Journal: Science Advances

Article Title: Trisomy 21–induced dysregulation of microglial homeostasis in Alzheimer’s brains is mediated by USP25

doi: 10.1126/sciadv.abe1340

Figure Lengend Snippet: ( A and B ) Quantitative proteomic analysis of the mouse cerebrum. (A) Proteomic analysis of the mouse cerebrum and a volcano plot illustrating down-regulated (blue) and up-regulated (red) proteins in the cerebra of 6-month-old 5×FAD; Usp25 −/− versus 5×FAD mice (fold change > 1.1, P < 0.05). n = 3 samples per group; each sample is a pool of three cerebra from the same genotype. (B) GO analysis of down-regulated proteins in 5×FAD; Usp25 −/− mice relative to 5×FAD mice. ( C ) Primary microglia from WT mice were transfected with siAtp6v0c or control siRNA (siNC) for 48 hours and subsequently treated with 10 μM oAβ 42 or vehicle control along with pHrodo Red–labeled synaptosomes (Syn) for another 24 hours, and the phagocytosis abilities of microglia were quantified on the basis of the pHrodo Red intensity in Iba1 + cells. Scale bar, 25 μm. n = 3 independent experiments; n = 207 to 211 cells per group were counted. ( D and E ) Coimmunoprecipitation (IP) between exogenously expressed USP25a-myc and HA-ATP6V0C (D) or HA-WDFY1 (E) proteins. ( F and G ) Immunoblot (IB) analysis of polyubiquitinated HA-ATP6V0C (F) and HA-WDFY1 (G) in HEK293T cells upon USP25 knockdown. n = 6. All data represent means ± SEM. P values were determined by the Kruskal-Wallis test with Dunn’s post hoc analysis in (C) and by the Mann-Whitney test in (F) and (G). * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: HEK293T cells transfected with siUSP25 or control siRNA were incubated with lysosomal inhibitor leupeptin (100 μg ml −1 ) (MCE, Monmouth Junction, NJ, USA; HY-18234A) or 10 μM proteasomal inhibitor MG132 (MCE, HY-13259).

Techniques: Transfection, Control, Labeling, Western Blot, Knockdown, MANN-WHITNEY

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 1. Exogenous PCSK9 can induce degradation of the LDLR in the absence of APLP2. HepG2 (A) and Huh7 (B) cells were transfected with a control non-target siRNA (Ctrl) or 3 different siRNAs targeting APLP2. Cells wereincubatedovernightwithserum-freeconditionedmedialackingorcon- taining 1 g/ml of PCSK9-V5. HepG2 and Huh7 cell lysates were then sub- jected to Western blotting using LDLR, APLP2, and -actin antibodies. LDLR and APLP2 signals were normalized to that of -actin. C, the input HEK293 conditioned medium was analyzed using mAb-V5 to detect PCSK9-V5. D, duplicate samples of Huh7 cells matching those in panel B were analyzed by FACS to assess the cell surface LDLR levels. Values were normalized to that of the first lane (control non-target siRNA in the absence of PCSK9). Error bars represent S.E. *, p 0.05 (Student’s t test). The data shown here are represen- tative of two to three independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Transfection, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 4. Sortilin and APLP2 are novel cellular targets of PCSK9. A, overexpressed PCSK9 induces sortilin and APLP2 degradation in HEK293 cells. Triplicate Western blot analyses revealing that both sortilin-Myc and APLP2-V5 expression levels in HEK293 cells were reduced by 90 and 40%, respectively, upon transfection with a PCSK9 plasmid, as compared with a control empty pIRES vector (V). Quantification of sortilin and APLP2 band intensities were normalized against those of -actin. B, HEK293 cells transfected with a cDNA coding for an empty vector control (pIRES; V) or individually with human sortilin or APLP2 tagged at the C terminus with a Myc or V5 epitope, respectively, or together in the absence or presence of a cDNA coding for untagged PCSK9. The following day the cells were washed and then pulsed for 4 h with [35S]Met Cys in the presence or absence of 5 mM NH4Cl. The cells were then extracted and their lysates immunoprecipitated (IP) with a mAb-V5 or mAb-Myc or a polyclonal antibody for PCSK9. The precipitates were separated on an 8% SDS-PAGE. The dried gel was then autoradiographed. Notice the co-precipitation of sortilin and APLP2 in the presence of NH4Cl. These data are representative of at least three independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Immunoprecipitation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 5. Sortilin, APLP2, and soluble APLP2 are degraded by both PCSK9 and ER-localized PCSK9-KDEL isoforms. HEK293 cells were transfected with indicated DNA amounts of vectors encoding a control protein 7B2, sortilin (no tag), APLP2-V5, soluble APLP2-V5 (sAPLP2-V5), PCSK9-V5, or PCSK9-V5-KDEL, as indicated. After 48 h, lysates and media were analyzed by Western blotting for the indicated proteins. The data show that overexpressed PCSK9 or PCSK9-KDEL induces degradation of sortilin (A), APLP2 (B), and sAPLP2 (C) in HEK293 cells. Quantification of sortilin and APLP2 band intensities were normalized against those of -actin or GAPDH. These data are representative of two independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Transfection, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 6. Co-expression of sortilin, APLP2, or both with PCSK9 has no major effect on LDLR degradation. Huh7 cells were transfected with a total of 3 g using 1 g of each vector encoding for either a control protein 7B2 (), sortilin (), APLP2 (), or PCSK9 (), as indicated. After 48 h, lysates were analyzed by Western blotting for expression of the LDLR, sortilin-Myc, APLP2- V5, intracellular pro- and mature-PCSK9-V5, and -actin. Media were ana- lyzed for secreted endogenous and overexpressed PCSK9-V5 using a rabbit polyclonal human PCSK9 antibody. Quantification of LDLR expression was normalized against that of -actin. These data are representative of at least 3 different experiments showing similar results.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 7. Sortilin binds APLP2. A, schematic diagram of sortilin and APLP2 fused to the G. princeps luciferase half-domains, Gluc-1 and Gluc-2, respectively. B, heat map generated by G. princeps luciferase complementation assay showing the interaction profile of 36 protein pairs. Normalized luminescence ratio ranging from strong to null interactions is displayed on a light blue to black scale. C, validation of sortilin-Myc and APLP2-V5 interaction was confirmed by co-expression in HEK293 cells and immunoprecipitation with mAb-Myc followed by Western blotting (WB) using a mAb-Myc or mAb-V5.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Luciferase, Generated, Biomarker Discovery, Expressing, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Cell-derived ECM loaded electrospun Polycaprolactone/Chitosan nanofibrous scaffolds for periodontal regeneration

doi: 10.1101/2023.03.30.534964

Figure Lengend Snippet: Characterization of decellularized extracellular matrix (dECM) derived from human periodontal ligament stem cells (PDLSCs). A) Immunofluorescence staining images of fibronectin (FIB, green), collagen I (COL I, red), laminin (LAM, red), asporin (ASP, red), osteopontin (OPN, red) and osteocalcin (OC, red) before and after decellularization DAPI staining (blue) revealed the absence of nuclei after decellularization. Scale bar 100 μm. B) DNA content present in PDLSCs and dECM. C) Quantification of sulphated glycosaminoglycans (sGAGs) in PDLSCs and dECM. D) Content of collagen present in PDLSCs and dECM. Three different samples were used in each quantification assay (N=3) for both conditions; *** p < 0.001.

Article Snippet: The primary antibodies, including rabbit polyclonal collagen I (1:200, Abcam), rabbit polyclonal asporin (1:100, Thermo Fisher Scientific), mouse monoclonal osteopontin (1:100, Thermo Fisher Scientific), mouse monoclonal osteocalcin (1:50, R&D Systems), rabbit polyclonal periostin (1:100, Abcam) and rabbit polyclonal cementum protein 1 (1:100, Thermo Fisher Scientific) prepared in a solution of 0.3% Triton X-100, 1% BSA, 10% FBS (in PBS) were added into the samples and incubated overnight at 4°C.

Techniques: Derivative Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Cell-derived ECM loaded electrospun Polycaprolactone/Chitosan nanofibrous scaffolds for periodontal regeneration

doi: 10.1101/2023.03.30.534964

Figure Lengend Snippet: Immunofluorescent staining images of collagen I (COL I, red), asporin (ASP, red), osteopontin (OPN, red), osteocalcin (OC, red), periostin (POSTN, red) and cementum protein 1 (CMP, red) expressed by PDLSCs cultured on PCL, PCL-CTS and PCL-CTS-ECM electrospun scaffolds for 21 days under osteogenic differentiation conditions. Nuclei were counterstained with DAPI (blue). Scale bar 100 μm.

Article Snippet: The primary antibodies, including rabbit polyclonal collagen I (1:200, Abcam), rabbit polyclonal asporin (1:100, Thermo Fisher Scientific), mouse monoclonal osteopontin (1:100, Thermo Fisher Scientific), mouse monoclonal osteocalcin (1:50, R&D Systems), rabbit polyclonal periostin (1:100, Abcam) and rabbit polyclonal cementum protein 1 (1:100, Thermo Fisher Scientific) prepared in a solution of 0.3% Triton X-100, 1% BSA, 10% FBS (in PBS) were added into the samples and incubated overnight at 4°C.

Techniques: Staining, Cell Culture

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 1 Coadministration of CD3/CD28 and CpG induce robust amounts of IL-10 expression in splenocytes when compared to either signal alone. (A) Splenocytes were treated with CpG, CD3/CD28, CD3/CD28/CpG, or left untreated for 72 hours and analyzed for IL0 production by ELISA. (B) Splenocytes were treated with CD3/CD28/CpG for 0, 48, or 72 hours, and IL-10 levels were measured by ELISA. (C) Supernatants of splenocytes treated with either CpG or control ODN (ctrl CpG) in the absence or presence of CD3/CD28 for 72 hours were analyzed for IL-10 production by ELISA. (D) Splenocytes were treated with CpG in the presence or absence of various T cell-activating antibodies (CD28, CD3, or CD3/CD28) for 72 hours, and IL-10 expression in the supernatants was measured via ELISA. (E) Supernatants from splenocytes treated as indicated for 72 hours were analyzed for IL-10 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control