elisa kit  (Bio-Techne corporation)

Journal: Clinical and Translational Medicine

Article Title: Glioblastoma‐educated mesenchymal stem‐like cells promote glioblastoma infiltration via extracellular matrix remodelling in the tumour microenvironment

doi: 10.1002/ctm2.997

Figure Lengend Snippet: Glioblastoma (GBM) secretes CD40L to upregulate LOX expression via CD40 in MSLCs. (A) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis in MSLC treatment control conditioned media (CM), X01 CM or astrocyte CM. (B) Cytokine array of X01 CM or astrocyte CM as indicated ( n = 2). (C) Densitometry analysis of cytokine array shown in (B). (D) qRT‐PCR for LOX and COL1A1 in MSLCs co‐cultured with X01 cells depleted of cytokine genes using siRNA. (E) qRT‐PCR for LOX and COL1A1 in MSLCs depleted of cytokine receptor gene using siRNA co‐cultured with X01. (F) Enzyme‐linked immunosorbent assay of LOX level in CM from (D) and (E). (G) Representative image of 3D collagen‐based matrix pre‐incubated with X01 cells transfected with siRNA‐control or si‐ CD40L and/or MSLCs transfected with siRNA‐control or si‐ CD40 stained with Picrosirius red. Image: bright field (top), collagen fibre (mid, polarized light), and analysis of polarized area (bottom). Collagen fibre area = polarized area/total area ( n = 4). Bright field scale bar: 100 μm; polarized light scale bar: 10 μm. (H) X01 spheroid infiltration in 3D collagen‐based matrix co‐cultured with MSLCs transfected with siRNA‐control or si‐ CD40 . Bottom graph shows calculation results of the infiltration area. Scale bar: 200 μm. * p < .05, ** p < .01, *** p < .001, **** p < .0001

Article Snippet: CM collected from each cell culture medium and the level of secreted LOX (MBS039099, MyBioSource, San Diego, CA, USA), Collagen1A1 (MBS763786, MyBioSource, San Diego, CA, USA) and CD40L (DCDL40, R&D system, MN, USA) were measured using ELISA according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Transfection, Staining

Journal: Clinical and Translational Medicine

Article Title: Glioblastoma‐educated mesenchymal stem‐like cells promote glioblastoma infiltration via extracellular matrix remodelling in the tumour microenvironment

doi: 10.1002/ctm2.997

Figure Lengend Snippet: CD40L increased LOX expression via CD40‐mediated NF‐κB2 nucleus translocation. (A) Western blot analysis to determine CD40 downstream effector activation status in MSLCs treated with control conditioned medium (CM) or X01 CM. (B) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) of LOX expression in MSLCs transfected with siRNA as indicated. (C) Western blot analysis of the nucleus translocation of NF‐κB1 or NF‐κB2 by X01 CM in the cytosol‐nuclear fraction of MSLCs. (D) Immunocytochemistry in MSLCs and nucleus translocation of NF‐κB1 or NF‐κB2 by X01 CM. Scale bar: 50 μm. (E) qRT‐PCR of LOX expression in MSLCs treated with X01 CM and transfected with siRNA as indicated. (F) Enzyme‐linked immunosorbent assay of LOX in CM after co‐culture with each transfected cell type. (G) Western blots of fractionated lysates of MSLCs transfected with siRNA‐control or si‐ CD40 after co‐culture with X01. (H) Nuclear translocation of NF‐κB2 in MSLC siRNA‐control or MSLC si‐ CD40 treated or not treated with X01 CM. Scale bar: 50 μm. (I) Chromatin immunoprecipitation for assessing NF‐κB2 binding to LOX promoter in MSLCs. (J) Representative image of 3D collagen‐based matrix pre‐incubated with MSLCs transfected with siRNA‐control or si‐ NF‐κB2 and/or X01 cells stained with Picrosirius red. Image: bright field (top), collagen fibre (mid, polarized light), and analysis of polarized area (bottom). Bottom graph shows collagen fibre area = polarized area/total area ( n = 4). Bright field scale bar: 100 μm; polarized light scale bar: 10 μm. (K) X01 spheroid infiltration in 3D collagen‐based matrix co‐cultured with MSLCs transfected with siRNA‐control or si‐ NF‐κB2 . Bottom graph shows calculation results of the infiltration area. Scale bar: 200 μm. ** p < .01, *** p < .001, **** p < .0001

Article Snippet: CM collected from each cell culture medium and the level of secreted LOX (MBS039099, MyBioSource, San Diego, CA, USA), Collagen1A1 (MBS763786, MyBioSource, San Diego, CA, USA) and CD40L (DCDL40, R&D system, MN, USA) were measured using ELISA according to the manufacturer's instructions.

Techniques: Expressing, Translocation Assay, Western Blot, Activation Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Chromatin Immunoprecipitation, Binding Assay, Incubation, Staining, Cell Culture

Journal: Clinical and Translational Medicine

Article Title: Glioblastoma‐educated mesenchymal stem‐like cells promote glioblastoma infiltration via extracellular matrix remodelling in the tumour microenvironment

doi: 10.1002/ctm2.997

Figure Lengend Snippet: CD40L neutralizing antibody and knockdown of CD40 suppressed ECM remodelling and GBM infiltration. (A) Schematic illustration of GBM model generation and the overall therapeutic procedure for orthotopic xenograft animal model. (B) H&E and ZEB1 staining of coronally sectioned mouse brain. Yellow dash represents tumour margin. Red triangles represent ZEB1‐positive infiltration cells ( n = 6 mouse/group). Scale bar: 100 μm. (C) The number of ZEB1‐positive cells infiltrated outside the tumour margin in (B). (D) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) of CD40L , CD40 and LOX expression in mouse brain tissue ( n = 3 mouse/group). (E) IHC of CD40, CD40L, NFκB2 and LOX in the indicated groups ( n = 6 mouse/group). Scale bar: 100 μm. (F) Representative image of Picrosirius red‐stained mouse brain tissue for each xenograft group. Collagen fibre area = polarized area/total area ( n = 6 mouse/group). Collagen fibre area (right) = polarized area/total area ( n = 6 mouse/group). Bright field scale bar: 100 μm; polarized light scale bar: 10 μm. *** p < .001, **** p < .0001

Article Snippet: CM collected from each cell culture medium and the level of secreted LOX (MBS039099, MyBioSource, San Diego, CA, USA), Collagen1A1 (MBS763786, MyBioSource, San Diego, CA, USA) and CD40L (DCDL40, R&D system, MN, USA) were measured using ELISA according to the manufacturer's instructions.

Techniques: Knockdown, Animal Model, Staining, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: Clinical and Translational Medicine

Article Title: Glioblastoma‐educated mesenchymal stem‐like cells promote glioblastoma infiltration via extracellular matrix remodelling in the tumour microenvironment

doi: 10.1002/ctm2.997

Figure Lengend Snippet: Correlation between tumour MSLC‐isolatable patients and clinical outcome. (A) IHC of LOX, COL1A1, CD40L, CD40 and NF‐κB2 in MSLC‐isolatable ( n = 4) and MSLC‐non‐isolatable ( n = 4) patient samples. Scale bar: 100 μm. (B) Picrosirius red‐stained MSLC‐isolatable ( n = 3) and MSLC‐non‐isolatable ( n = 3) GBM patient samples. Image: collagen fibre (top, polarized light) and analysis of polarized area (bottom). Graph shows collagen fibre area = polarized area/total area ( n = 3). (C) Gene set enrichment analysis (GSEA) of MSLC‐isolatable and non‐isolatable patients. Analysis of gene set related to ECM‐cell adhesion by each GBM subtype. (D) Kaplan–Meier survival curves of all glioma patients (REMBRANDT) with fixed high or low LOX and COL1A1 median expression. (E) GSEA of all GBMLGG patients with high and low median LOX expression. (F) GBM subtype‐specific expression of the indicated genes in patients with GBM in The Cancer Genome Atlas (TCGA). (G) Two‐gene scatter plots of the indicated genes in patients with glioma in TCGA. (H) Kaplan–Meier survival curves of all glioma patients (TCGA) with high or low indicated median expression of gene. (I) Schematic summarizing promotion of ECM remodelling and GBM infiltration by GBM‐educated MSLCs in the tumour microenvironment. n.s. = not significant, * p < .05, ** p < .01, **** p < .0001

Article Snippet: CM collected from each cell culture medium and the level of secreted LOX (MBS039099, MyBioSource, San Diego, CA, USA), Collagen1A1 (MBS763786, MyBioSource, San Diego, CA, USA) and CD40L (DCDL40, R&D system, MN, USA) were measured using ELISA according to the manufacturer's instructions.

Techniques: Staining, Expressing