protein 5 wdr5 inhibitor oicr 9429  (MedChemExpress)

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: Hypoxia-induced epigenetic regulation contributes to increased OPN expression. (A) Western blotting showing the levels of HIF-1α, H3K4me3, H3K9me3, H3K27me3, and H3 in THP-1 and U937 cells exposed to 1% O 2 for the indicated durations. (B) IF staining showing the levels of HIF-1α, H3K4me3, H3K9me3, and H3K27me3 in THP-1 cells exposed to 1% O 2 for the indicated durations. The target proteins are shown in green. DAPI was used to stain the nuclei, and F-actin was labeled to delineate the cytoplasmic region (scale bar = 40 μm). (C) qPCR analysis of H3K4me3 enrichment at SPP1 promoter area in THP-1 cells under indicated treatment conditions ( n = 3 per group; normoxia, normoxic culture for 24 h; hypoxia, hypoxic culture for 24 h; H + siCtrl, hypoxic culture for 24 h together with transfection of the negative control siRNA; H + siWDR5-2, hypoxic culture for 24 h together with WDR5 knockdown; H + DMSO, hypoxic culture for 24 h together with DMSO treatment; H + OICR-9429, hypoxic culture for 24 h together with OICR-9429 treatment). (D) qPCR analysis of WDR5 enrichment at SPP1 promoter area in THP-1 cells under indicated treatment conditions ( n = 3 per group). (E) Agarose gel electrophoresis assays showing ChIP-PCR (SPP1 promoter sequence) products from THP-1 cells immunoprecipitated with anti-H3K4me3, anti-WDR5, and IgG antibodies under indicated treatment conditions. (F) qPCR analysis of H3K4me3 enrichment at SPP1 promoter area in U937 cells under indicated treatment conditions ( n = 3 per group). (G) qPCR analysis of WDR5 enrichment at SPP1 promoter area in U937 cells under indicated treatment conditions ( n = 3 per group). (H) Agarose gel electrophoresis assays showing ChIP-PCR (SPP1 promoter sequence) products from U937 cells immunoprecipitated with anti-H3K4me3, anti-WDR5, and IgG antibodies under indicated treatment conditions. (I and J) qPCR analysis of SPP1 expression in THP-1 (I) and U937 (J) cells under indicated treatment conditions ( n = 3 per group). (K) Western blotting showing OPN expression in THP-1 and U937 cells subjected to the indicated treatments. The data are presented as the means ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001. HIF-1α, hypoxia-inducible factor α; H3K4me3, histone 3 lysine 4 trimethylation; H3K9me3, histone 3 lysine 9 trimethylation; H3K27me3, histone 3 lysine 27 trimethylation; IF, immunofluorescence; ChIP, chromatin immunoprecipitation; WDR5, WD40 repeat-containing protein 5; SPP1, secreted phosphoprotein 1; OPN, osteopontin; DMSO, dimethyl sulfoxide; qPCR, quantitative real-time PCR; SD, standard deviation; kDa, kilodaltons.

Article Snippet: THP-1 and U937 cells were treated with the WD40 repeat-containing protein 5 (WDR5) inhibitor OICR-9429 (20 μM, catalog no. HY-16993, MedChemExpress) for 24 h during hypoxic culture.

Techniques: Expressing, Western Blot, Staining, Labeling, Transfection, Negative Control, Knockdown, Agarose Gel Electrophoresis, Sequencing, Immunoprecipitation, Immunofluorescence, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: Targeting OPN increases the therapeutic effectiveness of TMZ in a C57BL/6J in vivo glioma model. (A) Schematic illustration of the in vivo experimental design in C57BL/6J mice. The blue arrow represents intracranial implantation; the red arrows represent treatments (control, treatment with PBS; OPNi-1, treatment with OPNi-1 alone; TMZ, treatment with TMZ alone; TMZ + OPNi-1, combined treatment with TMZ and OPNi-1); the green arrows represent MRI scans. (B) Representative MR images showing the intracranial tumor burden in C57BL/6J mice from the indicated treatment groups. (C) Quantification of the tumor volume in C57BL/6J mice from the indicated groups on days 0, 7, and 14 since initial treatment ( n = 7 mice per group). (D) Kaplan–Meier survival curves of glioma-bearing mice receiving the indicated treatments ( n = 7 mice per group). (E) Representative mIHC images of glioma tissues from mouse brain sections. Eight markers were divided into 2 staining panels and applied to serial tumor sections to preserve spatial consistency. Panel 1 includes PD-L1, F4/80, CD163, and OPN, and panel 2 includes CD20, NK1.1, CD8a, and CD4. Images for both panels were acquired from matched anatomical regions on adjacent serial sections. (F) Quantification of PD-L1 positivity based on mIHC staining ( n = 3 per group). (G) Quantification of macrophage (F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (H) Quantification of GAM (CD163 + F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (I) Quantification of OPN + GAM (OPN + CD163 + F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (J) Quantification of B cell (CD20 + cells) proportion based on mIHC staining ( n = 3 per group). (K) Quantification of NK cell (NK1.1 + cells) proportion based on mIHC staining ( n = 3 per group). (L) Quantification of CD8 + T cell (CD8a + cells) proportion based on mIHC staining ( n = 3 per group). (M) Quantification of CD4 + T cell (CD4 + cells) proportion based on mIHC staining ( n = 3 per group). Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01. PD-L1, programmed cell death ligand 1; F4/80, mouse EGF-like module-containing mucin-like hormone receptor-like 1; CD163, cluster of differentiation 163; OPN, osteopontin; GAM, glioma-associated macrophage; OPN + GAM, osteopontin positive glioma-associated macrophage; NK, natural killer; CD20, cluster of differentiation 20; SD, standard deviation. Hypoxia induces the emergence of OPN + GAMs through the epigenetic activation of the H3K4me3-WDR5 axis. The secreted OPN promotes the mesenchymal transition and PD-L1 expression in glioma cells via CD44-mediated activation of the NF-κB signaling pathway.

Article Snippet: THP-1 and U937 cells were treated with the WD40 repeat-containing protein 5 (WDR5) inhibitor OICR-9429 (20 μM, catalog no. HY-16993, MedChemExpress) for 24 h during hypoxic culture.

Techniques: In Vivo, Control, Staining, Standard Deviation, Activation Assay, Expressing

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: Schematic diagram showing mechanistic summary of OPN + GAM promoting mesenchymal transition of glioma cells under hypoxic conditions. Hypoxia induces the emergence of OPN + GAMs through the epigenetic activation of the H3K4me3-WDR5 axis. The secreted OPN promotes mesenchymal transition and PD-L1 expression in glioma cell axis. The secreted OPN promotes the mesenchymal transition and PD-L1 expression in glioma cells via CD44-mediated activation of the NF-κB signaling pathway. PD-L1, programmed cell death ligand 1; OPN, osteopontin; GAM, glioma-associated macrophage; H3K4me3, histone 3 lysine 4 trimethylation; MES, mesenchymal; PN, proneural; GBM, glioblastoma.

Article Snippet: THP-1 and U937 cells were treated with the WD40 repeat-containing protein 5 (WDR5) inhibitor OICR-9429 (20 μM, catalog no. HY-16993, MedChemExpress) for 24 h during hypoxic culture.

Techniques: Activation Assay, Expressing