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: EMBO Reports

Article Title: Mechanisms of axoneme and centriole elimination in Naegleria gruberi

doi: 10.1038/s44319-024-00329-w

Figure Lengend Snippet: ( A ) Schematic of reversion from flagellate to amoeba in Naegleria , highlighting the research question. Axonemal and intracellular microtubules: green; centrioles: magenta; nucleus: blue. ( B ) Fraction of cells with flagella assessed after Lugol’s iodine fixation (black), and with centrioles assessed by Centrin immunofluorescence (gray). Corresponding green and magenta dashed lines represent heat-shocked populations (heat shock from t = 95 min until t = 115 min). Three experimental replicates for each condition. For each replicate, time point and condition: N > 100 cells. Error bars represent standard deviations of the mean. Unless stated otherwise, in this and all other figures, t = 0 corresponds to the onset of transformation, whereas H.S. refers to heat shock. ( C – G ) Representative images of cells undergoing reversion after heat shock (from t = 95 min until t = 115 min). Cells were fixed at indicated times and immunostained for α/β tubulin (green), as well as Centrin (magenta); DNA is visible in blue. Arrowheads in ( C ) point to intracellular microtubules. Square marks position of inset magnified twofold on the right (Centrin channel); in ( G ), no centrioles are detected, such that a random area is magnified (indicated by a dashed box). Scale bars are 5 µm. .

Article Snippet: Naegleria gruberi (strain NEG, ATCC 30223) was grown on NM plates (1 liter: 2 g bacto-peptone (Difco), 11.1 mM glucose, 8.97 mM K 2 HPO 4 , 7.34 mM KH 2 PO 4 , 20 g bacto-agar (Difco), autoclaved).

Techniques: Immunofluorescence, Transformation Assay

Journal: EMBO Reports

Article Title: Mechanisms of axoneme and centriole elimination in Naegleria gruberi

doi: 10.1038/s44319-024-00329-w

Figure Lengend Snippet: ( A ) ClustalW alignment of human and Naegleria Centrin2. ( B ) Western blot with Centrin antibodies raised against the human protein Centrin-2 on Naegleria whole cell lysates harvested at t = 0, 70 and 150 min. Note single band at expected size appearing at t = 70 min. ( C , D ) Quantification of number ( A , N = 21 cell per time point) and brightness ( B , N = 100 tubulin spots per time point) of α/β tubulin spots per cell at t = 115 min and t = 135 min after transformation onset, as indicated. Cells were heat shocked from t = 95 min until t = 115 min. Boxes represent the interquartile range (IQR), containing the middle 50% of the data. Line represents the median, the cross the mean. Whiskers extend from the first (Q1) and third quartile (Q3) down and up to the smallest and largest value within 1.5 times the IQR below Q1 and above Q3. All data points including outliers are presented. Pairwise comparison by a two-tailed Student’s t-tests was conducted, with the resulting p values of 3.72E−10 for ( C ) and 1E−15 for ( D ). ( E ) ClustalW alignment of human and Naegleria ubiquitin. The K48 (magenta) and K63 (blue) poly-ubiquitinated residues are highlighted.

Article Snippet: Naegleria gruberi (strain NEG, ATCC 30223) was grown on NM plates (1 liter: 2 g bacto-peptone (Difco), 11.1 mM glucose, 8.97 mM K 2 HPO 4 , 7.34 mM KH 2 PO 4 , 20 g bacto-agar (Difco), autoclaved).

Techniques: Western Blot, Transformation Assay, Comparison, Two Tailed Test, Ubiquitin Proteomics

Journal: EMBO Reports

Article Title: Mechanisms of axoneme and centriole elimination in Naegleria gruberi

doi: 10.1038/s44319-024-00329-w

Figure Lengend Snippet: ( A ) Distribution of fragment length determined in the data (gray discs, binned into 0.05 μm bins; N = 98 fragments) or simulated considering random cutting (black triangles). ( B ) Fraction of axonemes in which the severed region coincides with the region of highest curvature. Two experimental replicates ( N = 74 and 52 axonemes, respectively). ( C ) Single section EM from a serial section series of an axonemal fragment in side view. Note that the fragment is ~500-nm long. Black arrowheads indicate the two ends of the fragment. Scale bar is 100 nm. ( D ) Top views of select 50 nm EM sections from 24 consecutive serial section series from an axonemal fragment, from the region shown in Fig. . Microtubule doublets are indicated by magenta arrowheads, central microtubules by green arrowhead. Note that microtubules are present only between sections 5 and 23 (covering ~900 nm), indicative of this element being larger than the average axonemal fragment. Scale bar is 100 nm. ( E ) Sequence logo diagram for residues in the nucleotide-binding site of AAA domains that was used to design a specific inhibitor for Spastin (top, from Cupido et al, ), as well as alignment of the relevant regions in human and Naegleria Spastin (bottom). Spastin Uniprot accession number: D2VS83 (Fritz-Laylin et al, ). Note that the N-terminal part of the protein was not correctly annotated and is thus missing in the accession number entry; Uniprot was contacted to correct this. Note also that mutating the “A” in “DIAGO” (highlighted in red) abolishes Spatozoline action on human Spastin (Cupido et al, ).

Article Snippet: Naegleria gruberi (strain NEG, ATCC 30223) was grown on NM plates (1 liter: 2 g bacto-peptone (Difco), 11.1 mM glucose, 8.97 mM K 2 HPO 4 , 7.34 mM KH 2 PO 4 , 20 g bacto-agar (Difco), autoclaved).

Techniques: Sequencing, Binding Assay

Journal: EMBO Reports

Article Title: Mechanisms of axoneme and centriole elimination in Naegleria gruberi

doi: 10.1038/s44319-024-00329-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Naegleria gruberi (strain NEG, ATCC 30223) was grown on NM plates (1 liter: 2 g bacto-peptone (Difco), 11.1 mM glucose, 8.97 mM K 2 HPO 4 , 7.34 mM KH 2 PO 4 , 20 g bacto-agar (Difco), autoclaved).

Techniques: Software