full length human sstr4 construct  (OriGene)

Journal: British Journal of Cancer

Article Title: Regulation of somatostatin receptor 4-mediated cytostatic effects by CD26 in malignant pleural mesothelioma

doi: 10.1038/bjc.2014.151

Figure Lengend Snippet: CD26 interacts with somatostatin receptor 4 (SSTR4) in MPM cells. ( A ) Immunoaffinity purification of CD26-containing proteins. Membrane fractions of MSTO-Mock, MSTO-CD26WT or MSTO-CD26/10Chi were extracted in a native condition. After being precleared with normal goat immunoglobulin (Ig) crosslinked Dynabeads, the native membrane extracts were immunoprecipitated with anti-CD26 pAb-crosslinked Dynabeads and eluted. The eluates were resolved by the Blue Native PAGE and sliver stained. The protein bands were retrieved and analysed by tandem mass spectrometry. The determined proteins were indicated in the right of the panel. Similar results were obtained in three independent experiments. ( B ) Immunoprecipitation assay using membrane extractions from MSTO-Mock, MSTO-CD26WT or MSTO-CD26/10Chi. After being precleared with normal goat Ig crosslinked Dynabeads, the membrane extracts were immunoprecipitated by the same method as shown in ( A ). The eluates were resolved by SDS–PAGE, and immunoblotted using anti-CD26 or anti-SSTR4 antibodies (upper two panels). Total lysates from each cell type were resolved by SDS–PAGE, and immunoblotted using anti-CD26 or anti-SSTR4 antibodies (lower two panels). SSTR4 is co-precipitated with CD26WT, but not with CD26/CD10 chimaeric protein (upper panel). Similar results were obtained in three independent experiments. ( C ) Immunoprecipitation assay using membrane extractions from parental MSTO-H211 (MSTO-P), JMN or H-MESO-1 (MESO1) cell lines. The membrane extracts were immunoprecipitated and immunoblotted by the same method as shown in ( B ). SSTR4 is co-precipitated with natively expressing CD26 molecules (upper panel). Similar results were obtained in three independent experiments. ( D ) Colocalisation of CD26 with SSTR4. JMN or MESO1 cells were fixed using 4% paraformaldehyde-PBS, and stained with Alexa Fluor 488-conjugated anti-CD26 pAb and Alexa Fluor 594-conjugated anti-SSTR4 pAb. Stained cells were mounted using Prolong Gold antifade reagent with DAPI. Observations were made on 10–15 cells in each of three different experiments using confocal laser microscopy. The micrographs are representative of >75% of the cells observed. CD26 and SSTR4 are observed as punctate coexpression on the cell surface. Original magnification, × 200. Scale bars indicate 25 μ m. ( E ) Association of CD26 with SSTR4 via their respective C-terminal region. After being precleared using normal mouse Ig and protein G resin, membrane extracts from HEK293 cells transiently expressing full-length CD26 (CD26WT), CD26-CD10 chimaeric receptor (CD26/10Chi), FLAG-tagged full-length SSTR4 (SSTR4WT-FLAG) or FLAG-tagged SSTR4 with deleted C-terminal cytoplasmic tail (SSTR4ΔC-FLAG) were immunoprecipitated with anti-FLAG affinity resin. Immunocomplexes were then immunoblotted using anti-CD26 or anti-FLAG antibodies (upper two panels). Total lysates from each cell were resolved by SDS–PAGE, and immunoblotted using anti-CD26 or anti-FLAG antibodies (lower two panels). Co-precipitation is observed in CD26WT and SSTR4WT, but not in CD26WT and SSTR4ΔC, CD26/10Chi and SSTR4WT, or CD26/10Chi and SSTR4ΔC (upper panel). Similar results were obtained in three independent experiments. The full colour version of this figure is available at British Journal of Cancer online.

Article Snippet: Full-length human SSTR4 construct in pCMV6-Entry vector (SSTR4WT-FLAG) was obtained from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Immunoaffinity Purification, Immunoprecipitation, Blue Native PAGE, Staining, Mass Spectrometry, SDS Page, Expressing, Microscopy

Journal: British Journal of Cancer

Article Title: Regulation of somatostatin receptor 4-mediated cytostatic effects by CD26 in malignant pleural mesothelioma

doi: 10.1038/bjc.2014.151

Figure Lengend Snippet: SSTR4-mediated cytostatic effects are inhibited in the presence of CD26. ( A ) MSTO-Mock, MSTO-CD26WT or MSTO-CD26/10Chi cells were transfected with two different siRNAs against SSTR4 (si-1 or si-2) or control siRNA (csi). After 48 h of transfection, cell migration (panel a), invasion (panel b) or colony formation assays (panel c) were conducted by the same methods as in (mean±s.e.m.; n =5 experiments with triplicates), with results being shown in bar graphs (MSTO-Mock (red bar), MSTO-CD26WT (blue bar) or MSTO-CD26/10Chi (green bar)). A significant increase in si-1 or si-2 in MSTO-Mock or MSTO-CD26/10Chi cells is indicated as P <0.0001 ( vs the corresponding csi). A significant increase in csi of MSTO-CD26WT cells (*) is indicated as P <0.0001 ( vs csi of MSTO-Mock or MSTO-CD26/10Chi). NS denotes ‘not significant'. P -values are calculated by ANOVA with Tukey–Kramer post hoc test. ( B ) Using MSTO-Mock, MSTO-CD26WT or MSTO-CD26/10Chi cells adapted to the serum-reduced condition, cell migration (panel a), invasion (panel b) or colony formation assays (panel c) were conducted by the same methods as in (mean±s.e.m.; n =5 experiments with triplicates). The SSTR4 agonist L803087 at the indicated concentrations or DMSO as a solvent control was added to the assays. P <0.0001 vs those in MSTO-Mock or MSTO-CD26/10Chi at respective concentrations of L803087 is calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. ( C , D ) Knockdown of endogenous CD26 induces inhibitory effects of SSTR4 agonist in JMN or MESO1 cells. After being adapted to the serum-reduced condition, endogenous CD26-expressing JMN ( C ) or MESO1 ( D ) cells were transfected with two different siRNAs against CD26 (si-1 or si-2), control siRNA (csi) or transfection reagent alone (none). After 48 h of transfection, cell migration (panels a), invasion (panels b) or colony formation assays (panels c) were conducted by the same methods as in (mean±s.e.m.; n =5 experiments with triplicates). The SSTR4 agonist L803087 at the indicated concentrations or DMSO as a solvent control was added to the assays. A significant decrease in si-1 or si-2 is indicated as P <0.0001 ( vs the corresponding csi or none), as calculated by ANOVA with Tukey–Kramer post hoc test. The full colour version of this figure is available at British Journal of Cancer online.

Article Snippet: Full-length human SSTR4 construct in pCMV6-Entry vector (SSTR4WT-FLAG) was obtained from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Transfection, Migration, Expressing

Journal: British Journal of Cancer

Article Title: Regulation of somatostatin receptor 4-mediated cytostatic effects by CD26 in malignant pleural mesothelioma

doi: 10.1038/bjc.2014.151

Figure Lengend Snippet: Downstream signalling with SHP-2 has a role in SSTR4-mediated cytostatic effects. ( A ) Endogenous CD26-deficient parental MSTO cells (MSTO-P) adapted to the serum-reduced condition were incubated with 1 μ M of SHP-1/2 inhibitor (black bars) or PBS as a solvent control (grey bars) for 30 min, followed by cell migration (panel a), invasion (panel b) or colony formation assays (panel c) in the presence of the SSTR4 agonist L803087 (10 μ M ) or DMSO as a solvent control (vehicle) (mean±s.e.m.; n =5 experiments with triplicates). A significant suppression in L803087 treatment without SHP-1/2 inhibitor ( P <0.0001, grey bars) was restored in the presence of SHP-1/2 inhibitor ( P <0.0001, grey and black bars of L803087), as calculated by ANOVA with Tukey–Kramer post hoc test. ( B ) MSTO-P cells adapted to the serum-reduced condition were transfected with two different siRNAs against SHP-2 (si-1 or si-2), control siRNA (csi) or transfection reagent alone (none). After 48 h of transfection, cell migration (panel a), invasion (panel b) or colony formation assays (panel c) were conducted by the same methods as in (mean±s.e.m.; n =5 experiments with triplicates). The SSTR4 agonist L803087 at the indicated concentrations or DMSO as a solvent control was added to the assays. A significant increase in si-1 or si-2 is indicated as P <0.0001 ( vs the corresponding csi or none), as calculated by ANOVA with Tukey–Kramer post hoc test.

Article Snippet: Full-length human SSTR4 construct in pCMV6-Entry vector (SSTR4WT-FLAG) was obtained from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Incubation, Migration, Transfection

Journal: British Journal of Cancer

Article Title: Regulation of somatostatin receptor 4-mediated cytostatic effects by CD26 in malignant pleural mesothelioma

doi: 10.1038/bjc.2014.151

Figure Lengend Snippet: Humanised anti-CD26 mAb ligation promotes SSTR4 clustering with CD26 in lipid rafts and enhances SSTR4-mediated cytostatic effects. ( A ) MSTO-CD26WT cells were stimulated for 10 min with humanised anti-CD26 mAb alone (5.0 μ g ml −1 ), or with control human Ig (5.0 μ g ml −1 ) in the presence of anti-Fc γ (5.0 μ g ml −1 ). Lipid raft or cytosolic fractions were prepared by sucrose gradient ultracentrifugation. The distribution of CD26, SSTR4, SHP-2 and transferrin receptor (TfR) was determined by immunoblotting with specific antibodies. TfR is a representative of non-lipid raft proteins, and used as a quantity control indicating equal amounts in the experiments. Fraction number (Fr#) 3–5 or 9–11 contains lipid raft or cytosolic fractions, respectively. CD26, SSTR4 and SHP-2 molecules are recruited into the lipid raft fractions after anti-CD26 mAb treatment (upper three in the right panels). Similar results were obtained in three independent experiments. ( B ) MSTO-CD26WT cells were stimulated, and sucrose gradient ultracentrifugation was conducted by the same methods as in ( A ). Lipid raft fractions were pooled by collection of corresponding Fr#3–5. After being precleared with normal rabbit IgG, immunoprecipitation of lipid rafts with anti-SSTR4 rabbit pAb was performed, followed by resolution with SDS–PAGE, and immunoblotted with the indicated antibodies. After stimulation by anti-CD26 mAb, SSTR4 molecules were increased in lipid rafts (lane 2 of the lower panel), while the association of SSTR4 with CD26 molecules was decreased (lane 2 of the upper panel). Similar results were obtained in three independent experiments. * corresponds to the protein bands of immunoglobulin heavy chain, and arrow head, those of SSTR4. ( C ) MSTO-CD26WT cells adapted to the serum-reduced condition were incubated with 1 μ M of cytochalasin D (Cytoch. D) or DMSO as a solvent control for 30 min, followed by cell migration (panel a), invasion (panel b) or colony formation assays (panel c) in the presence of the SSTR4 agonist L803087 (10 μ M) or DMSO as a solvent control (mean±s.e.m.; n =5 experiments with triplicates). A significant decrease in anti-CD26 mAb-treated cell in control reagents was observed ( P <0.0001, white bars), and a more profound decrease was observed in the presence of L803087 (*, P <0.0001). This reduction in the anti-CD26 mAb treatment group was restored in the presence of Cytoch. D ( P <0.0001). P -values are calculated by ANOVA with Tukey–Kramer post hoc test.

Article Snippet: Full-length human SSTR4 construct in pCMV6-Entry vector (SSTR4WT-FLAG) was obtained from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Ligation, Western Blot, Immunoprecipitation, SDS Page, Incubation, Migration

Journal: British Journal of Cancer

Article Title: Regulation of somatostatin receptor 4-mediated cytostatic effects by CD26 in malignant pleural mesothelioma

doi: 10.1038/bjc.2014.151

Figure Lengend Snippet: The inhibitory effect of combination of SSTR4 agonist and humanised anti-CD26 mAb on tumour growth in a murine xenograft model. ( A ) JMN cells (1 × 10 6 ) were inoculated s.c. into the left flank of mice. Mice were treated with i.p. injection of control IgG (10 μ g per dose) alone, SSTR4 agonist L803087 (20 μ M per dose) alone, L803087 (20 μ M per dose) plus control IgG (10 μ g per dose), humanised anti-CD26 mAb (10 μ g per dose) alone or L803087 (20 μ M per dose) plus humanised anti-CD26 mAb (10 μ g per dose) on the seventh day post cancer cell inoculation, when the tumour mass became visible (5 mm in size). Each antibody or agonist was given three times per week. Each cohort was examined with n =20. A significant decrease in the anti-CD26 mAb treatment cohort (blue line) was observed ( P <0.0001 vs control Ig, L803087 alone or L803087 plus control Ig cohort), and more profound decrease was observed in the L803087 plus anti-CD26 mAb treatment cohort (red line, P <0.0001 vs control Ig, L803087 alone, L803087 plus control Ig or anti-CD26 mAb alone cohort). P -values are calculated by ANOVA with Tukey–Kramer post hoc test. ( B ) Representative macroscopic photo of resected specimens in an s.c. tumorigenicity model on the 28th day post first treatment. Scale bar indicates 1 cm. ( C ) After 1 day of i.p. injection of luciferase-expressing JMN cells (1 × 10 5 ), mice were treated with i.p. injection of control IgG (10 μ g per dose), L803087 (20 μ M per dose) plus control IgG (10 μ g per dose), humanised anti-CD26 mAb (10 μ g per dose) alone or L803087 (20 μ M per dose) plus humanised anti-CD26 mAb (10 μ g per dose). Each antibody or agonist was given three times per week. Tumour growth was measured by in vivo bioluminescence photometry, with imaging data of each cohort being indicated as total flux of photons per second (mean±s.e.m.; n =20). A significant decrease in anti-CD26 mAb treatment cohort (blue line) was observed ( P <0.0001 vs control Ig, L803087 alone or L803087 plus control Ig cohort), and a more profound decrease was observed in the L803087 plus anti-CD26 mAb treatment cohort (red line, P <0.0001 vs control Ig, L803087 alone, L803087 plus control Ig or anti-CD26 mAb alone cohort). P -values are calculated by ANOVA with Tukey–Kramer post hoc test. Representative optical bioluminescence imaging of each cohort mice was shown with intensity of luminescence as heat maps in the right panels. The full colour version of this figure is available at British Journal of Cancer online.

Article Snippet: Full-length human SSTR4 construct in pCMV6-Entry vector (SSTR4WT-FLAG) was obtained from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Injection, Luciferase, Expressing, In Vivo, Imaging

Journal: British Journal of Cancer

Article Title: Regulation of somatostatin receptor 4-mediated cytostatic effects by CD26 in malignant pleural mesothelioma

doi: 10.1038/bjc.2014.151

Figure Lengend Snippet: Coexpression of CD26 and SSTR4 in surgical specimens of MPM patients. ( A ) Representative serial sections of resected specimens of epithelioid type of malignant mesothelioma. Panel a, Haematoxylin and eosin (H&E) stain, panel b, anti-CD26 and panel c, anti-SSTR4 immunohistochemistry. Original magnification, x100. ( B ) CD26 and SSTR4 expression profile among 50 patient tissues. Fifty consecutive surgically resected specimens from the primary sites were examined for membranous expression of SSTR4 and CD26 by the same method as in ( A ). CD26 was highly expressed on epithelioid or biphasic type of MPM (purple bars), and SSTR4 was detected on epithelioid or biphasic type of MPM (red bars), predominantly coexpressed with membranous CD26 (black bars). The full colour version of this figure is available at British Journal of Cancer online.

Article Snippet: Full-length human SSTR4 construct in pCMV6-Entry vector (SSTR4WT-FLAG) was obtained from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Staining, Immunohistochemistry, Expressing