Journal: Experimental and therapeutic medicine

Article Title: miR-130b suppresses the invasion and migration of prostate cancer via inhibiting DLL1 and regulating the PI3K/Akt pathways.

doi: 10.3892/etm.2021.11021

Figure Lengend Snippet: Figure 3. DLL1 expression is upregulated in prostate cancer. (A) Western blotting and (B) analysis of DLL1 expression. In prostate cancer cells, DLL1 expres‑ sion level was significantly increased compared with the control group. Data are presented as the mean ± standard deviation. **P<0.01, tumor group vs. control group. DLL, Delta‑like 1.

Article Snippet: The membrane was then incubated overnight at 4 ̊C with primary antibodies: DLL1 (cat. no. 2588; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p)‐PI3K (cat. no. 17366; 1:1,000; Cell Signaling Technology, Inc.), PI3K (cat. no. 4255; 1:1,000; Cell Signaling Technology, Inc.), p‐Akt (cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.), Akt (cat. no. 9272; 1:1,000; Cell Signaling Technology, Inc.), GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.) and MMP9 (cat. no. 3852; 1:1,000; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot, Control, Standard Deviation

Journal: Experimental and therapeutic medicine

Article Title: miR-130b suppresses the invasion and migration of prostate cancer via inhibiting DLL1 and regulating the PI3K/Akt pathways.

doi: 10.3892/etm.2021.11021

Figure Lengend Snippet: Figure 4. miR‑130b inhibits the protein level of DLL1. (A) Western blotting and (B) analysis of DLL1 expression. The protein level of DLL1 treated with miR‑130b mimic was significantly decreased compared with the non‑control and control groups. Data are presented as the mean ± standard deviation. **P<0.01, mimic group vs. NC group. miR, microRNA; DLL, Delta‑like 1; NC, negative control.

Article Snippet: The membrane was then incubated overnight at 4 ̊C with primary antibodies: DLL1 (cat. no. 2588; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p)‐PI3K (cat. no. 17366; 1:1,000; Cell Signaling Technology, Inc.), PI3K (cat. no. 4255; 1:1,000; Cell Signaling Technology, Inc.), p‐Akt (cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.), Akt (cat. no. 9272; 1:1,000; Cell Signaling Technology, Inc.), GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.) and MMP9 (cat. no. 3852; 1:1,000; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Expressing, Control, Standard Deviation, Negative Control

Journal: Experimental and therapeutic medicine

Article Title: miR-130b suppresses the invasion and migration of prostate cancer via inhibiting DLL1 and regulating the PI3K/Akt pathways.

doi: 10.3892/etm.2021.11021

Figure Lengend Snippet: Figure 5. miR‑130b may regulate DLL1 mRNA stability by directly targeting the 3'UTR in prostate cancer cells. (A) The possible binding site of miR‑130b and DLL1 was predicted by TargetScan 7.2. (B) The luciferase activity of the WT of DLL1 was significantly decreased after the transfection with the miR‑130b mimic, while there was no significant difference between the non‑control and miR‑130b mimic groups cloned with MUT DLL1. Data are presented as the mean ± standard deviation. **P<0.01. miR, microRNA; DLL, Delta‑like 1; UTR, untranslated region; MUT, mutant; WT, wild type; NC, negative control.

Article Snippet: The membrane was then incubated overnight at 4 ̊C with primary antibodies: DLL1 (cat. no. 2588; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p)‐PI3K (cat. no. 17366; 1:1,000; Cell Signaling Technology, Inc.), PI3K (cat. no. 4255; 1:1,000; Cell Signaling Technology, Inc.), p‐Akt (cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.), Akt (cat. no. 9272; 1:1,000; Cell Signaling Technology, Inc.), GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.) and MMP9 (cat. no. 3852; 1:1,000; Cell Signaling Technology, Inc.).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Clone Assay, Standard Deviation, Mutagenesis, Negative Control

Journal: Experimental and therapeutic medicine

Article Title: miR-130b suppresses the invasion and migration of prostate cancer via inhibiting DLL1 and regulating the PI3K/Akt pathways.

doi: 10.3892/etm.2021.11021

Figure Lengend Snippet: Figure 6. miR‑130b inhibits the mRNA level of the PI3K/Akt signaling pathway and MMP9 expression. The mRNA expression levels of (A) DLL1, (B) PI3K, (C) Akt and (D) MMP9 were significantly decreased after miR‑130b treatment, compared with those of the non‑control and control groups. Data are pre‑ sented as the mean ± standard deviation. **P<0.01, mimic group vs. NC group. p‑AKT, phosphorylated‑protein kinase B; MMP9, matrix metalloproteinase‑9; miR, microRNA; PI3K, phosphatidylinositol 3 kinase; DLL, Delta‑like 1.

Article Snippet: The membrane was then incubated overnight at 4 ̊C with primary antibodies: DLL1 (cat. no. 2588; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p)‐PI3K (cat. no. 17366; 1:1,000; Cell Signaling Technology, Inc.), PI3K (cat. no. 4255; 1:1,000; Cell Signaling Technology, Inc.), p‐Akt (cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.), Akt (cat. no. 9272; 1:1,000; Cell Signaling Technology, Inc.), GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.) and MMP9 (cat. no. 3852; 1:1,000; Cell Signaling Technology, Inc.).

Techniques: Expressing, Control, Standard Deviation

Journal: Experimental and therapeutic medicine

Article Title: miR-130b suppresses the invasion and migration of prostate cancer via inhibiting DLL1 and regulating the PI3K/Akt pathways.

doi: 10.3892/etm.2021.11021

Figure Lengend Snippet: Figure 7. miR‑130b downregulates the protein level of p‑PI3K, p‑AKT, MMP9 and DLL1. miR‑130b mimic or DLL1 siRNA significantly decreased the protein level of p‑PI3K, p‑AKT, MMP9 and DLL1. Data are presented as the mean ± standard deviation. *P<0.05, mimic group vs. NC group. p‑AKT, phosphory‑ lated‑protein kinase B; MMP9, matrix metalloproteinase‑9; miR, microRNA; PI3K, phosphatidylinositol 3 kinase; DLL, Delta‑like 1.

Article Snippet: The membrane was then incubated overnight at 4 ̊C with primary antibodies: DLL1 (cat. no. 2588; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p)‐PI3K (cat. no. 17366; 1:1,000; Cell Signaling Technology, Inc.), PI3K (cat. no. 4255; 1:1,000; Cell Signaling Technology, Inc.), p‐Akt (cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.), Akt (cat. no. 9272; 1:1,000; Cell Signaling Technology, Inc.), GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.) and MMP9 (cat. no. 3852; 1:1,000; Cell Signaling Technology, Inc.).

Techniques: Standard Deviation

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Effects of Delta1 overexpression on Notch activation and keratinocytes differentiation. (A) Schematic representation of the Delta constructs. (B) Anti DeltaD immunofluorescence staining of human keratinocytes transduced with DeltaFl. Scale bar: 20μm. (C) Hes1 luciferase reporter activity (upper panel) in J2-3T3 cells transduced with empty vector (ev), or Delta constructs. Fold induction relative to Renilla control is shown. Mean of 4 experiments ± SEM. (lower panels) Western blot of lysates of J2-3T3 cells used in the luciferase assay, probed with antibodies to DeltaD and Actin (loading control). (D) Clonal growth of keratinocytes transduced with empty vector or Delta constructs. (E) Clonal growth of wild type keratinocytes on a feeder layer of J2-3T3 cells transduced with empty vector (ev) or Delta constructs. (F-H) Epidermis reconstituted on DED by keratinocytes transduced with empty vector (ev,F,I), DeltaFl (G,J) or DeltaDS (H,K). Sections were stained with H&E (F-H) or antibody to Keratin 10 (green) with DAPI nuclear counterstain (blue) (I-K). Scale bar: 50μm.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Over Expression, Activation Assay, Construct, Immunofluorescence, Staining, Transduction, Luciferase, Activity Assay, Plasmid Preparation, Control, Western Blot

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Effect of Delta1 expression on clonal growth of keratinocytes Keratinocytes were transduced with the retroviral vectors indicated and cultured on a wild type feeder layer. WT: cells transduced with empty vector. Data are mean ± s.d. of triplicate dishes from at least 3 experiments. CFE: colony forming efficiency.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Expressing, Transduction, Retroviral, Cell Culture, Plasmid Preparation, Clone Assay

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Effect of Delta1 expressing feeders on clonal growth of wild type keratinocytes Wild type keratinocytes (K-WT) were cultured on feeder layer s of J2 3T3 cells transduced with the retroviral vectors shown. Data are mean ± s.d. of triplicate dishes in at least 3 experiments. CFE: colony forming efficiency.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Expressing, Cell Culture, Transduction, Retroviral, Clone Assay

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: The Delta1 PDZ domain binds syntenin and promotes keratinocyte cohesiveness. (A) GFP labelled clones formed by keratinocytes expressing the Delta constructs shown 5 days after seeding within a confluent sheet of unlabelled WT keratinocytes. Scale bar: 20 μm. (B) Percentage of cohesive colonies. Data are mean of 5 experiments ± SD. Values significantly different from WT control are marked with asterisks (student’s t-test: P<0.001*** or P<0.05**). (C) Western blots of A431 cells transfected with empty vector (ev) or the Delta constructs shown following pull down with GST (control; right hand panel) or GST-syntenin (left hand panel). Blots were probed with anti-ZfDelta or anti-GST. (D) Western blot of untransfected A431 cell lysates following pull down with GST or GST-syntenin (GST-syn). Blot was probed with antibody to human Delta1. Total cell lysates (Lys) served as positive control. (E) A431 cell lysates immunoprecipitated with antibodies against syntenin (α-syn) or laminin5 (α-Lam) followed by western blotting with anti Delta 1 antibody. Lys: lysate subjected to western blotting without prior immunoprecipitation. (F) Immunofluorescence staining of human primary keratinocytes with anti syntenin antibody (green). DAPI was used as a nuclear counterstain (blue). Scale bar: 10 μm. (G) Immunofluorescence staining of human interfollicular epidermis with an anti syntenin antibody. Horizontal lines mark position of putative stem cell clusters. Scale bar: 50 μm.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Clone Assay, Expressing, Construct, Control, Western Blot, Transfection, Plasmid Preparation, Positive Control, Immunoprecipitation, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Functional consequences of syntenin knockdown. (A) Western blot of keratinocytes transduced with empty vector (ev) or syntenin RNAi (Rnai1) probed with anti syntenin (α-syn) or, as a loading control, anti tubulin (α-tub). (B) Hes1 luciferase reporter activity in J2-3T3 cells transduced with empty vector (ev) or the Delta constructs shown, in the presence or absence of syntenin RNAi. Fold induction relative to Renilla control is shown. Mean of 3 experiments ± SEM. Values significantly different from ev control or Fl are marked with asterisks (student’s t-test: P<0.001*** or P<0.05**). (C) Clonal growth of keratinocytes transduced with DeltaFl or Delta DS and control (Ctrl) or syntenin Rnai1. (D-G) GFP labelled clones formed by human keratinocytes expressing DeltaFl alone (Fl) (D) or in combination with syntenin Rnai1(E), or formed by mouse keratinocytes with endogenous Delta1 levels (Dll1flox/flox)(F) or lacking Delta1 (K5Cre × Dll1flox/flox)(G). Clones were photographed 5 days after seeding within a confluent sheet of unlabelled WT keratinocytes (human or mouse). Scale bar: 100 μm. (H) Percentage of cohesive colonies formed by GFP labelled keratinocytes. Data are mean of 3 experiments ± SD (student’s t-test: P < 0.001***).

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Functional Assay, Knockdown, Western Blot, Transduction, Plasmid Preparation, Control, Luciferase, Activity Assay, Construct, Clone Assay, Expressing

Journal: Journal of cell science

Article Title: Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

doi: 10.1242/jcs.016253

Figure Lengend Snippet: Effects of syntenin knock down or Delta1 mutation on cell surface expression of Delta mutants. (A-F) Keratinocytes transduced with DeltaFl and pRetrosuper control vector (A-C) or syntenin Rnai (D-F) were double labelled with anti DeltaD (green A,D) and anti syntenin (red B,E). Right hand panels are merged images of middle and left hand panels (C,F). Scale bars: 20μm. (G-O) Primary human keratinocytes transduced with DeltaFl, DeltaDS or DeltaVA were incubated with an anti-Delta antibody at 4°C (t = 0), transferred to fresh medium at 37°C for the times indicated. Scale bar: 50μm. (P) Quantitation of % cells with Delta antibody at the plasma membrane. Data are means of 3 independent experiment ±SEM. 100 cells of each type were counted per experiment. (Q) Western blot of keratinocytes transduced with ev, DeltaFl, DeltaDS and DeltaVA constructs, probed with antibodies to Zf-Delta, syntenin and actin.

Article Snippet: Antibodies The following antibodies were used: Delta D, Zdd2 (kind gifts of J. Lewis), α6 integrin (GoH3; Serotec MCA699), keratin10 (Covance), transglutaminase1 (BC1; kind gift of R. Rice), loricrin (DH11; rabbit antiserum to NH2-HQTQQKQAPTWPCK-COOH peptide), Anti-laminin5 (Polykal; kind gift of P.Marinkovitch), Human Delta1 (biotinylated; R&D Systems; BAF1818) and syntenin (SYSY; 133002).

Techniques: Knockdown, Mutagenesis, Expressing, Transduction, Control, Plasmid Preparation, Incubation, Quantitation Assay, Clinical Proteomics, Membrane, Western Blot, Construct