lipopeptide AB36_Notch1 siRNA complex and MDA-MB-231 cells treated with a combination of lipopeptide AB36_Notch1 siRNA and metformin (200 μM) showed significant reduction in cell proliferation and micrometastasis in the in vivo zebrafish model compared to untreated MDA-MB-231 cells. (c) Quantification of cell proliferation of MDA-MB-231 in the in vivo zebrafish xenograft model by measuring the fluorescence intensity of CM-Dil labelled MDA-MB-231 cells on the fifth day compared to the day of injection. Data are represented as mean ± SEM of n = 10 at each data point. The experiment was repeated twice. Quantification of fluorescence intensity was carried out using ImageJ software and is represented as % mean fluorescence intensity, where the reading of day 0 was taken as the baseline. Image stitching was also achieved using ImageJ software. (d) Graphical representation of the immunogenicity study calculating the stimulation index of peripheral blood mononuclear cells (PBMCs) isolated from goat blood by MTT studies. Designed lipopeptide AB36 and HiPerFect are non-immunogenic till 72 h. Data are represented as mean ± SEM of n = 3 at each data point. Lipopeptide AB36 (having a similar peptide sequence to lipopeptide AB18) was used to avoid interference induced by FAM.

Article Snippet: Notch1 siRNA (Cat. No.: sc-36095) was acquired from Santacruz Biotechnology.

Techniques: In Vivo, Injection, Fluorescence, Software, Immunopeptidomics, Isolation, Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: GRWD1 affects the proliferation, apoptosis, invasion and migration of triple negative breast cancer through the Notch signaling pathway

doi: 10.3892/etm.2022.11400

Figure Lengend Snippet: Notch1 overexpression reverses the effect of GRWD1 knockdown on triple negative breast cancer cell proliferation and apoptosis. (A) The mRNA and (B) protein expression of Notch1 in MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was detected by reverse transcription-quantitative PCR and western blotting, respectively. *** P<0.001 vs. Control group. ### P<0.001 vs. Ov-NC group. (C) The proliferation of MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was determined by Cell Counting Kit-8 assay. (D) The protein expression of proliferation-related proteins in MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was detected by western blotting. (E) The proliferation of MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was also determined by Edu staining (magnification, x200). (F) The protein expression of apoptosis-related proteins in MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was detected by western blot analysis. * P<0.05 and *** P<0.001 vs. Control group. # P<0.05 and ### P<0.001 vs. siRNA-GRWD1 + Ov-NC group. GRWD1, glutamate-rich WD-repeat-containing protein 1; siRNA, small interfering RNA; NC, negative control; Ov, overexpression.

Article Snippet: Small interfering RNA (siRNA)-negative control (NC) (siB06525141922-1-5), siRNA-GRWD1 (siG000083743A-1-5), overexpression (Ov)-NC and Ov-Notch1 were synthesized and purchased from Guangzhou RiboBio Co., Ltd. A total of 50 nM siRNA-NC, siRNA-GRWD1, Ov-NC and Ov-Notch1 were transfected into MDA-MB-231 and HCC1937 cells when their confluence reached 80% using Lipofectamine ® 3000 kit (cat. no. L3000015; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol.

Techniques: Over Expression, Knockdown, Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, Cell Counting, Staining, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: GRWD1 affects the proliferation, apoptosis, invasion and migration of triple negative breast cancer through the Notch signaling pathway

doi: 10.3892/etm.2022.11400

Figure Lengend Snippet: Notch1 overexpression reverses the effect of GRWD1 knockdown on triple negative breast cancer cell invasion and migration. (A and B) The invasion and migration of MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was detected by Matrigel and wound healing assays. (C) The expression of metastasis-related proteins in MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was detected by western blotting. (D) The expression of epithelial-mesenchymal transition-related proteins in MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was detected by western blotting. *** P<0.001 vs. Control group. # P<0.05, ## P<0.01 and ### P<0.001 vs. siRNA-GRWD1+ Ov-NC group. GRWD1, glutamate-rich WD-repeat-containing protein 1; siRNA, small interfering RNA; NC, negative control; Ov, overexpression.

Article Snippet: Small interfering RNA (siRNA)-negative control (NC) (siB06525141922-1-5), siRNA-GRWD1 (siG000083743A-1-5), overexpression (Ov)-NC and Ov-Notch1 were synthesized and purchased from Guangzhou RiboBio Co., Ltd. A total of 50 nM siRNA-NC, siRNA-GRWD1, Ov-NC and Ov-Notch1 were transfected into MDA-MB-231 and HCC1937 cells when their confluence reached 80% using Lipofectamine ® 3000 kit (cat. no. L3000015; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol.

Techniques: Over Expression, Knockdown, Migration, Transfection, Expressing, Western Blot, Control, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: GRWD1 affects the proliferation, apoptosis, invasion and migration of triple negative breast cancer through the Notch signaling pathway

doi: 10.3892/etm.2022.11400

Figure Lengend Snippet: Notch1 overexpression reverses the effect of GRWD1 knockdown on the Notch signaling pathway in triple negative breast cancer cells. The expression of p21, c-Myc, CCND1, HER2 and NF-κB in MDA-MB-231 and HCC1937 cells co-transfected with siRNA-GRWD1 and Ov-Notch1 was detected by western blotting. ** P<0.01 and *** P<0.001 vs. Control group. # P<0.05, ## P<0.01 and ### P<0.001 vs. siRNA-GRWD1 + Ov-NC group. GRWD1, glutamate-rich WD-repeat-containing protein 1; CCND1, cyclin D1; HER2, human epidermal growth factor 2 receptor; siRNA, small interfering RNA; NC, negative control; Ov, overexpression.

Article Snippet: Small interfering RNA (siRNA)-negative control (NC) (siB06525141922-1-5), siRNA-GRWD1 (siG000083743A-1-5), overexpression (Ov)-NC and Ov-Notch1 were synthesized and purchased from Guangzhou RiboBio Co., Ltd. A total of 50 nM siRNA-NC, siRNA-GRWD1, Ov-NC and Ov-Notch1 were transfected into MDA-MB-231 and HCC1937 cells when their confluence reached 80% using Lipofectamine ® 3000 kit (cat. no. L3000015; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol.

Techniques: Over Expression, Knockdown, Expressing, Transfection, Western Blot, Control, Small Interfering RNA, Negative Control