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

Journal: Theranostics

Article Title: Neuron-specific WDR5 epigenetically upregulates ARID5B to impair GABAergic synaptic transmission and promotes epileptogenesis

doi: 10.7150/thno.122246

Figure Lengend Snippet: Inhibition of WDR5-KMT2 complex reduces seizure susceptibility and severity. (A) qPCR analysis of indicated mRNA expression levels in ipsilateral hippocampal tissues from sham control versus IHKA-2d mice. n = 5 biologically independent mice per group. (B) Representative immunoblots (top) and quantitative analysis (bottom) of KMT2A, SETD1A, and SETD1B protein expression in ipsilateral hippocampal tissues from sham control versus IHKA-2d mice. n = 4-5 biologically independent mice per group. (C) Schematic diagram of experimental design and timeline. ( D-E ) Pharmacological effects of OICR-9429 (2.5, 5, 10 mg/kg, i.p.) versus vehicle control on latency to GTCS ( D ) and mortality rates ( E ) in the PTZ-induced acute seizure model. n = 13 biologically independent mice per group. (F) Representative EEG traces showing seizure activity in PTZ-injected mice pretreated with either vehicle or OICR-9429 (10 mg/kg). The boxed region indicates the magnified epileptiform discharges. (G) Representative immunoblots (top) and quantitative analysis (bottom) of nuclear H3K4me3, H3K27me3, and WDR5 protein levels in hippocampal lysates from PTZ-kindled mice pretreated with OICR-9429 (0, 2.5, 5, 10 mg/kg). n = 6 biologically independent mice per group. (H) Schematic diagram of experimental design and timeline. (I) Representative EEG traces showing typical SRS electrographic activity recorded from chronic IHKA-TLE mice treated with either vehicle or OICR-9429 (10 mg/kg). The boxed region indicates the magnified epileptiform discharges. (J-K) Bar graph denotes electroclinical SRS ( J ) daily frequency (SRSs/day) and ( K ) mean duration in chronic IHKA-TLE mice treated with vehicle or OICR-9429 (10 mg/kg, i.p.). n = 5 biologically independent mice per group. No mortality occurred in any group during the behavioral observation period following SE induction. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analyses were performed as follows: two-tailed unpaired Student's t-tests with Holm-Šidák correction for multiple comparisons (Figure A-B); one-way ANOVA with Tukey's post hoc test (Figure D); two-tailed unpaired Student's t-tests (Figure J-K). In Figure G, H3K27me3 protein levels were analyzed using the Kruskal-Wallis test, while other data were assessed with one-way ANOVA with Tukey's post hoc test.

Article Snippet: The following chemicals were used in this study: Kainic acid (KA; MedChemExpress, catalog no. HY-N2309), pentylenetetrazole (PTZ; Sigma‒Aldrich, catalog no. P6500), OICR-9429 (MedChemExpress, catalog no. HY-16993, purity: 99.71%), tetrodotoxin (TTX; Tocris Bioscience, catalog no. 1078), picrotoxin (PTX; Sigma‒Aldrich, catalog no. P1675), D-AP5 (Tocris, catalog no. 0106), NBQX disodium salt (Tocris, catalog no. 1044), and Sulfobutylether-β-Cyclodextrin (SBE-β-CD; MedChemExpress, catalog no. HY-17031).

Techniques: Inhibition, Expressing, Control, Western Blot, Activity Assay, Injection, Two Tailed Test

Journal: Theranostics

Article Title: Neuron-specific WDR5 epigenetically upregulates ARID5B to impair GABAergic synaptic transmission and promotes epileptogenesis

doi: 10.7150/thno.122246

Figure Lengend Snippet: Arid5b as a potential direct target of WDR5-H3K4me3 under epileptic condition . (A) Volcano plot of RNA-seq analysis showing DEGs in the ipsilateral hippocampus of IHKA-2d mice pretreated with vehicle control or OICR-9429. Mice received intraperitoneal injections of OICR-9429 or vehicle control every 24 hours beginning 3 days prior to IHKA induction. Hippocampal tissues were collected for RNA-seq 2 days post-modeling. n = 3 biologically independent mice per group. Cutoff criteria: fold change > 1.5, P < 0.05. FC, fold change. (B) Venn diagram showing overlapping genes between H3K4me3-enriched genes in IHKA-2d mice and genes with reduced expression following OICR-9429 treatment. (C) Heatmap displaying the top 8 most significantly downregulated genes selected from the 19 overlapping genes identified in ( B ). (D) Genome browser snapshots of H3K4me3 ChIP-seq signals at the Arid5b locus in sham control versus IHKA-2d groups. Purple shading highlights H3K4me3 peak enrichment at the Arid5b promoter region. (E) qPCR analysis of indicated mRNA expression levels in ipsilateral hippocampal tissues IHKA-2d mice pretreated with vehicle control or OICR-9429. n=5 biologically independent mice per group. (F) qPCR analysis of Arid5b mRNA expression in ipsilateral hippocampal tissues from sham control versus IHKA-2d mice. n=5 biologically independent mice per group. (G-I) ChIP-qPCR analysis of H3K4me3 enrichment at the Arid5b promoter in hippocampal tissues from indicated groups. Data represent fold-enrichment over IgG control calculated by 2 -ΔΔCT method. n = 4 biologically independent mice per group. (J-L) ChIP-qPCR analysis of WDR5 enrichment at the Arid5b promoter in hippocampal tissues from indicated groups. n = 4 biologically independent mice per group. *p < 0.05, ***p < 0.001. Statistical analyses were performed as follows: two-tailed unpaired Student's t-tests with Holm-Šidák correction for multiple comparisons (Figure E); two-tailed unpaired Student's t-tests (Figure F); one-way ANOVA with Dunnett's post hoc test (Figure G-L).

Article Snippet: The following chemicals were used in this study: Kainic acid (KA; MedChemExpress, catalog no. HY-N2309), pentylenetetrazole (PTZ; Sigma‒Aldrich, catalog no. P6500), OICR-9429 (MedChemExpress, catalog no. HY-16993, purity: 99.71%), tetrodotoxin (TTX; Tocris Bioscience, catalog no. 1078), picrotoxin (PTX; Sigma‒Aldrich, catalog no. P1675), D-AP5 (Tocris, catalog no. 0106), NBQX disodium salt (Tocris, catalog no. 1044), and Sulfobutylether-β-Cyclodextrin (SBE-β-CD; MedChemExpress, catalog no. HY-17031).

Techniques: RNA Sequencing, Control, Expressing, ChIP-sequencing, ChIP-qPCR, Two Tailed Test

Journal: Theranostics

Article Title: Neuron-specific WDR5 epigenetically upregulates ARID5B to impair GABAergic synaptic transmission and promotes epileptogenesis

doi: 10.7150/thno.122246

Figure Lengend Snippet: ARID5B is essential for WDR5-mediated regulation of epileptogenesis through inhibitory synaptic receptors. (A-B) GSEA enrichment plot of the GABAergic ( A ) and glutamatergic ( B ) synapse pathway showing significant differential enrichment in vehicle- versus OICR-9429-pre-treated IHKA-2d mice. (C-G) Representative immunoblots ( C ) and quantitative analysis ( D-G ) of WDR5, ARID5B and other synaptic protein levels in ipsilateral hippocampal lysates from indicated groups. n = 5 biologically independent mice per group. (H-K) Representative immunoblots ( H ) and quantitative analysis ( I-K ) of WDR5, ARID5B and other synaptic protein levels in hippocampal lysates from indicated groups. n = 5 biologically independent mice per group. (L) Representative EEG traces: (top) Control (oeCtrl) mouse showing physiological activity; (bottom) oeWDR5 mouse exhibiting spike waves. The boxed region indicates the magnified epileptiform discharges. (M) Representative EEG traces showing typical SRS electrographic activity recorded from chronic IHKA-TLE mice transduced with indicated AAVs. (N-O) Daily frequency (N) and mean duration (O) of electroclinical SRSs in chronic IHKA-TLE mice transduced with indicated AAVs. n = 7 biologically independent mice per group, following the exclusion of mortalities in oeCtrl+shScr (2 mice: 1d and 20d post-SE), oeWDR5+shScr (2 mice: 1d and 4d post-SE), and oeWDR5+shARID5B (1 mouse: 2d post-SE). *p < 0.05, **p < 0.01, ***p < 0.001. Statistical comparisons were performed using one-way ANOVA with Tukey's post hoc test.

Article Snippet: The following chemicals were used in this study: Kainic acid (KA; MedChemExpress, catalog no. HY-N2309), pentylenetetrazole (PTZ; Sigma‒Aldrich, catalog no. P6500), OICR-9429 (MedChemExpress, catalog no. HY-16993, purity: 99.71%), tetrodotoxin (TTX; Tocris Bioscience, catalog no. 1078), picrotoxin (PTX; Sigma‒Aldrich, catalog no. P1675), D-AP5 (Tocris, catalog no. 0106), NBQX disodium salt (Tocris, catalog no. 1044), and Sulfobutylether-β-Cyclodextrin (SBE-β-CD; MedChemExpress, catalog no. HY-17031).

Techniques: Western Blot, Control, Activity Assay, Transduction