Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2 redistributes EZH2 in the DNMT3A promoter to suppress its expression. A Immunoblot detection of DNMT3A and H3 p.K27me3 in U87-EGFRvIII cells treated with GSK126 (EZH2 inhibitor: 2.5 µM) and GSKJ4 (JmjC inhibitor: 10 µM) for 48 h. B , C ChIP-qPCR analysis on H3 p.K27me3 ( B ) and EZH2 ( C ) enrichment in DNMT3A promoter regions of U87-EGFRvIII cells transfected with siRNAs against Scramble sequence or Rictor. D Immunoblot analyses of acetylated EZH2 (Ac-EZH2) in U87-EGFRvIII cells with shScramble or shRictor. Ac-K, acetylated-lysine; IB, immunoblotting; IP, immunoprecipitation. E Immunoblot analyses of acetylated EZH2 (Ac-EZH2) in U87 cells with overexpression of GFP or Rictor. Ac-K, acetylated-lysine; IB, immunoblotting; IP, immunoprecipitation. F Analyses on acetylation and redistribution of EZH2 on the DNMT3A promoter in U87-EGFRvIII cells with addition of TSA (1.0 µM) and acetate (10 mM) for 48 h. Ac, acetate. G mRNA expression of DNMT3A in U87-EGFRvIII cells treated by PP242 (mTORC1/C2 inhibitor: 5 uM) along with supplementation of TSA (1.0 µM) and acetate (10 mM) for 48 h. H mTORC2 drives protein acetylation to redistribute EZH2 into the DNMT3A promoter region, and increases H3 p.K27me3 to suppress the expression of DNMT3A in GBM. Ac, acetyl-group

Article Snippet: Overexpressing lentiviral vectors encoding GFP and human Rictor were established by VectorBuilder Inc (Chicago, IL).

Techniques: Expressing, Western Blot, ChIP-qPCR, Transfection, Sequencing, Immunoprecipitation, Over Expression

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: Reprogramming of glutamate metabolism drives invasive phenotypes in GBM. A Lower methylation signals by MeDIP-qPCR on the GRIA1 promoter and higher expression of GRIA1 transcripts were observed in U87 cells with lentivirus-mediated overexpression of human Rictor (Rictor OE) in comparison with control (GFP). Met, methylation (5-mC). B GBM tumors from rats infected with PDGFB-HA-IRES-EGFP retroviral vectors (n = 4) were probed by immunohistochemistry against GRIA1. Note intratumoral heterogeneity of GRIA1 immunoreactivity in accordance with the status of mTORC2 activation (pAkt) and 5-mC expression. Peri-necrotic area indicates pAkt_low/5-mC_high region, and non-necrotic to pAkt_high/5-mC_low region. Refer to Fig. B. Nec, necrosis. Scale bar, 20 µm. C Scratch assays using the co-culture of U87-EGFRvIII (GBM) cells with siRNA-mediated knockdown of GRIA1 and SH-SY5Y (neuroblastoma) cells. The area of gap was calculated 24 h after scratch. Cells were colored in red with the binary mode (red) of ImageJ software. Scale bar, 100 µm. D Knockdown of GRIA1 significantly ( p < 0.01) affected GBM cell migration in the co-culture of U87-EGFRvIII and SH-SY5Y cells. E Measurement of glutamate (Glu) indicated that GRIA1 knockdown increased extracellular Glu (reduced intracellular Glu) in U87-EGFRvIII GBM cells. Conc, concentration. F Wound healing/migration assay on the co-culture of SH-SY5Y neuroblastoma cells with U87-EGFRvIII GBM cells treated by PhTx-74 (GRIA1/GRIA2 inhibitor: 20 μM) or a combination of PhTx-74 (20 μM) with GSK2256098 (FAK inhibitor: 100 nM). Cells were colored in red with the binary mode (red) of ImageJ software. Scale bar, 100 µm. G PhTx-74 treatment increased extracellular Glu (reduced intracellular Glu), and FAK inhibitor decreased phosphorylation of FAK (Tyr397) in U87-EGFRvIII GBM cells. Conc, concentration. H TCGA datasets on overall survival and progression free survival of GBM cases stratified by the expression level of GRIA1 transcripts

Article Snippet: Overexpressing lentiviral vectors encoding GFP and human Rictor were established by VectorBuilder Inc (Chicago, IL).

Techniques: Methylation, Methylated DNA Immunoprecipitation, Expressing, Over Expression, Comparison, Control, Infection, Retroviral, Immunohistochemistry, Activation Assay, Co-Culture Assay, Knockdown, Software, Migration, Concentration Assay, Phospho-proteomics

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2 activation correlates with global DNA hypomethylation phenotypes in RTK-mutated GBM. A Immunohistochemical (IHC) staining of human GBM tissue (n = 20) with antibodies against mutant EGFR (EGFRvIII) and a DNA methylation marker (5-mC). EGFR amplification was assessed by FISH with probes for EGFR (7p11.2, Red) and CEP7 (7p11.1-q11.1, Green). Scale bar, 40 µm. B Cerebral and brainstem tissue with GBM tumors was harvested from rats infected with PDGFB-HA-IRES-EGFP retroviral vectors (n = 4). Immunohistochemistry was performed on paraffin-embedded tissue sections against a DNA methylation marker (5-mC) and an mTORC2 marker (p-Akt S473). Nec, necrosis. Scale bars, 50 µm (upper panels) and 20 µm (lower panels). C Immunofluorescent staining of 5-mC in U87-EGFRvIII cells transfected with shRNAs against control sequence (scramble) or Rictor (shRictor#1). Scale bar, 10 µm. D Dot blot analysis of 5-mC in U87-EGFRvIII cells transfected with shScramble versus shRictor#1 (upper panel), or overexpressed (OE) with GFP versus Rictor (lower panel). Total DNA for each sample was determined by methylene blue staining. E Detection of global DNA methylation (ELISA-based assay), represented by methylation of LINE-1 retrotransposable elements in U87-EGFRvIII cells transfected with shScramble or shRictor. OD, optical density; STD, standard

Article Snippet: Overexpressing lentiviral vectors encoding GFP and human Rictor were established by VectorBuilder Inc (Chicago, IL).

Techniques: Activation Assay, Immunohistochemical staining, Immunohistochemistry, Mutagenesis, DNA Methylation Assay, Marker, Amplification, Infection, Retroviral, Staining, Transfection, Control, Sequencing, Dot Blot, Enzyme-linked Immunosorbent Assay, Methylation

Journal: Bioactive Materials

Article Title: HtrA3 paves the way for MSC migration and promotes osteogenesis

doi: 10.1016/j.bioactmat.2024.05.016

Figure Lengend Snippet: HtrA3 is upregulated in the leading edge of bone defect repair during the early stage of bone healing in response to the inflammatory environment. (A) Illustration of the rat mandible bone defect model and the various timepoints for harvesting speciments post-surgery. (B) Immunohistofluorescence staining results showed that HtrA3 expression in strol1 + BMSCs increased after surgery around the 3rd day and decreased by the 7th day (Scale bar: left 100 μm, right 25 μm). (C) Quantification analysis of relative fluorescence intensity of HtrA3 staining in (B). Immunohistochemistry staining showed HtrA3 (D) and TNF-α (E). (Scale bar: 100 μm) (F) Quantification analysis of HtrA3 and TNF-α, indicating a similar expression pattern in the defect area. (G) Schematic of TNF-α treatment and related experiments. (H–I) qRT-PCR (H) and immunocytochemical staining (I) showed HtrA3 were significantly increased by TNF-α treatment in a concentration dependent manner (Scale bar: 25 μm). *P < 0.05.

Article Snippet: All lentiviral vectors encoding green fluorescent protein (GFP) for knockdown of HtrA3 were purchased from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Immunohistofluorescence, Staining, Expressing, Fluorescence, Immunohistochemistry, Quantitative RT-PCR, Concentration Assay

Journal: Bioactive Materials

Article Title: HtrA3 paves the way for MSC migration and promotes osteogenesis

doi: 10.1016/j.bioactmat.2024.05.016

Figure Lengend Snippet: HtrA3 enhances formation of cellular cortical protrusions, MSCs migration and osteogenic differentiation in vitro . (A) Representative microscopy images of control and HtrA3 over-expressing hBMSCs cultured in gel for 1 and 3 days. Images show cell nuclei (blue) and GFP expression in cytosol (green). Insets demonstrated 3D-reconstructed micrographs of individual cells (Scale bar: 20 μm). (B) Quantitative analysis of cell surface area, volume and sphericity were performed using Imaris software. (C) Single cell tracking showed that HtrA3 promoted the cell migration speed of hBMSCs for 24 h. (D) Immunocytochemical staining showed that RUNX2 and its nuclear translocation were significantly increased after rhHtrA3 treatment (Scale bar: 25 μm). (E) qPCR showed that treatment of hBMSCs with rhHtrA3 for 3 days promoted expression of osteogenic related markers. (F–I) ALP (F and G) and ARS (H and I) staining also showed enhanced osteogenic differentiation of MSCs upon treatment with rhHtrA3 for 3 days (Scale bar: 0.5 mm). *P < 0.05.

Article Snippet: All lentiviral vectors encoding green fluorescent protein (GFP) for knockdown of HtrA3 were purchased from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Migration, In Vitro, Microscopy, Control, Expressing, Cell Culture, Software, Single Cell Tracking, Staining, Translocation Assay

Journal: Bioactive Materials

Article Title: HtrA3 paves the way for MSC migration and promotes osteogenesis

doi: 10.1016/j.bioactmat.2024.05.016

Figure Lengend Snippet: HtrA3 continuous overexpression inhibited BMSC osteogenic differentiation. (A) qPCR showed that HtrA3 overexpression decreased expression of osteogenic related markers. ALP (B) and ARS (C) staining showed inhibitory effects on osteogenic differentiation in the HtrA3 overexpression group (Scale bar: 0.5 mm). *P < 0.05.

Article Snippet: All lentiviral vectors encoding green fluorescent protein (GFP) for knockdown of HtrA3 were purchased from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Expressing, Staining

Journal: Bioactive Materials

Article Title: HtrA3 paves the way for MSC migration and promotes osteogenesis

doi: 10.1016/j.bioactmat.2024.05.016

Figure Lengend Snippet: HtrA3 promoted MSC migration and osteogenesis in vivo . (A) Representative micro-CT images of critical-sized rat calvarial full-thickness defects at week 4 and week 8 after treatment with rhHtrA3 for 3 days, indicating more new bone formation in defect area treated with rHtrA3. (B–C) Quantitative analysis of bone volume (B) and bone mineral density (C). (E-F) HE and Masson's trichrome staining of histological sections. (NB, nascent bone; FT, fibrous tissue; MC, medullary cavity; OT, osteoid tissue; MT, mineralized tissue). (F) Immunohistofluorescence staining showed that rHtrA3 promoted MSCs migration during the early stage of bone defect repair (Scale bar: up 3 mm, down 50 μm). *P < 0.05.

Article Snippet: All lentiviral vectors encoding green fluorescent protein (GFP) for knockdown of HtrA3 were purchased from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Migration, In Vivo, Micro-CT, Staining, Immunohistofluorescence

Journal: Bioactive Materials

Article Title: HtrA3 paves the way for MSC migration and promotes osteogenesis

doi: 10.1016/j.bioactmat.2024.05.016

Figure Lengend Snippet: HtrA3 selectively degrades ECM and increased expression of Itgβ1 in hBMSCs. (A) SEM images showed the fibrillar structure of collagen I and the cloud-like structure of Matrigel. The far right SEM images demonstrated that the fibrillar structure of the collagen-Matrigel mixture gel was exposed after HtrA3 recombinant protein treatment (Scale bar: 1 μm). (B) Co-localization of HtrA3 fusion protein of MSCs and degraded green fluorescent collagen IV (Scale bar: 50 μm). (C) Proteolysis assay results of collagen IV - HtrA3 co-culture showed the degradation of collagen IV by rhHtrA3. (D) Quantitative analysis of proteolysis assay in (C). (D) Immunocytochemical staining results of Matrigel-cultured hBMSCs showed that HtrA3 over-expression promoted the expression of Itgβ1 (Scale bar: 100 μm). *P < 0.05.

Article Snippet: All lentiviral vectors encoding green fluorescent protein (GFP) for knockdown of HtrA3 were purchased from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Expressing, Recombinant, Proteolysis Assay, Co-Culture Assay, Staining, Cell Culture, Over Expression

Journal: Bioactive Materials

Article Title: HtrA3 paves the way for MSC migration and promotes osteogenesis

doi: 10.1016/j.bioactmat.2024.05.016

Figure Lengend Snippet: HtrA3 promotes the migration and osteogenic differentiation of MSCs through mechanotransduction. (A) The diagram of detecting traction forces with TFM. (B) The curves of total traction force exerted by hBMSCs in different groups at different time durations from adhesion. The maximum of total traction force and the slope of total traction force-time curves at the first 2 h ( l 2h ) were shown in (C) and (D), respectively. (E) The typical mechanics heatmaps of celluar stiffness detected through AFM. (F) Quantitative analysis showed that celluar stiffness was increased by HtrA3 overexpression, which was abrogated by the Itg β1 antibody. (G-I) Immunocytochemical staining of YAP showed HtrA3 overexpression significantly increased YAP expression and its nuclear translocation, which were abrogated by the neutralizing Itg β1 antibody (Scale bar: 25 μm). *P < 0.05.

Article Snippet: All lentiviral vectors encoding green fluorescent protein (GFP) for knockdown of HtrA3 were purchased from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Migration, Over Expression, Staining, Expressing, Translocation Assay

Journal: Bioactive Materials

Article Title: HtrA3 paves the way for MSC migration and promotes osteogenesis

doi: 10.1016/j.bioactmat.2024.05.016

Figure Lengend Snippet: Itgβ1 blocking diminishes the cellular cortical protrusions, the increased mobility and osteogenic differentiation of BMSCs induced by HtrA3 over-expression. (A) Itg β1 antibody treatment decreased the cell surface area and volume, while increasing sphericity in the HtrA3 overexpression group. (B–C) Single cell tracking showed that neutralizing Itgβ1 antibody decreased cell migration speed of HtrA3 overexpression hBMSCs. (D) The representative RUNX2 immunofluorescence staining of hBMSCs in different groups (Scale bar: 25 μm). (E-F) Quantitative analysis of RUNX2 nuclear transloaction and its fluorescence intensity. (G) qPCR analysis showed that expression of osteogenic-related genes in the rhHtrA3 group were diminished after Itg β1 antibody treatment. *P < 0.05.

Article Snippet: All lentiviral vectors encoding green fluorescent protein (GFP) for knockdown of HtrA3 were purchased from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Blocking Assay, Over Expression, Single Cell Tracking, Migration, Immunofluorescence, Staining, Fluorescence, Expressing

Journal: Bioactive Materials

Article Title: HtrA3 paves the way for MSC migration and promotes osteogenesis

doi: 10.1016/j.bioactmat.2024.05.016

Figure Lengend Snippet: Schematic diagram illustrating the underlying mechanisms by which HtrA3 promotes MSCs migration and osteogenesis. During the early stage of bone healing, HtrA3 is upregulated by inflammatory factors in MSCs and then secreted to ECM. It breaks down ECM to make room for cell movement and increases expression of integrin β1, thus enhancing BMSC-ECM crosstalk and osteogenic differentiation of MSCs.

Article Snippet: All lentiviral vectors encoding green fluorescent protein (GFP) for knockdown of HtrA3 were purchased from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Migration, Expressing

Journal: Cell Cycle

Article Title: CUL4B regulates autophagy via JNK signaling in diffuse large B-cell lymphoma

doi: 10.1080/15384101.2018.1560718

Figure Lengend Snippet: Correlation between CUL4B expression and clinical characteristics of DLBCL patients.

Article Snippet: The green fluorescent protein (GFP)-tagged lentivirus vectors either encoding CUL4B (shCUL4B, lvCUL4B) or control were purchased from Genechem (Shanghai, China).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Therapeutic effects of hepatocyte growth factor-overexpressing dental pulp stem cells on liver cirrhosis in a rat model

doi: 10.1038/s41598-017-14995-5

Figure Lengend Snippet: Isolation and characterization of DPSCs. ( A , B ) Phase-contrast images of primary DPSCs cultured for 48 h ( A ) and DPSCs at the 3 rd passage ( B ). ( C – G ) FACS analyses of the expression of CD29 ( C ), CD90 ( D ), CD44 ( E ), CD34 ( F ) and CD45 ( G ) in DPSCs. ( H , I ) Images of Alizarin Red ( C ) and Oil Red O staining ( D ) in DPSCs cultured in osteogenic and adipogenic differentiation medium for 21 d. ( J – K ) Gross ( J ) and phase contrast ( K ) images of PAS staining in DPSCs grown in hepatogenic differentiation medium for 17 d. ( L ) Fluorescence microscopic image of DPSCs grown in hepatogenic differentiation medium for 17 d, immunostained with anti-albumin (red) and counterstained with DAPI (blue). ( M ) Fluorescence microscopic image of GFP (green)-encoding lentiviral vector-transduced DPSCs. ( N , O ) Images from Western blot analyses for HGF and hHGF in DPSCs ( N ) and related quantitative analysis ( O ). ( P ) ELISA of HGF levels in the medium of DPSCs. Scale bars ( A , B ) = 50 μm; ( H , L , M ) = 20 μm; ( J ) = 5 μm; ( J ) = 10 mm; ( K ) = 25 μm. ** P < 0.01; *** P < 0.001.

Article Snippet: Two lentiviral vectors (a blank lentiviral vector encoding only green fluorescent protein (GFP) and a hHGF-expressing lentiviral vector encoding GFP and hHGF) were purchased from Shanghai Genechem Company ( GOSL49905 ) .

Techniques: Isolation, Cell Culture, Expressing, Staining, Fluorescence, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay