Journal: Anti-Cancer Drugs

Article Title: Serum heat shock protein family A member 9 protein as a biomarker for bortezomib resistance and poor prognosis in patients with multiple myeloma

doi: 10.1097/CAD.0000000000001764

Figure Lengend Snippet: Expression levels of HSPA9, DKK1, PSMD14, and TRIM21 proteins in MM patients. Serum samples were collected from 46 MM patients and 52 healthy controls, and ELISA was performed to detect the levels of HSPA9 (a), DKK1 (b), TRIM21 (c), and PSMD14 (d) proteins. Data are expressed as mean ± SD from three independent experiments. DKK1, dickkopf Wnt signaling pathway inhibitor 1; HSPA9, heat shock protein family A member 9; MM, multiple myeloma; PSMD14, proteasome 26S subunit non-ATPase 14; TRIM21, tripartite motif containing 21.

Article Snippet: The following ELISA kits were used in the study: HSPA9 (order no. D711242; Sangon Biotech, Shanghai, China), DKK1 (order no. D711029; Sangon Biotech), PSMD14 (order no. CSB-PA018904OB01HU; CUSABIO, Wuhan, China), and TRIM21 (order no. ml106271; Enzyme-linked Biotechnology, Shanghai, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Aging Cell

Article Title: Synaptic Vesicle Glycoprotein 2A Suppresses Amyloidogenesis Beyond Its Synaptic Role: A Novel Mechanism Disrupting BACE1 Binding and Altering APP Localization

doi: 10.1111/acel.70379

Figure Lengend Snippet: Amyloid deposition in SV2A‐upregulated APP/PS1 mice. (a) Thioflavin S staining of the Aβ plaques in the hippocampus and cortex of APP/PS1 mice injected with AAV‐SV2Aoe or AAV‐Con, Scale bar: 50 μm. (b) Quantification of amyloid plaques per mm 2 area, amyloid plaque size (μm 2 ), and the percentage of Aβ plaques area in brain sections, ( n = 9 brain sections, from three mice per group). (c) The fluorescence intensity of 6E10 in hippocampus of the APP/PS1 mice injected with AAV‐SV2Aoe or AAV‐Con, Scale bar: 50 μm. (d) Quantification of amyloid plaques per mm 2 area, amyloid plaque size (μm 2 ), the ratio of fluorescence intensity of 6E10 to DAPI and the percentage of 6E10 fluorescence intensity in hippocampal region, ( n = 6, from three mice per group). (e) The fluorescence intensity of 6E10 in cortex of the APP/PS1 mice injected with AAV‐SV2Aoe or AAV‐Con, Scale bar: 50 μm. (f) Quantification of amyloid plaques per mm 2 area, amyloid plaque size (μm 2 ), the ratio of fluorescence intensity of 6E10 to DAPI and the percentage of 6E10 fluorescence intensity in cortical region, ( n = 15, from three mice per group). * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: For SV2A Simoa assay, the polyclonal antibody specifically recognizing SV2A was used (CSB‐PA022978LA01HU, CUSABIO, Wuhan, China) as the capture antibody, with identifying sequence of 36–149, and another rabbit anti‐SV2A antibody (bs‐2407R, BIOSS, Beijing, China) used as the detection antibody with the recognition site of 451–550.

Techniques: Staining, Injection, Fluorescence

Journal: Aging Cell

Article Title: Synaptic Vesicle Glycoprotein 2A Suppresses Amyloidogenesis Beyond Its Synaptic Role: A Novel Mechanism Disrupting BACE1 Binding and Altering APP Localization

doi: 10.1111/acel.70379

Figure Lengend Snippet: Effect of SV2A on APP degradation in vitro and in vivo. (a–d) Levels of Aβ40, Aβ42, sAPPβ, sAPPα in the hippocampus and cortex of APP/PS1 mice injected with AAV‐SV2Aoe or AAV‐Con. (e–h) Levels of Aβ40, Aβ42, sAPPβ, sAPPα in the serum of APP/PS1 mice injected with AAV‐SV2Aoe or AAV‐Con. (i–l) Levels of Aβ40, Aβ42, sAPPβ, and sAPPα in the supernatants of primary hippocampal neuronal cells infected with SV2Aoe or control lentiviruses. (m–p) Levels of Aβ40, Aβ42, sAPPβ, and sAPPα in the supernatants of SH‐SY5Y‐APP cells transfected with SV2Aoe or control plasmid. Data are presented as the mean ± SD. All dot plots: t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For SV2A Simoa assay, the polyclonal antibody specifically recognizing SV2A was used (CSB‐PA022978LA01HU, CUSABIO, Wuhan, China) as the capture antibody, with identifying sequence of 36–149, and another rabbit anti‐SV2A antibody (bs‐2407R, BIOSS, Beijing, China) used as the detection antibody with the recognition site of 451–550.

Techniques: In Vitro, In Vivo, Injection, Infection, Control, Transfection, Plasmid Preparation

Journal: Aging Cell

Article Title: Synaptic Vesicle Glycoprotein 2A Suppresses Amyloidogenesis Beyond Its Synaptic Role: A Novel Mechanism Disrupting BACE1 Binding and Altering APP Localization

doi: 10.1111/acel.70379

Figure Lengend Snippet: Experiments of SV2A binding with APP. (a) Co‐localization of SV2A and APP in HT22 cells; scale bar: 10 μm. (b) Visualization of SV2A and APP co‐localization in HT22 cells. (c) Co‐localization of SV2A and APP in primary hippocampal neurons isolated from APP/PS1 fetal mice; scale bar: 10 μm. (d) Visualization of SV2A and APP co‐localization in primary hippocampal neurons isolated from APP/PS1 fetal mice. (e) Co‐localization of APP with SV2A in the cortical regions of 3‐month‐old APP/PS1 mice; scale bar: 10 μm. (f) Visualization of SV2A and APP co‐localization in the cortical tissues of APP/PS1 mice. (g) Co‐IP test results of SV2A and APP in brain tissue of wild‐type mice and APP/PS1 mice. Co‐localization analysis was performed by Image J by measuring fluorescence intensity alongside the drawn line. Data are presented as the mean ± SD. All dot plots: t ‐test.

Article Snippet: For SV2A Simoa assay, the polyclonal antibody specifically recognizing SV2A was used (CSB‐PA022978LA01HU, CUSABIO, Wuhan, China) as the capture antibody, with identifying sequence of 36–149, and another rabbit anti‐SV2A antibody (bs‐2407R, BIOSS, Beijing, China) used as the detection antibody with the recognition site of 451–550.

Techniques: Binding Assay, Isolation, Co-Immunoprecipitation Assay, Fluorescence

Journal: Aging Cell

Article Title: Synaptic Vesicle Glycoprotein 2A Suppresses Amyloidogenesis Beyond Its Synaptic Role: A Novel Mechanism Disrupting BACE1 Binding and Altering APP Localization

doi: 10.1111/acel.70379

Figure Lengend Snippet: Effect of SV2A overexpression on the binding of BACE1 to APP. (a) Co‐localization of BACE1 with APP in SV2A overexpressed SH‐SY5Y cells; scale bar: 10 μm. (b) Co‐localization of BACE1 with APP in SV2A‐overexpressed N2a cells; scale bar: 10 μm. (c) Quantification of BACE1 co‐localization with APP by Manders overlap coefficient (MOC) in SV2A‐overexpressed SH‐SY5Y cells. (d) Quantification of BACE1 co‐localization with APP by Manders overlap coefficient (MOC) in SV2A‐overexpressed N2a cells. (e) Schematic depiction of bimolecular fluorescence complementation assay (BiFC), wherein APP and BACE1 are tagged with complementary fragments of Venus fluorescent protein (VN and VC, respectively). The reconstitution of Venus fluorescence occurs upon the interaction of APP and BACE1. (f) Representative images of Venus fluorescence in SH‐SY5Y cells transfected with SV2Aoe or control plasmids; scale bar: 10 μm. (g) Integrated density of Venus fluorescence in SH‐SY5Y cells transfected with SV2Aoe or control plasmids. (h) Percentage of area of Venus fluorescence in SH‐SY5Y cells transfected with SV2Aoe or control plasmids. (i) Co‐localization of BACE1 and APP in the hippocampal tissues of APP/PS1 mice injected with AAV‐SV2Aoe or AAV‐Con; scale bar: 50 μm. (j, k) Quantification of BACE1 co‐localization with APP by pearson correlation coefficient (PCC) and manders overlap coefficient (MOC). (l) Co‐localization of BACE1 with APP performed by Image J by measuring fluorescence intensity alongside the drawn line. The co‐localization analysis was performed using Image J software. Data are presented as the mean ± SD. All dot plots: t ‐test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: For SV2A Simoa assay, the polyclonal antibody specifically recognizing SV2A was used (CSB‐PA022978LA01HU, CUSABIO, Wuhan, China) as the capture antibody, with identifying sequence of 36–149, and another rabbit anti‐SV2A antibody (bs‐2407R, BIOSS, Beijing, China) used as the detection antibody with the recognition site of 451–550.

Techniques: Over Expression, Binding Assay, Bimolecular Fluorescence Complementation Assay, Fluorescence, Transfection, Control, Injection, Software

Journal: Aging Cell

Article Title: Synaptic Vesicle Glycoprotein 2A Suppresses Amyloidogenesis Beyond Its Synaptic Role: A Novel Mechanism Disrupting BACE1 Binding and Altering APP Localization

doi: 10.1111/acel.70379

Figure Lengend Snippet: Effect of SV2A overexpression on the distribution of APP. (a, b) Expressions of EEA1, Rab7, Rab11, and LAMP1 in SV2A‐overexpressed SH‐SY5Y cells (N2a cells) and their control cells, as well as the relative expression analysis. (c, d) Fluorescence intensity of EEA1 and APP in SV2A‐overexpressed SH‐SY5Y cells (N2a cells) and the co‐localization analysis of EEA1 and APP. (e, f) Fluorescence intensities of Rab7 and APP in SV2A‐overexpressed SH‐SY5Y cells (N2a cells) and the co‐localization analysis of Rab7 and APP. (g, h) Fluorescence intensity of Rab11 and APP in SV2A‐overexpressed SH‐SY5Y cells (N2a cells) and the co‐localization analysis of Rab11 and APP. (i–j) Fluorescence intensities of LAMP1 and APP in SV2A‐overexpressed SH‐SY5Y cells (N2a cells) and the co‐localization analysis of LAMP1 and APP. (k) Quantification of surface APP expression in SH‐SY5Y cells transfected with SV2Aoe or control plasmids. (l) Quantification of surface APP fluorescence intensity (APP/DAPI) in SH‐SY5Y cells transfected with SV2Aoe or control plasmids, scale bar: 10 μm. (m) Fluorescence level of APP on the cell surface in hippocampal regions of APP/PS1 mice injected with AAV‐SV2A or AAV‐Con; scale bar: 50 μm. (n) Relative fluorescence intensity of APP (APP/DAPI) on the cell surface in the hippocampal regions of APP/PS1 mice injected with AAV‐SV2A or AAV‐Con. (o) Fluorescence level of APP on the cell surface in cortical regions of APP/PS1 mice injected with AAV‐SV2A or AAV‐Con; scale bar: 50 μm. (p) Relative fluorescence intensity of APP (APP/DAPI) on the cell surface in the cortical regions of APP/PS1 mice injected with AAV‐SV2A or AAV‐Con. The protein levels of APP on the cell surface were measured by the cell surface biotin assay. Data are presented as the mean ± SD. All dot plots: t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For SV2A Simoa assay, the polyclonal antibody specifically recognizing SV2A was used (CSB‐PA022978LA01HU, CUSABIO, Wuhan, China) as the capture antibody, with identifying sequence of 36–149, and another rabbit anti‐SV2A antibody (bs‐2407R, BIOSS, Beijing, China) used as the detection antibody with the recognition site of 451–550.

Techniques: Over Expression, Control, Expressing, Fluorescence, Transfection, Injection

Journal: Aging Cell

Article Title: Synaptic Vesicle Glycoprotein 2A Suppresses Amyloidogenesis Beyond Its Synaptic Role: A Novel Mechanism Disrupting BACE1 Binding and Altering APP Localization

doi: 10.1111/acel.70379

Figure Lengend Snippet: The diagram on the mechanism by which SV2A reduces amyloid plaque deposition. The molecular mechanism by which SV2A affects Aβ deposition is as follows: (1) SV2A overexpression reduces the localization of APP in early endosomes, late endosomes, and lysosomes, while increasing its localization in recycling endosomes and the plasma membrane; (2) as a binding per of APP, SV2A overexpression reduces the level of BACE1‐APP interaction. Through these mechanisms, SV2A overexpression inhibits the amyloidogenic pathway of APP, consequently leading to a reduction in Aβ protein.

Article Snippet: For SV2A Simoa assay, the polyclonal antibody specifically recognizing SV2A was used (CSB‐PA022978LA01HU, CUSABIO, Wuhan, China) as the capture antibody, with identifying sequence of 36–149, and another rabbit anti‐SV2A antibody (bs‐2407R, BIOSS, Beijing, China) used as the detection antibody with the recognition site of 451–550.

Techniques: Over Expression, Clinical Proteomics, Membrane, Binding Assay