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: Nature Communications

Article Title: Two high-risk susceptibility loci at 6p25.3 and 14q32.13 for Waldenström macroglobulinemia

doi: 10.1038/s41467-018-06541-2

Figure Lengend Snippet: Association statistics for two independent SNP genotypes and WM/LPL risk

Article Snippet: The EXOC2 3′UTR Lenti-reporter-GFP (green fluorescent protein) vector was purchased from Applied Biological Materials Inc. (Richmond, BC, Canada Catalog # MT-h57118).

Techniques: Variant Assay

Journal: Nature Communications

Article Title: Two high-risk susceptibility loci at 6p25.3 and 14q32.13 for Waldenström macroglobulinemia

doi: 10.1038/s41467-018-06541-2

Figure Lengend Snippet: Genomic position and alignments of rs116446171 to miRs. a Schematic representation of the position of rs116446171 relative to the 3′UTR of EXOC2 on chromosome 6 and b alignments of rs116446171 wild type and risk variants with the binding sites of microRNAs, miR-378a-5p and miR-324-3p

Article Snippet: The EXOC2 3′UTR Lenti-reporter-GFP (green fluorescent protein) vector was purchased from Applied Biological Materials Inc. (Richmond, BC, Canada Catalog # MT-h57118).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Two high-risk susceptibility loci at 6p25.3 and 14q32.13 for Waldenström macroglobulinemia

doi: 10.1038/s41467-018-06541-2

Figure Lengend Snippet: The rs116446171 variants affect reporter activity and cell proliferation. a EGFP reporter activity in HEK293T stably transduced cell lines. Cells transduced with the risk variant (G) showed significantly increased fluorescence levels of EGFP compared to the cell lines transduced with the wild type (WT, (C); P = 0.012). Cells transduced with the Null (Δ) had decreased EGFP fluorescence ( P = 0.054, n = 14), and cells transduced with the commercial 3′UTR of EXOC2 showed significantly decreased EGFP fluorescence ( P < 0.0001, n = 14). Data are expressed as mean fold change relative to the cells transduced with the vector, ±standard error of the mean (s.e.m.), n = 14 replicates. ** P < 0.01, **** P < 0.0001. b Quantitative PCR analysis of EGFP transcripts in HEK293T stably transduced cell lines. Significant changes of EGFP mRNA levels were detected in cells harboring the variant allele compared to the cells harboring the wild-type allele ( P = 0.031). Cells harboring the Null allele had reduced EGFP transcripts levels ( P = 0.036). Data are expressed as mean % change relative to the endogenous controls, ±s.e.m., n = 9 replicates for each experiment. * P < 0.05. c Proliferation assay of cells harboring rs116446171, the deletion (Null) of an 18-bp segment centered on rs116446171, and the commercial 3′UTR reporter. The cell line transduced with the variant allele showed significantly increased cell proliferation compared to the cell lines transduced with the EXOC2 3′UTR, the WT and the Null. Data are expressed as mean fold change of the cell line in the day seeded, ±s.e.m., n = 9 replicates. **** P < 0.0001. All P -values were calculated with unpaired t -test

Article Snippet: The EXOC2 3′UTR Lenti-reporter-GFP (green fluorescent protein) vector was purchased from Applied Biological Materials Inc. (Richmond, BC, Canada Catalog # MT-h57118).

Techniques: Activity Assay, Stable Transfection, Transduction, Variant Assay, Fluorescence, Plasmid Preparation, Real-time Polymerase Chain Reaction, Proliferation Assay

Journal: Nature Communications

Article Title: Two high-risk susceptibility loci at 6p25.3 and 14q32.13 for Waldenström macroglobulinemia

doi: 10.1038/s41467-018-06541-2

Figure Lengend Snippet: Dose-dependent effect of rs116446171 variants on reporter transcription and cell proliferation. a Quantitative PCR analysis of EGFP transcripts in stably transduced cells with tandem repeats of the rs116446171 variant allele. The variant allele was inserted within the EXOC2 3′UTR region as a single copy or as two, four and eight repeats in either cis or trans orientation. Data are expressed as mean fold change of the endogenous controls, ±s.e.m., n = 9 replicates. **** P < 0.0001. b Proliferation assay of cells transduced with tandem repeats of the variant allele. Cells harboring eight tandem repeats proliferate significantly faster than cells harboring four tandem repeats, and cells harboring four tandem repeats proliferate significantly faster than cells harboring two or one repeat. Data are expressed as mean fold change of the cell line in the day seeded, ±s.e.m., n = 9 replicates. **** P < 0.0001. All P -values were calculated with unpaired t -test

Article Snippet: The EXOC2 3′UTR Lenti-reporter-GFP (green fluorescent protein) vector was purchased from Applied Biological Materials Inc. (Richmond, BC, Canada Catalog # MT-h57118).

Techniques: Real-time Polymerase Chain Reaction, Stable Transfection, Variant Assay, Proliferation Assay, Transduction

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Table 1 Sequences of primers used in this study

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Fut9 is upregulated in the process by which Wnt4 promotes neuronal differentiation in NSCs (A,B) RNA sequencing of NSCs treated with Wnt4 for 3 days. (A) Volcano plots of differentially expressed genes (DEGs) in NSCs treated with Wnt4 in comparison with the untreated group. (B) Heatmap of DEGs of NSCs treated with Wnt4 in comparison with the untreated group. (C,D) RT-qPCR and western blot analysis of Fut9 expression in NSCs stimulated with Wnt4 for 3 days. (E) Quantification of Fut9 protein expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: RNA Sequencing, Comparison, Quantitative RT-PCR, Western Blot, Expressing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Wnt4 upregulates Fut9 expression through the β-catenin signaling pathway (A) GO pathway enrichment analysis of the differentially expressed genes between the NT group and Wnt4 group. (B,C) RT-qPCR and western blot analysis of Fut9 expression in NSCs treated with a specific pathway inhibitor and then stimulated with Wnt4 for 3 days. (D) Quantification of Fut9 protein expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group; #P<0.05 compared with the Wnt4 group. IWR-1: Wnt/β-catenin inhibitor.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Overexpression of Fut9 promotes neuronal differentiation (A,B) mRNA and protein expression levels of Fut9 in the NT, LV-Con, and LV-Fut9 groups. (C) Immunofluorescence staining for neural-differentiated markers of NSCs in the NT, LV-Con, and LV-Fut9 groups. Scale bar: 100 μm. (D,E) Quantification of dendritic length and neural-differentiated marker-positive cells of NSCs. (F,G) mRNA and protein expression levels of NF200, β3-tubulin and MAP2 in the NT, LV-Con, and LV-Fut9 groups. (H) Quantification of neural-differentiated marker expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the nontreatment group and LV-Con group.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Over Expression, Expressing, Immunofluorescence, Staining, Marker, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Fut9 rescues the negative effect of Notch signaling to promote neuronal differentiation (A) Immunofluorescence staining for neural-differentiated markers of NSCs in the NT, LV-Con+Jag1, and LV-Fut9+Jag1 groups. Scale bar: 100 μm. (B, C) Quantification of dendritic length and neural-differentiated marker-positive cells of NSCs. (D,E) mRNA and protein expression levels of NF200, β3-tubulin and MAP2 in the NT, LV-Con+Jag1, and LV-Fut9+Jag1 groups. (F) Quantification of neural-differentiated marker expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the LV-Con+Jag1 group.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Immunofluorescence, Staining, Marker, Expressing, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Fut9 inhibits the furin enzyme activity of S1 cleavage to suppress the activation of the Notch signaling pathway (A) Western blot analysis of Notch1 and NICD expression in NSCs treated with furin. (B) Quantification of Notch1 and NICD expression by western blot analysis. (C) Furin-like enzyme activity was measured in NSCs in different groups. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group. (D,E) RT-qPCR and western blot analysis of Hes1 and Hes5 in the LV-Con, LV-Con+furin and LV-Fut9+furin groups. (F) RT-qPCR analysis of neural transcription factor expression in the LV-Con, LV-Con+furin and LV-Fut9+furin groups. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the LV-Con+furin group.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Activity Assay, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Fut9 promotes functional recovery and tissue repair (A) Images showing hind limb climb from Sham, SCI, LV-Con and LV-Fut9 groups at the eighth week postinjury; white arrows point to the hind limb. (B) BBB scores of the different groups. (C) H&E, MRI and Nissl staining analyses of the spinal cord in different groups. Sections at 2 mm rostral and caudal to the lesion epicenter were counted for each rat. (D,F) Quantification of H&E, MRI and Nissl staining analyses in different groups. Data are presented as the mean±SD. *P<0.05 compared with the sham group; #P<0.05 compared with the SCI group. (G) Immunofluorescence staining of the spinal cord in different groups. Scale bar: 50 μm. (H) Quantification of immunofluorescence staining. Data are presented as the mean±SD from two independent experiments. **P<0.001.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Functional Assay, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Talaromyces marneffei promotes M2 polarization of human macrophages by downregulating SOCS3 expression and activating TLR9 pathway

doi: 10.1101/2021.02.17.431726

Figure Lengend Snippet: (A) Construction of a SOCS3-overexpressed THP-1 macrophage cell line using lentivirus vector with confirmation by qPCR and Western Blot. (B) Levels of TNF-α and IL-10 in SOCS3 - overexpressed THP-1 macrophages and control cells as measured by qPCR and CBA. (C-E) Expression of CD163 (C), CD200R (D) and p-STAT6 (E) in SOCS3-overexpressed THP-1 macrophages and control cells as measured by flow cytometry. (F, G) The effect of SOCS3 overexpression on antifungal function of THP-1 macrophages. SOCS3 overexpression THP-1 macrophages and control cells were incubated with T.marneffei spores (MOI =10) for 24h, respectively. (F) Phagocytic activity, as measured by phagocytic index, of SOCS3-overexpressed macrophages. (G) Antifungal activity of SOCS3-overexpressed macrophages as measured by CFUs using microdilution spot assay. (H-J) Effects of T. marneffei infection on the expression of TNF-α, IL-10, p-STAT6, SOCS3 in SOCS3-overexpressed THP-1 macrophages. SOCS3-overexpressed THP-1 macrophages were infected with T. marneffei spores for 24hr. The levels of TNF-α (H) and IL-10 (H) were measured by qPCR and CBA. The level of p-STAT6 (I) was measured by flow cytometry. The level of SOCS3 (J) was measured by western blot. Levels of mRNA or protein expression were normalized to GAPDH or ACTIN, respectively, and compared with controls.

Article Snippet: The lentivirus vector containing green fluorescent protein (GFP)-SOCS3 sequences was constructed by Genechem Company (Shanghai, China).

Techniques: Plasmid Preparation, Western Blot, Expressing, Flow Cytometry, Over Expression, Incubation, Activity Assay, Spot Test, Infection