Journal: International Journal of Molecular Sciences

Article Title: Influence of the Cholinergic System on the Pathogenesis of Glioblastoma: Impact of the Neutrophil Granulocytes

doi: 10.3390/ijms27010321

Figure Lengend Snippet: Influence of the cholinergic system on IL-8 production by U251 tumor cells. ( A ) IL-8 levels in culture supernatants measured by ELISA after culture. Ten experiments were performed in the 2D model (** p < 0.005, mean ± SD). ( B ) Correlation of CHRM1 (upper panels) and CHRM3 (lower panels) and of ( C ) ChAT (upper panels) and AChE (lower panels) with IL-8 expression in LGG (n = 516) and GBM (n = 153) samples, estimated by TIMER2.0 ( http://timer.cistrome.org/ ). ( D – G ) PD-L1 expression in U251 tumor cells. ( D ) PD-L1 expression in U251 cells in 2D model (monolayer grown for 24 h with cholinergic system agonists ACh (10 −8 –10 −9 M) or Carb (10 −8 –10 −9 M) and ( F ) 3D model (spheroids grown for 9 days in 1.5% agar, 5 × 10 4 cells/mL, treated with ACh (10 −8 –10 −9 M) or Carb (10 −8 –10 −9 M). * p < 0.05. ( E ) Representative experiment on 2D cultures and ( G ) 3D cultures. ( H ) Correlation of CHRM1 (upper panels) and CHRM3 (lower panels), and of ( I ) ChAT (upper panels) and AChE (lower panels), with PD-L1 expression in LG (n = 516) and GBM (n = 153) samples, estimated by TIMER2.0.

Article Snippet: Human IL-8 ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA), and VEGF ELISA kit was obtained from Biolegend (San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Influence of the Cholinergic System on the Pathogenesis of Glioblastoma: Impact of the Neutrophil Granulocytes

doi: 10.3390/ijms27010321

Figure Lengend Snippet: Relationship between VEGF expression and the cholinergic system in GBM. ( A ) Correlation of CHRM1 (upper panels) and CHRM3 (lower panels) with VEGF-A expression in LGG (n = 516) and GBM (n = 153) samples, estimated by TIMER2.0 ( http://timer.cistrome.org/ ). ( B ) Correlation of ChAT (upper panels) and AChE (lower panels) with VEGF-A expression in LGG and GBM samples, estimated by TIMER2.0. ( C – G ) VEGF production in U251 cells cultured as 3D spheroids (9 days, 1.5% agar, 5 × 10 4 cells/mL) and treated with ACh (10 −8 –10 −9 M), Carb (10 −8 –10 −9 M), Tio (30 nM, 15 min pretreatment followed by ACh or Carb 10 −8 M), or Riv (5 μM). Supernatants were collected and VEGF levels measured by ELISA. Nine independent experiments were performed (** p < 0.005, mean ± SD). ( H – L ) VEGF production in U251 cells cultured as 2D monolayers (24 h) with the same treatments described above. Ten independent experiments were performed (* p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001, mean ± SD). ( M ) ICAM-1 expression in 3D cultures measured by flow cytometry. A representative experiment is shown.

Article Snippet: Human IL-8 ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA), and VEGF ELISA kit was obtained from Biolegend (San Diego, CA, USA).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Influence of the Cholinergic System on the Pathogenesis of Glioblastoma: Impact of the Neutrophil Granulocytes

doi: 10.3390/ijms27010321

Figure Lengend Snippet: Functional evaluation of the cholinergic system in PMN-p, PMN-h and cocultures with the U251 cell line. ( A – C ) PD-L1 expression in PMN-h cultured (5 × 10 6 cells/mL) with or without U251 cells for 24 h in the presence of ACh (10 −8 M) or Carb (10 −8 M). PD-L1 levels were measured by flow cytometry using CD11b as PMN marker. ( D , E ) PD-L1 expression in PMN-p cultured for 24 h with ACh (10 −8 –10 −9 M). Seven independent experiments were performed (** p < 0.005). ( J , K ) PD-L1 expression in PMN-h cultured under the same conditions. ( E , K ) Representative experiment. ( F , G , L , M ) CD11b expression in ( F , G ) PMN-p and ( L , M ) PMN-h after 15 min stimulation with ACh (10 −8 –10 −9 M). CD11b levels were evaluated by flow cytometry. Seven independent experiments were performed (* p < 0.05). ( G , M ) Representative experiments. ( H , I , N , O ) IL-8 and VEGF production in ( H , I ) PMN-p ( N , O ) and PMN-h after 24 h stimulation with ACh (10 −8 –10 −9 M). Cytokine levels were measured in culture supernatants by ELISA. Seven independent experiments were performed (* p < 0.05).

Article Snippet: Human IL-8 ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA), and VEGF ELISA kit was obtained from Biolegend (San Diego, CA, USA).

Techniques: Functional Assay, Expressing, Cell Culture, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay