Journal: Scientific Reports

Article Title: PTH/SDF-1α cotherapy induces CD90+CD34− stromal cells migration and promotes tissue regeneration in a rat periodontal defect model

doi: 10.1038/srep30403

Figure Lengend Snippet: ( A ) H&E staining of the periodontal defects at 1, 2, 4 and 8 weeks post-surgery. The visual fields framed by the black line were magnified in the images below. The black arrows represented the border of the defect. B indicated new bone. D displayed the dentine of the native root. F represented newly formed fibers. ( B ) Quantitative analysis of newly formed bone areas in the four groups at four time points. * P < 0.05 compared with control group; ▲ P < 0.05 PTH group compared with SDF-1α group; § P < 0.05 PTH + SDF-1α group compared with other experimental groups.

Article Snippet: Left-side defects were implanted with collagen membranes loaded with SDF-1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), while the right-side defects were implanted with collagen membranes loaded with PBS.

Techniques: Staining

Journal: Scientific Reports

Article Title: PTH/SDF-1α cotherapy induces CD90+CD34− stromal cells migration and promotes tissue regeneration in a rat periodontal defect model

doi: 10.1038/srep30403

Figure Lengend Snippet: ( A ) Immunofluorescence staining of CXCR4+ cells in four groups at four time points. ( B ) Quantitative analysis of the number of CXCR4+ cells showed that PTH + SDF-1α promoted the recruitment of CXCR4+ cells than that in the other three groups at the early stage of healing process (day 3, week 1 and week 2). The number of CXCR4+ cells in SDF-1α group was larger than that in PTH group at day 3 and week 1, and there was no difference at week 2. At week 4, CXCR4+ cells were hardly detected. * P < 0.05 compared with control group; ▲ P < 0.05 PTH group compared with SDF-1α group; § P < 0.05 PTH + SDF-1α group compared with other experimental groups.

Article Snippet: Left-side defects were implanted with collagen membranes loaded with SDF-1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), while the right-side defects were implanted with collagen membranes loaded with PBS.

Techniques: Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: PTH/SDF-1α cotherapy induces CD90+CD34− stromal cells migration and promotes tissue regeneration in a rat periodontal defect model

doi: 10.1038/srep30403

Figure Lengend Snippet: ( A ) Immunofluorescence double staining of the four groups at four time points. ( B ) Quantitative analysis of the number of CD90+CD34− stromal cells showed that SDF-1α combined with PTH increased the recruitment of CD90+CD34− stromal cells than the other three groups at day 3, week 1 and 2. The number of CD90+CD34− stromal cells peaked at week 1 and reduced at week 2. At week 4, MSCs were hardly detected. White arrow indicated CD90+CD34− stromal cells. * P < 0.05 compared with control group; ▲ P < 0.05 PTH group compared with SDF-1α group; § P < 0.05 PTH + SDF-1α group compared with other experimental groups.

Article Snippet: Left-side defects were implanted with collagen membranes loaded with SDF-1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), while the right-side defects were implanted with collagen membranes loaded with PBS.

Techniques: Immunofluorescence, Double Staining

Journal: Scientific Reports

Article Title: PTH/SDF-1α cotherapy induces CD90+CD34− stromal cells migration and promotes tissue regeneration in a rat periodontal defect model

doi: 10.1038/srep30403

Figure Lengend Snippet: ( A ) TRAP (red) staining at day 3, week 1, 2 and 4. ( B ) TRAP+ cells in four groups. * P < 0.05 compared with control group; ▲ P < 0.05 PTH group compared with SDF-1α group; § P < 0.05 PTH + SDF-1α compared with other experimental groups.

Article Snippet: Left-side defects were implanted with collagen membranes loaded with SDF-1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), while the right-side defects were implanted with collagen membranes loaded with PBS.

Techniques: Staining

Journal: Scientific Reports

Article Title: PTH/SDF-1α cotherapy induces CD90+CD34− stromal cells migration and promotes tissue regeneration in a rat periodontal defect model

doi: 10.1038/srep30403

Figure Lengend Snippet: ( A,B ) Immunohistochemical staining of Runx2 (brown) and ALP (brown) at week 1, 2 and 4. ( C ) Immunohistochemical staining of Col I (brown) at week 2, 4 and 8. ( D–F ) Quantitative analyses of Runx2+ cells, ALP expression and Col I expression in four groups. * P < 0.05 compared with control group; ▲ P < 0.05 PTH group compared with SDF-1α group; § P < 0.05 PTH + SDF-1α compared with other experimental groups.

Article Snippet: Left-side defects were implanted with collagen membranes loaded with SDF-1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), while the right-side defects were implanted with collagen membranes loaded with PBS.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cancers

Article Title: Targeting HIF-1α Regulatory Pathways as a Strategy to Hamper Tumor-Microenvironment Interactions in CLL

doi: 10.3390/cancers13122883

Figure Lengend Snippet: The CXCL12/CXCR4 axis plays a central role in the SC-mediated triggering of HIF-1α regulatory pathways. Primary CLL cells were cultured for 48 h in presence of M2-10B4 SC or CXCL12. In selected conditions, the CXCR4 antagonist AMD3100 was added. Both SC and CXCL12 induced an increase in the amount of GTP-bound RAS (RAS-GTP) and of the active phosphorylated form of ERK1-2 (pERK1-2) ( A ), and in the phosphorylation and activity of AKT ( B , C ). Accordingly, CLL cells cultured with SC or CXCL12 displayed an increase in the cytosolic and nuclear amount of HIF-1α ( D ), and in HIF-1α activity ( E ). The addition of the CXCR4 antagonist AMD3100 abrogated the inducing effects mediated both by SC and CXCL12 at all levels. In ( A , B , D ) a representative blot (with relative Unique Patient Number, UPN), together with the corresponding cumulative band intensity data of 6 independent experiments, respectively, is shown. Box and whiskers plots show median values, 25–75% percentiles, and minimum and maximum values for each group. In ( C , E ) bar graphs represent mean results and SEM ( n = 6). Repositioned gel lanes are indicated by vertical lines. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05. Please find the whole western blot in the .

Article Snippet: CLL cells (10 cells) were cultured in the presence or absence of M2-10B4 SC (5 × 10 5 cells) and exposed to CXCL12 100 ng/mL (Human SDF-1α/CXCL12; Miltenyi Biotec, Bologna, Italy), CXCR4 antagonist AMD3100 5 µg/mL (Genzyme, Europe B.V., Naarden, The Netherlands), simvastatin 1 μM (Sigma Aldrich, Milan, Italy), ERK1-2 kinase inhibitor PD98059 1 μM (Sigma Aldrich, Milan, Italy), PI3K inhibitor idelalisib 10 μM, unless otherwise specified (Selleckchem, Munich, Germany), HIF-1α inhibitor BAY87-2243 1 μM (Selleckchem, Munich, Germany) and fludarabine 10 µM (arabinosyl-2-fluoroadenine, dephosphorylated nucleoside form of fludarabine; Sigma Aldrich, Milan, Italy) for 30 min, 6 h or 48 h. The medium of M2-10B4 SC cultured for 2 days was collected and used in CLL cells-SC 30 min co-culture.

Techniques: Cell Culture, Activity Assay, Western Blot

Journal: Cancers

Article Title: Targeting HIF-1α Regulatory Pathways as a Strategy to Hamper Tumor-Microenvironment Interactions in CLL

doi: 10.3390/cancers13122883

Figure Lengend Snippet: Targeting the RAS/ERK1-2 and PI3K/AKT signalling inhibits SC- and CXCL12-induced HIF-1α upregulation. A schematic representation of RAS/ERK1-2 and PI3K/AKT HIF-1α regulatory pathways, and the target protein of each inhibitor used in the following experimental setting (i.e., simvastatin, Sim; PD98059, PD; idelalisib, Ide; BAY87-2243, BAY) is depicted in ( A ). CLL cells cultured for 48 h in the absence ( B ) and in presence of M2-10B4 SC ( C ) or CXCL12 ( D ) were exposed to simvastatin, PD98059, idelalisib or BAY87-2243 and evaluated for HIF-1α expression and activity. Treatment with targeted inhibitors reduced the cytosolic and nuclear amount of HIF-1α and downregulated its activity, both in the absence and in the presence of SC or CXCL12. In ( B – D ) a representative blot with UPN and cumulative band intensity data obtained from the analysis of 9, 9 and 5 independent experiments, respectively, is shown. Repositioned gel lanes are indicated by vertical lines. Box and whiskers plots show median values, 25–75% percentiles, and minimum and maximum values for each group; each point represents a single sample. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05. Please find the whole western blot in the .

Article Snippet: CLL cells (10 cells) were cultured in the presence or absence of M2-10B4 SC (5 × 10 5 cells) and exposed to CXCL12 100 ng/mL (Human SDF-1α/CXCL12; Miltenyi Biotec, Bologna, Italy), CXCR4 antagonist AMD3100 5 µg/mL (Genzyme, Europe B.V., Naarden, The Netherlands), simvastatin 1 μM (Sigma Aldrich, Milan, Italy), ERK1-2 kinase inhibitor PD98059 1 μM (Sigma Aldrich, Milan, Italy), PI3K inhibitor idelalisib 10 μM, unless otherwise specified (Selleckchem, Munich, Germany), HIF-1α inhibitor BAY87-2243 1 μM (Selleckchem, Munich, Germany) and fludarabine 10 µM (arabinosyl-2-fluoroadenine, dephosphorylated nucleoside form of fludarabine; Sigma Aldrich, Milan, Italy) for 30 min, 6 h or 48 h. The medium of M2-10B4 SC cultured for 2 days was collected and used in CLL cells-SC 30 min co-culture.

Techniques: Cell Culture, Expressing, Activity Assay, Western Blot

Journal: Cancers

Article Title: Targeting HIF-1α Regulatory Pathways as a Strategy to Hamper Tumor-Microenvironment Interactions in CLL

doi: 10.3390/cancers13122883

Figure Lengend Snippet: The targeting of PI3K/AKT signalling pathway downmodulates HIF-1α and its target genes expression in SC. M2-10B4 SC and patient-derived BMSC were cultured in absence or in presence of idelalisib (Ide) or BAY87-2243 (BAY) for 48 h. Idelalisib effectively inhibits pAKT expression in SC ( A ). Both drugs induced a decrease in cytosolic and nuclear HIF-1α expression ( B ), and in HIF-1α activity ( C ) after 48 h culture. CXCL12 , CA9 , ENOA , GLUT1 and VEGF mRNA were also quantified, showing that idelalisib and BAY87-2243 significantly reduced HIF-1α target genes’ expression in SC ( D ). In ( A ), a representative blot (with relative UPN, when applicable) with cumulative band intensity data obtained from the analysis of 4 replicates for M2-10B4 SC and 7 patient-derived BMSC independent experiments is shown. In ( B ), a representative blot (with UPN, when applicable) with cumulative band intensity data obtained from the analysis of 6 replicates for M2-10B4 SC and 11 patient-derived BMSC independent experiments is shown. In ( A – C ) box and whiskers plots show median values, 25–75% percentiles and minimum and maximum values for each group. In ( C ) each point represents one experiment with M2-10B4 SC ( n = 5) or one patient-derived SC sample ( n = 6). In ( D ), bar graphs represent mean results obtained from the analysis of 4 replicates for M2-10B4 SC and 7 patient-derived BMSC independent experiments, together with SEM. Repositioned gel lanes are indicated by vertical lines. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05. Please find the whole western blot in the .

Article Snippet: CLL cells (10 cells) were cultured in the presence or absence of M2-10B4 SC (5 × 10 5 cells) and exposed to CXCL12 100 ng/mL (Human SDF-1α/CXCL12; Miltenyi Biotec, Bologna, Italy), CXCR4 antagonist AMD3100 5 µg/mL (Genzyme, Europe B.V., Naarden, The Netherlands), simvastatin 1 μM (Sigma Aldrich, Milan, Italy), ERK1-2 kinase inhibitor PD98059 1 μM (Sigma Aldrich, Milan, Italy), PI3K inhibitor idelalisib 10 μM, unless otherwise specified (Selleckchem, Munich, Germany), HIF-1α inhibitor BAY87-2243 1 μM (Selleckchem, Munich, Germany) and fludarabine 10 µM (arabinosyl-2-fluoroadenine, dephosphorylated nucleoside form of fludarabine; Sigma Aldrich, Milan, Italy) for 30 min, 6 h or 48 h. The medium of M2-10B4 SC cultured for 2 days was collected and used in CLL cells-SC 30 min co-culture.

Techniques: Expressing, Derivative Assay, Cell Culture, Activity Assay, Western Blot

Journal: Cancers

Article Title: Targeting HIF-1α Regulatory Pathways as a Strategy to Hamper Tumor-Microenvironment Interactions in CLL

doi: 10.3390/cancers13122883

Figure Lengend Snippet: Idelalisib treatment downregulates HIF-1α in CLL cells, reduces serum CXCL12 and modifies the BM microenvironment. Leukemic cells isolated from CLL patients treated for one month with idelalisib were evaluated for HIF-1α expression by WB and for the expression of the HIF-1α target genes CXCR4, CA9, ENOA, GLUT1 and VEGF by RT-PCR. Compared to the baseline, a reduced cytosolic and nuclear expression of HIF-1α ( A ), and a decreased expression of the analyzed target genes ( B ) were detected. In patients’ sera, CXCL12 concentration decreased after 6 months of treatment with idelalisib ( C ). Immunohistochemical analyses performed on BM sections collected from CLL patients during idelalisib treatment showed, compared to the baseline, a reduced vessel density, highlighted by anti-CD34 immunostaining, and an enrichment in the CD68+ monocytes/macrophages characterized by a round shape morphology and no cellular extensions ( D ). In ( A ), blots from the analysis of 5 independent experiments (with UPN) and cumulative band intensity data obtained are shown. In ( B ), bar graphs represent mean results obtained from 6 experiments together with SEM. In ( C ), a line graph represents individual data values for the same sample in each timepoint. In ( D ), immunohistochemistry of a representative experiment out of 3 is shown. ** p < 0.01 and * p < 0.05. Please find the whole western blot in the .

Article Snippet: CLL cells (10 cells) were cultured in the presence or absence of M2-10B4 SC (5 × 10 5 cells) and exposed to CXCL12 100 ng/mL (Human SDF-1α/CXCL12; Miltenyi Biotec, Bologna, Italy), CXCR4 antagonist AMD3100 5 µg/mL (Genzyme, Europe B.V., Naarden, The Netherlands), simvastatin 1 μM (Sigma Aldrich, Milan, Italy), ERK1-2 kinase inhibitor PD98059 1 μM (Sigma Aldrich, Milan, Italy), PI3K inhibitor idelalisib 10 μM, unless otherwise specified (Selleckchem, Munich, Germany), HIF-1α inhibitor BAY87-2243 1 μM (Selleckchem, Munich, Germany) and fludarabine 10 µM (arabinosyl-2-fluoroadenine, dephosphorylated nucleoside form of fludarabine; Sigma Aldrich, Milan, Italy) for 30 min, 6 h or 48 h. The medium of M2-10B4 SC cultured for 2 days was collected and used in CLL cells-SC 30 min co-culture.

Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Immunohistochemical staining, Immunostaining, Immunohistochemistry, Western Blot