bst2 expression  (Proteintech)

Journal: Cancer Gene Therapy

Article Title: Serum small extracellular vesicles-derived BST2 as a biomarker for papillary thyroid microcarcinoma promotes lymph node metastasis

doi: 10.1038/s41417-024-00854-9

Figure Lengend Snippet: A Venn diagram of differentially expressed proteins from different comparisons. B BST2 levels according to proteomic analysis ( n = 10 for NLNM-sEVs and LNM-sEVs; *BN-sEVs vs. other groups, * P < 0.05, **** P < 0.0001; # NLNM-sEVs vs. LNM-sEVs, ## P < 0.01). C Western blot analysis showed BST2 levels in serum sEVs ( n = 3 for BN-sEVs, NLNM-sEVs, and LNM-sEVs, *BN-sEVs vs. other groups, ** P < 0.01, *** P < 0.001; # NLNM-sEVs vs. LNM-sEVs, # P < 0.05); Flotillin-1 as sEVs protein marker was used as the control. D ELISA results revealed BST2 protein concentrations in serum sEVs of the validation cohort (** P < 0.01, **** P < 0.0001). E ROC curve analysis of the sensitivity, specificity, and AUC of serum sEVs BST2 level in the validation cohort. The T refers to the whole group of PTMC samples (LNM and NLNM). Error bars represent standard deviations.

Article Snippet: A BST2 polyclonal antibody (1:200, Proteintech, 13560-1-AP) was used to visualize BST2 expression via an IHC assay.

Techniques: Western Blot, Marker, Control, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Cancer Gene Therapy

Article Title: Serum small extracellular vesicles-derived BST2 as a biomarker for papillary thyroid microcarcinoma promotes lymph node metastasis

doi: 10.1038/s41417-024-00854-9

Figure Lengend Snippet: A The proliferation rate of BCPAP cells was downregulated after the knockdown of BST2 (* P < 0.05, ** P < 0.01). B The cell migration of BCPAP cells was downregulated after the knockdown of BST2 (*** P < 0.001). NC: negative control. C The proliferation rate of BCPAP cells was upregulated after overexpression of BST2 (** P < 0.01, *** P < 0.001, **** P < 0.0001). D The cell migration of BCPAP cells was upregulated after overexpression of BST2 (**** P < 0.0001). E , F The morphology and size of BCPAP-derived sEVs were determined by TEM and NTA; scale bar: 100 nm. G SEVs markers CD81, CD63, ALIX, Calnexin, and sEVs BST2 levels were measured using western blot. H Lymph tube formation response to OE-BST2 sEVs is determined (*** P < 0.001). I–J The cell migration and proliferation of HLEC cells in response to OE-BST2 sEVs are shown (* P < 0.05, ** P < 0.01, **** P < 0.0001). Error bars represent standard deviations.

Article Snippet: A BST2 polyclonal antibody (1:200, Proteintech, 13560-1-AP) was used to visualize BST2 expression via an IHC assay.

Techniques: Knockdown, Migration, Negative Control, Over Expression, Derivative Assay, Western Blot

Journal: Cancer Gene Therapy

Article Title: Serum small extracellular vesicles-derived BST2 as a biomarker for papillary thyroid microcarcinoma promotes lymph node metastasis

doi: 10.1038/s41417-024-00854-9

Figure Lengend Snippet: A Representative gross anatomical image of the lower limb of a nude mouse with footpad tumor (left arrow) and metastatic popliteal lymph node (right arrow). B Images of the popliteal lymph nodes in control-sEVs-BCPAP or BST2-sEVs-BCPAP groups ( n = 12). C Statistical comparison of the volume of popliteal lymph nodes in each group ( n = 12, ** P < 0.01). D Representative images of HE staining and BST2 IHC staining for popliteal lymph nodes in each group with different magnifications. Scale bars: 100 μm for the 10 × magnified images and 50 μm for the 20 × magnified images. E The IHC staining was assessed as the sum of the percentage and intensity scores (* P < 0.05). Error bars represent standard deviations.

Article Snippet: A BST2 polyclonal antibody (1:200, Proteintech, 13560-1-AP) was used to visualize BST2 expression via an IHC assay.

Techniques: Control, Comparison, Staining, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: ITIM motif of the cytoplasmic tail domain in BST2.

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Sequencing, Residue

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: PolyI:C stimulated Bst2 -/- NK cells have a higher cytotoxicity to tumor cells than Bst2 +/+ NK cells. B6-origin Bst2 wildtype (+/+) and Bst2 knockout (-/-) mice were intraperitoneally injected with polyI:C. At 16 hrs after injections, splenic NK cells were isolated using DX-5 microbeads. ( A ) Bst2 +/+ and Bst2 -/- NK cells were stained with anti-BST2 antibody and analyzed by flow cytometry. NK1.1 + TCRβ - cells were indicated as NK cells. Data are representative of two independent experiments. ( B ) Calcein-AM stained target cells (YAC-1 and RMA-S) were co-cultured with Bst2 +/+ and Bst2 -/- NK cells for 4 hrs at 37 °C in a humidified incubator. Released calcein was measured by SpectraMAX with excitation/emission wavelengths at 485 nm/535 nm. Graphs showed a mean ± S.D. ( n = 3 per group). Data are representative of three independent experiments. (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Knock-Out, Injection, Isolation, Staining, Flow Cytometry, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: Bst2 -/- lymphokine activated killer cells have higher cytotoxicity to tumor cells than Bst2 +/+ LAK cells. Naïve splenocytes of B6-origin Bst2 +/+ and Bst2 -/- mice were stimulated with IL-2 for seven days to generate LAK cells. ( A ) Bst2 +/+ and Bst2 -/- LAK cells were stained with anti-BST2 antibody and analyzed by flow cytometry. ( B ) Calcein-AM stained target cells (YAC-1) were co-cultured with Bst2 +/+ and Bst2 -/- LAK cells for 1 hr at 37 °C in a humidified incubator. Released calcein was measured to analyze cytotoxicity of effector cells as described in . Graphs showed a mean ± S.D. ( n = 3–4 per group). Data are representative of three independent experiments. ( C ) After detecting calcein released in media, released granzyme B in the same media was measured by enzyme-linked immunosorbent assay (ELISA). Graphs showed a mean ± S.D. ( n = 3–4 per group). Data are representative of two independent experiments. ( D ) Bst2 +/+ and Bst2 -/- LAK cells were co-cultured with target cells (YAC-1) in culture media containing Golgistop and anti-CD107a antibody. NK1.1 and TCRβ were stained after 1 hr of cultivation at 37 °C in a humidified incubator. Percentages of CD107a positive cells from LAK cells (NK1.1 + TCRβ - ) were indicated. Graphs showed a mean ± S.D. ( n = 3 per group). Data are representative of two independent experiments. ( E ) CFSE stained Bst2 +/+ LAK cells were treated with isotype control or anti-BST2 antibodies and co-cultured with target cells (RMA-S and B16) for 4 hrs at 37 °C in a humidified incubator. Cells were stained with 7-AAD after cultivation. These cells were then fixed with 1% paraformaldehyde and analyzed by flow cytometry. CFSE - , 7-AAD + population is designated as lysed target cells. The word ‘iso’ indicates isotype control antibody treatment and ‘αBST2′ indicates anti-BST2 antibody treatment. Graphs showed a mean ± S.D. ( n = 3–5 per group). (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Staining, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: Galectin-9 is associated with BST2-dependent NK cell activity. ( A ) Recombinant BST2 protein was pulled down with GST-tagged Gal-3 (GST-Gal3), as well as the GST-tagged long isoform of Gal-9 (GST-Gal9L). Precipitates were then analyzed by western blotting with anti-BST-2 antibody. ( B ) Recombinant BST2 protein was pulled down with either GST-tagged NCRD or CCRD of Gal9L (GST-Gal9L-NCRD or GST-Gal9L-CCRD). Precipitates were then analyzed by western blotting with anti-BST-2 antibody. ( C ) Naïve splenocytes of B6-origin Bst2 +/+ and Bst2 -/- mice were stimulated by IL-2 for seven days to generate LAK cells. CFSE stained Bst2 +/+ and Bst2 -/- LAK cells were co-cultured with target cells (B16) for 4 hrs at 37 °C in a humidified incubator in the presence of isotype control antibody or anti-Gal-9 antibody. Cells were stained with 7-AAD after cultivation. These cells were then fixed with 1% paraformaldehyde and analyzed by flow cytometry. CFSE - , 7-AAD + population is designated as lysed target cells. Graphs showed a mean ± S.D. ( n = 2–4 per group). ( D ) Naïve splenocytes of B6-origin Bst2 +/+ and Bst2 -/- mice were stimulated by IL-2 for seven days to generate LAK cells. Calcein-AM stained target cells (Wild type (WT) YAC-1 and Lgals9 -/- YAC-1) were co-cultured with Bst2 +/+ and Bst2 -/- LAK cells for 3 hrs in a 96-well round plate. Released calcein was measured to analyze cytotoxicity of effector cells as described in . Graphs showed a mean ± S.D. ( n = 4–6 per group). Data are representative of two independent experiments. ( E ) Western blotting was performed on whole cell lysates of tumor target cell lines with anti-galectin-9 antibody and anti-GAPDH antibody. GAPDH was used as a loading control. The upper panel showed the result of wildtype tumor cell lines (RMA-S, B16, and YAC-1) and the lower panel showed the comparison of wildtype YAC-1 and Lgals9 -/- YAC-1 clone used in this study. (*, p < 0.05; **, p < 0.01; ****, p < 0.0001; n.s., not significant).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Activity Assay, Recombinant, Western Blot, Staining, Cell Culture, Control, Flow Cytometry, Comparison

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: Long isoform, but not short isoform, of BST2 on NK cells impedes cytotoxicity. ( A ) F1 hybrids were generated by crossing B6-origin Bst2 +/- mice and NZW mice ( Bst2 S/S ). ( B ) Naïve splenocytes of F1 hybrids and B6-origin Bst2 -/- mice were stimulated by IL-2 for seven days to generate LAK cells. Expression of BST2 was detected through flow cytometry staining. ( C ) CFSE stained Bst2 S/+ and Bst2 S/- LAK cells were co-cultured with target cells (YAC-1, RMA-S, and B16) for 4 hrs at 37 °C in a humidified incubator. Cells were stained with 7-AAD after cultivation. These cells were then fixed with 1% paraformaldehyde and analyzed by FACS. CFSE-, 7-AAD+ population is designated as lysed target cells. Graphs showed a mean ± S.D. ( n = 4 per group). ( D ) CFSE stained Bst2 S/+ and Bst2 S/- LAK cells from F1 hybrids were co-cultured with target cells (RMA-S) cells for 4 hrs at 37 °C in a humidified incubator in absence or presence of anti-BST2 antibody. Graphs showed mean ± S.D. ( n = 3–4 per group). (**, p < 0.01; ****, p < 0.0001; n.s., not significant).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Generated, Expressing, Flow Cytometry, Staining, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: NK cells with short isoform of BST2 show enhanced cytotoxicity as well as BST2 deficient NK cells. ( A ) Backcross from NZW ( Bst2 S/S ) background to B6 background. ( B ) BST2 expression of LAK cells generated from splenocytes of B6-background Bst2 +/- , Bst2 S/- , and Bst2 -/- mice. ( C ) Calcein-AM stained target cells (YAC-1, RMA-S, and B16) were co-cultured with Bst2 +/- , Bst2 S/- , and Bst2 -/- LAK cells for 4 hrs at 37 °C in a humidified incubator. Released calcein was measured to analyze cytotoxicity of effector cells as described in . Data are representative of two independent experiments. Graphs showed mean ± S.D. ( n = 4 per group). ( D ) Naïve splenocytes of B6-origin Bst2 +/+ , Bst2 S/- , and Bst2 -/- mice were stimulated by IL-2 for seven days to generate LAK cells. Expression level of BST2 was measured through flow cytometry staining. ( E ) Calcein-AM stained target cells (YAC-1 and Lgals9 -/- YAC-1) were co-cultured with Bst2 +/+ , Bst2 S/- , and Bst2 -/- LAK cells for 4 hrs at 37 °C in a humidified incubator. Released calcein was measured to analyze cytotoxicity of effector cells as described in . Graphs showed a mean ± S.D. ( n = 6 per group). (*, p < 0.05; **, p < 0.01; n.s., not significant).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Expressing, Generated, Staining, Cell Culture, Flow Cytometry

Journal: Virology

Article Title: Japanese encephalitis virus counteracts BST2 restriction via its envelope protein E.

doi: 10.1016/j.virol.2017.07.008

Figure Lengend Snippet: Fig. 1. Overexpression of BST2 decreases the release of JEV progeny virions. (A) 293T cells were transfected with pBST2 or pcDNA3.1, harvested at different time points, lysed and the total expression of BST2 was detected by western blot. (B and C) 293T cells were transfected with pBST2 or pcDNA3.1. At 6 h post transfection, cells were infected with JEV at an MOI of 10. The titers of supernatant virus (SV) or cell-associated virus (CV) were determined by plaque assay at the indicated time points post infection. Data shown are mean ± SD of three independent experiments with each condition performed in triplicate. Compared to pcDNA3.1, *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: BST2 expressing plasmid (pBST2) was from Origene.

Techniques: Over Expression, Transfection, Expressing, Western Blot, Infection, Virus, Plaque Assay

Journal: Virology

Article Title: Japanese encephalitis virus counteracts BST2 restriction via its envelope protein E.

doi: 10.1016/j.virol.2017.07.008

Figure Lengend Snippet: Fig. 2. Downregulation of endogenous BST2 enhances the release of JEV progeny virions. (A) HeLa cells were transfected with BST2-specific shRNA or control shRNA. At 48 h post infection, cells were harvested and lysed for western blot analysis. (B and C) HeLa cells transfected with BST2 shRNA or non-targeting shRNA were collected at different time points and analyzed by western blot (B) and flow cytometry (C). (D and E) HeLa cells were infected with JEV at an MOI of 25 following 6 h transfection with BST2-specific shRNA or control shRNA, and the titers of supernatant virus (SV) or cell-associated virus (CV) were determined by plaque assay at the indicated time points. (F) HeLa cells were infected with JEV at an MOI of 25 following 6 h transfection with BST2 shRNA or control shRNA. 293T cells were transfected with pBST2 or pcDNA3.1. The levels of protein E in SV or CV were determined by western blot assay at 24 h post infection. Data shown are mean ± SD of three independent experiments with each condition performed in triplicate. Compared to control shRNA, *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: BST2 expressing plasmid (pBST2) was from Origene.

Techniques: Transfection, shRNA, Control, Infection, Western Blot, Cytometry, Virus, Plaque Assay

Journal: Virology

Article Title: Japanese encephalitis virus counteracts BST2 restriction via its envelope protein E.

doi: 10.1016/j.virol.2017.07.008

Figure Lengend Snippet: Fig. 3. JEV infection downregulates the expression of BST2. (A) HeLa cells were mock-infected or infected with JEV at an MOI of 25. At 48 h post infection, cells were fixed and probed with PE-conjugated anti-BST2 antibody. Cells were then analyzed by flow cytometry. The bar graph is mean ± SD of MFI from three independent experiments with one representative histogram being shown. (B) At 48 or 72 h post infection, HeLa cells infected or uninfected with JEV were collected and analyzed by western blot. (C) 293T cells were transfected with plasmid expressing BST2 or empty plasmid. At 6 h post transfection, cells were infected with JEV, collected at 24 h post infection and stained with anti-BST2 antibody for flow cytometry analysis. The bar graph is mean ± SD of MFI from three independent experiments with one representative histogram being shown. (D) At 24 or 48 h post infection, 293T cells infected or uninfected with JEV were collected and analyzed by western blot. One representative experiment out of three is shown. (E) HeLa cells were transfected with control shRNA or BST2 specific shRNA, infected with JEV at an MOI of 25. 293T cells were transfected with empty plasmid or plasmid expressing BST2, infected with JEV at an MOI of 10. The total RNA of the samples was extracted, and the BST2 mRNA was determined by RT-PCR. The mRNA level of GAPDH was scored in parallel and used as an internal control. Data shown are mean ± SD of three independent experiments.

Article Snippet: BST2 expressing plasmid (pBST2) was from Origene.

Techniques: Infection, Expressing, Cytometry, Western Blot, Transfection, Plasmid Preparation, Staining, Control, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: Virology

Article Title: Japanese encephalitis virus counteracts BST2 restriction via its envelope protein E.

doi: 10.1016/j.virol.2017.07.008

Figure Lengend Snippet: Fig. 4. JEV envelope protein E antagonizes BST2. (A) HeLa cells were transfected with plasmid expressing the structural or non-structural proteins of JEV. The surface expression of BST2 was analyzed by flow cytometry (left), while the total expression of JEV proteins was analyzed by western blot (right). (B) HeLa cells were transfected with plasmid expressing HIV- 1 Vpu, JEV envelope protein E and control plasmid pcDNA3.1(+), and the cell surface expression of BST2 was analyzed by flow cytometry. The bar graph is mean ± SD of MFI from three independent experiments with one representative histogram being shown. (C) The total expression level of BST2 in the parallel samples of (B) was analyzed by western blot. (D) The cell surface expression level of BST2 on 293T cells transfected with plasmid expressing BST2 alone or cotransfected with plasmids expressing BST2 and JEV protein E or HIV-1 Vpu. The bar graph is mean ± SD of MFI from three independent experiments with one representative histogram being shown. (E) The total expression level of BST2 in the parallel samples of (D) was analyzed by western blot. (F) Examination of protein E-mediated BST2 degradation. HeLa cells were transfected with pcDNA3.1 or plasmid expressing gE-flag. The prepared cell extracts were ultracentrifuged by density gradient centrifugation and the lysosome band was located in the top 2 mL of the gradient. The corresponding bands were collected and the finally harvested lysosome pellets were detected by western blot. One representative experiment out of three is shown.

Article Snippet: BST2 expressing plasmid (pBST2) was from Origene.

Techniques: Transfection, Plasmid Preparation, Expressing, Cytometry, Western Blot, Control, Gradient Centrifugation

Journal: Virology

Article Title: Japanese encephalitis virus counteracts BST2 restriction via its envelope protein E.

doi: 10.1016/j.virol.2017.07.008

Figure Lengend Snippet: Fig. 5. JEV protein E physically interacts with BST2. 293T cells were cotransfected with pBST2 and plasmid expressing E-flag, ME-flag or M-HA. At 48 h post transfection, cell lysates were analyzed by co-IP. Co-IP was pulled down using the anti-BST2, anti-flag or anti-HA antibody. Proteins were immunoprecipitated with the anti-flag (A) or anti-BST2 antibody (B) as indicated. (C) Proteins were immunoprecipitated with the anti-HA or anti-BST2 antibody as indicated. (D) HeLa cells were infected with JEV at a MOI of 25. At 48 h post infection, cell lysates were analyzed by co-IP. Co-IP was pulled down using the anti-BST2 antibody. One representative experiment out of three is shown. (E) Colocalization of BST2 with JEV E-flag or ME-flag. 293T cells cotransfected with pBST2 and plasmid expressing E-flag or ME-flag were costained with anti-flag (red) and anti-BST2 (green) antibodies. Nuclei were counterstained with DAPI (blue). Representative confocal images from three independent experiments are shown. Scale bars in all panels represent 10 µm. (F) HeLa cells were transfected with plasmid expressing protein E. The surface expression of BST2 was analyzed by flow cytometry.

Article Snippet: BST2 expressing plasmid (pBST2) was from Origene.

Techniques: Plasmid Preparation, Expressing, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Infection, Cytometry

Journal: Virology

Article Title: Japanese encephalitis virus counteracts BST2 restriction via its envelope protein E.

doi: 10.1016/j.virol.2017.07.008

Figure Lengend Snippet: Fig. 6. Mapping of BST2 domains essential for its anti-JEV activity. (A) Schematic representation of the BST2 mutants. (B) 293T cells were cotransfected with pME-flag and plasmid expressing wild type BST2 or BST2 mutants. At 48 h post transfection, cells were collected and lysed. The expression of BST2 mutants in 293T cells was confirmed by Western blot. (C) and (D) The interaction between protein E and BST2 mutants in the parallel samples of (B) was analyzed by Co-IP. (E) 293T cells were transfected with pcDNA3.1 or wild type BST2 or BST2 mutants. At 6 h post transfection, cells were infected with JEV at an MOI of 10 and the relative viral production of JEV was assessed by viral release assay. One representative experiment out of three is shown.

Article Snippet: BST2 expressing plasmid (pBST2) was from Origene.

Techniques: Activity Assay, Plasmid Preparation, Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Infection, Release Assay