Journal: Journal of Cellular and Molecular Medicine

Article Title: CD44 Participates to Extramedullary Haematopoiesis Onset by Mediating the Interplay Between Monocytes and Haematopoietic Stem Cells in Myelofibrosis

doi: 10.1111/jcmm.70720

Figure Lengend Snippet: CD44 is highly expressed in MF monocytes and HSPCs and its expression is increased in the spleen of MF mice. (a) Immunophenotypic evaluation of α4β1, αvβ3 and CD44 in circulating CD14 + monocytes in HDs ( n = 15) and MF patients ( n = 8). (b) Immunophenotypic evaluation of α4β1, αvβ3 and CD44 in circulating CD34 + cells in HDs ( n = 15) and MF patients ( n = 8). (c, d) Flow cytometry evaluation of CD44 , α4β1 and αvβ3 expression in macrophages (c) and LSK cells (d) in the spleen of NT ( n = 2) and TPO ‐ RA ( n = 4) treated mice. Each dot represents a mouse. (e, f) Flow cytometry evaluation of the frequency of CD44 + macrophages (e) and LSK cells (f) in the spleen of NT ( n = 2), TPO ‐ RA ( n = 4) and TPO ‐ RA + Ruxolitinib ( n = 5) treated mice. Each dot represents a mouse. In graphs, each dot represents a patient or a mouse, and each column represents group mean ± standard deviation. Comparisons were performed by means of unpaired T ‐test for panels (a–d) and one‐way ANOVA for panels (e, f). p ‐values are reported only when < 0.05.

Article Snippet: For in vitro extravasation assay the following antibodies and corresponding isotype controls were used: monoclonal anti‐CD44 antibody (clone IM7, cat. #14‐0441‐82), rat IgG2a kappa Isotype Control as control IgG for the monoclonal anti‐CD44 antibody (clone eBR2a, cat. #14‐4321‐82; both from eBioscience, Invitrogen, ThermoFisher, Waltham, Massachussets, USA); Polyclonal anti‐CD44 antibody (clone HCAM DF1485, cat. #sc‐7297), anti‐αvβ3 antibody (clone 23C6, cat. #sc‐7312), normal mouse IgG as control IgG for the polyclonal anti‐CD44 and anti‐αvβ3 antibodies (cat. #sc‐2025, all from Santa Cruz Biotechnology Inc., Dallas, Texas, USA); anti‐α4β1 antibody, InVivoMAb anti‐mouse/human VLA‐4 (CD49d) (clone PS/2, cat. #BE0071), InVivoMAb rat IgG2b isotype control as control IgG for the anti‐α4β1 antibody (clone LTF‐2, cat. #BE0090, all from BioXCell, Lebanon, New Hampshire, USA); anti‐human OPN Antibody (cat. AF1433, Biotechne, R&D systems, Minnesota, USA); Hyaluronan inhibitor (cat. #AS‐62622, Anaspec, California, USA).

Techniques: Expressing, Flow Cytometry, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD44 Participates to Extramedullary Haematopoiesis Onset by Mediating the Interplay Between Monocytes and Haematopoietic Stem Cells in Myelofibrosis

doi: 10.1111/jcmm.70720

Figure Lengend Snippet: CD44 regulates HD and MF CD14 + monocyte migration. Panel (a) shows the effect of anti‐αvβ3 antibody on migration of HD CD14 + monocyte in vitro in presence of HUVEC coating while panel (b) displays results for the anti‐α4β1 antibody treatment. Panel (c) shows the effect of the polyclonal anti‐CD44 antibody on migration of HD CD14 + monocyte in vitro in presence of HUVEC endothelium while panel (d) displays results for the monoclonal anti‐CD44 antibodies. (e) Graph displays the difference in migration between HD ( n = 6) and MF ( n = 3) CD14 + monocytes in the established in vitro extravasation model. (f) The effect of anti‐CD44 antibody on migration of MF CD14 + monocyte in vitro ( n = 3). (g) Bar plot represents results of HD CD14 + monocyte migration assays with and without HUVEC for the monoclonal anti‐CD44 antibody; Transwell samples with HUVEC are represented with purple bars while Transwell samples without HUVEC are illustrated with pink bars; the percentage of migration is normalised considering control IgG samples, not shown in this graph. In all bar plots the red dashed line represents 100% migration. On X axis are reported antibodies doses, Y axis reports the normalised cell count (see Section ). In graphs each dot represents a replicate, each column represents group mean ± standard deviation. Comparisons between samples treated with the specific antibody or control IgG were performed by means of paired T ‐test. p ‐values are reported only if < 0.05.

Article Snippet: For in vitro extravasation assay the following antibodies and corresponding isotype controls were used: monoclonal anti‐CD44 antibody (clone IM7, cat. #14‐0441‐82), rat IgG2a kappa Isotype Control as control IgG for the monoclonal anti‐CD44 antibody (clone eBR2a, cat. #14‐4321‐82; both from eBioscience, Invitrogen, ThermoFisher, Waltham, Massachussets, USA); Polyclonal anti‐CD44 antibody (clone HCAM DF1485, cat. #sc‐7297), anti‐αvβ3 antibody (clone 23C6, cat. #sc‐7312), normal mouse IgG as control IgG for the polyclonal anti‐CD44 and anti‐αvβ3 antibodies (cat. #sc‐2025, all from Santa Cruz Biotechnology Inc., Dallas, Texas, USA); anti‐α4β1 antibody, InVivoMAb anti‐mouse/human VLA‐4 (CD49d) (clone PS/2, cat. #BE0071), InVivoMAb rat IgG2b isotype control as control IgG for the anti‐α4β1 antibody (clone LTF‐2, cat. #BE0090, all from BioXCell, Lebanon, New Hampshire, USA); anti‐human OPN Antibody (cat. AF1433, Biotechne, R&D systems, Minnesota, USA); Hyaluronan inhibitor (cat. #AS‐62622, Anaspec, California, USA).

Techniques: Migration, In Vitro, Control, Cell Counting, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Figure 2. Apelin enhances integrin αvβ3-dependent prostate cancer motility. (A) Cells were treated with apelin for 24 hours, the indicated integrin expression was examined by qPCR (n=3). (B&C) Cells were treated with integrin αvβ3 or α5β1 antibody then with apelin, the wound healing and cell migration was examined (n=3). (D) Integrin αv and β3 gene levels in normal and prostate cancer patients retrieved from the GEO database. (E&F) Representative images showing the results of IHC staining for integrin αvβ3 in tissue samples from healthy individuals (n=3) and prostate cancer patients (n=3). (G&H) Cells were co-transfected with integrin αv and β3 siRNA then with apelin, the wound healing and cell migration was examined (n=3). * p < 0.05 compared with the control group. # p < 0.05 compared with the apelin-treated group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Migration, Immunohistochemistry, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Figure 3. MAPK pathway is regulated apelin-induced integrin expression and prostate cancer migration. (A&B) IPA pathway enrichment figure showing pathways that were changed in the GSE7930 dataset (Orange color indicated upregulated gene profile; blue color indicated downregulated gene profile). (C-H) Cells were treated with ERK (ERK II inhibitor; 10 μM), p38 (SB203580; 10 μM) and JNK (SP600125; 10 μM) inhibitors or transfected with ERK, p38 and JNK siRNA for 24 hours then with apelin, the wound healing, cell migration and integrin mRNA expression was examined (n=3). (I) PC3 cells were stimulated with apelin and ERK, p38 and JNK phosphorylation was examined by Western blotting (n=3). * p < 0.05 compared with the control group. # p < 0.05 compared with the apelin-treated group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Migration, Transfection, Phospho-proteomics, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Figure 4. Apelin promotes integrin production and cell motility via the STAT3 pathway. (A-F) Cells were treated with STAT3 inhibitor (10 μM) or transfected with STAT3 siRNA for 24 hours then with apelin, the wound healing, cell migration and integrin mRNA expression was examined (n=3). (G&H) PC3 cells were treated with apelin or pretreated with ERK, p38 and JNK inhibitor then with apelin, the STAT3 phosphorylation was examined by Western blotting (n=3). * p < 0.05 compared with the control group. # p < 0.05 compared with the apelin-treated group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Transfection, Migration, Expressing, Phospho-proteomics, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Figure 5. Apelin enhances integrin expression and promotes cell migration by inhibiting miR-8070 expression. (A) The diagrams depict the selection of miRNA candidates targeting integrin αv and β3. (B) PC3 cells were treated with apelin, the miRNAs expression was examined by qPCR (n=3). (C) Cells were treated with apelin for 24 hours, the miR-8070 expression was examined by qPCR (n=3). (D-F) Cells were transfected with miR-8070 mimic for 24 hours then with apelin, the wound healing, cell migration and integrin mRNA expression was examined (n=3). (G-I) Cells were treated with ERK, p38 and JNK inhibitor or siRNA then with apelin, the 3’UTR activity and miRNA expression was examined by luciferase activity and qPCR (n=3). * p < 0.05 compared with the control group. # p < 0.05 compared with the apelin-treated group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Migration, Selection, Transfection, Activity Assay, Luciferase, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Figure 6. Apelin blockade inhibits prostate cancer metastasis in vivo. (A-C) Cells were transfected with apelin siRNA, the integrin expression, wound healing and cell migration was examined (n=3). (D&E) IHC analysis of prostate cancer tissue samples stained with integrin αvβ3 antibody (n=3). (F) IHC analysis of leg bone (n=3), liver (n=3), and lung (n=3) sections stained with apelin and integrin αvβ3 antibodies. * p < 0.05 compared with the control group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: In Vivo, Transfection, Expressing, Migration, Staining, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Figure 7. Schematic diagram illustrating the mechanism underlying the effects of apelin in prostate cancer metastasis. Apelin stimulation enhances integrin αvβ3-dependent prostate cancer migration and metastasis. The activation of STAT3 and inhibition of miR-8070 via the ERK, p38 and JNK pathways mediate apelin-facilitated integrin synthesis and cell motility.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Migration, Activation Assay, Inhibition