Journal: Human Molecular Genetics

Article Title: Frontotemporal dementia non-sense mutation of progranulin rescued by aminoglycosides

doi: 10.1093/hmg/ddz280

Figure Lengend Snippet: G418 and gentamicin induced readthrough of progranulin PTC mutation R493X. (A) Schematic diagram of WT progranulin and PTC mutations examined in this study. Each construct has a C-terminal FLAG tag. (B) Examination of potential readthrough effect of 11 aminoglycosides and PTC124 on R493X progranulin. N2A cells were transfected with WT or R493X progranulin, allowed to recover and treated with two different concentrations of each compound for 24 h. Cell lysates were generated, separated by SDS-PAGE and analyzed by western analysis using anti-FLAG (top), anti-progranulin (middle) and anti-actin (lower) antibodies. Among 12 compounds tested, only G418 and gentamicin induced FLAG bands, demonstrating positive readthrough effect. WT progranulin was included as a positive control. No transfection or no compound treatment was included as negative controls. For the anti-progranulin blot, the lower band is the R493X truncated protein and the higher band is full-length progranulin. (C) G418 had no readthrough effect on two other FTD mutations Q125X and Y229X. No full-length readthrough protein was observed in the FLAG blot. In the anti-progranulin blot, the higher band is WT progranulin and the lower band is the Q229Y truncated protein. The Q125X truncated protein was visible with longer exposure. (D) Examination of G418 on the R495X mutation of FUS responsible for familial ALS. WT or R495X FUS was tagged with FLAG at the N-terminus. N2A cells were transfected with WT or R495X FUS, allowed to recover and treated with three concentrations of G418 for 24 h. Cells were harvested and cell lysates were subjected to SDS-PAGE and western analysis using anti-FLAG (top) and anti-actin (lower) antibodies. The slightly higher band is WT FUS and the lower band is R495X truncated FUS. G418 did not induce readthrough of R495X FUS.

Article Snippet: Plasmids The WT progranulin plasmid with a C-terminal FLAG tag (pC-Flag-PGRN-WT) was purchased from Sino Biological Inc. (Cat.HG10826-CF).

Techniques: Mutagenesis, Construct, FLAG-tag, Transfection, Generated, SDS Page, Western Blot, Positive Control

Journal: Human Molecular Genetics

Article Title: Frontotemporal dementia non-sense mutation of progranulin rescued by aminoglycosides

doi: 10.1093/hmg/ddz280

Figure Lengend Snippet: G418 and gentamicin induced dose-dependent R493X progranulin readthrough. (A) Gentamicin and G418 induced readthrough of progranulin R493X in a dose-dependent manner. N2A cells were transfected with WT or R493X progranulin, allowed to recover and treated with increasing concentrations of G418 or gentamicin for 24 h. Cell lysates were generated, separated by SDS-PAGE and analyzed by western analysis using anti-FLAG (top), anti-progranulin (middle) and anti-actin (lower) antibodies. For the anti-progranulin blot, the lower band is the R493X truncated protein and the higher band is full-length progranulin. (B) Band intensities were quantified to determine the dose response of readthrough efficiency. All FLAG bands were normalized against corresponding actin bands and the individual readthrough band was subsequently compared to the WT progranulin (the last lane in A). *P < 0.1, **P < 0.01, ***P < 0.001.

Article Snippet: Plasmids The WT progranulin plasmid with a C-terminal FLAG tag (pC-Flag-PGRN-WT) was purchased from Sino Biological Inc. (Cat.HG10826-CF).

Techniques: Transfection, Generated, SDS Page, Western Blot

Journal: Human Molecular Genetics

Article Title: Frontotemporal dementia non-sense mutation of progranulin rescued by aminoglycosides

doi: 10.1093/hmg/ddz280

Figure Lengend Snippet: Time dependent readthrough effect by gentamicin and G418 on progranulin R493X. (A) G418 induced readthrough of R493X mutation of progranulin in a time-dependent manner. Cells were treated as in Figure 1 and lysates analyzed by western analysis using an anti-FLAG (top blot), anti-progranulin (middle blots) and anti-actin (lower blot) antibodies. The two middle blots are a short and long exposure of the anti-progranulin analysis. (B) Band intensities were quantified to determine readthrough efficiency of the time-course. All FLAG bands were normalized against corresponding actin bands and the individual readthrough band was compared to the WT progranulin (the last lane in A). **P < 0.01, ***P < 0.001. (C) The time-course of gentamicin-induced readthrough effect on R493X.

Article Snippet: Plasmids The WT progranulin plasmid with a C-terminal FLAG tag (pC-Flag-PGRN-WT) was purchased from Sino Biological Inc. (Cat.HG10826-CF).

Techniques: Mutagenesis, Western Blot

Journal: Human Molecular Genetics

Article Title: Frontotemporal dementia non-sense mutation of progranulin rescued by aminoglycosides

doi: 10.1093/hmg/ddz280

Figure Lengend Snippet: Subcellular localization of the G418-induced R493X readthrough and WT progranulin. (A) A schematic of N- and C-terminal tagged WT progranulin and expected proteins in the absence and presence of G418. All G418 treatment was 1000 μg/ml for 24 h in this figure. (B) Top: The N-terminal HA-tagged and C-terminal FLAG-tagged WT progranulin are largely co-localized in N2A cells. Middle: N2A cells expressing HA-tagged WT progranulin and FLAG-tagged R493X-progranulin in the absence of G418. No FLAG signal was observed in the absence of G418. Bottom: N2A cells expressing HA-tagged WT progranulin and FLAG-tagged R493X-progranulin in the presence of G418. The FLAG-tagged R493X readthrough protein co-localizes with the HA-tagged WT progranulin. A histogram shows green (FLAG) and red (HA) signals along the cross section line drawn in the zoom view for each row. The concurrence of green and red signals demonstrates the co-localization of FLAG- and HA-tagged proteins. (C) The overexpressed WT progranulin (top), the G418-induced readthrough full-length protein (third row) and the endogenous progranulin (bottom) are partially co-localized with lysosome marker Lamp1. No FLAG signal was observed in the absence of G418 (second row). (D) The overexpressed WT progranulin (top), the G418-induced readthrough full-length protein (third row) and the endogenous progranulin (bottom) are partially co-localized with Golgi marker GM130. No FLAG signal was observed in the absence of G418 (second row). Scale bars, 20 μm.

Article Snippet: Plasmids The WT progranulin plasmid with a C-terminal FLAG tag (pC-Flag-PGRN-WT) was purchased from Sino Biological Inc. (Cat.HG10826-CF).

Techniques: Expressing, Marker

Journal: The Journal of biological chemistry

Article Title: Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

doi: 10.1016/j.jbc.2022.102348

Figure Lengend Snippet: Figure 1. Physical interaction between CD68 and granulin E. A, COS7 cells transfected with CD68-GFP were incubated with conditioned media containing AP-tagged granulin peptides. Positive interaction between CD68 and the granulin E was observed after incubation with AP substrate. The scale bar represents 100 μm. B, Myc-His-tagged CD68 and GFP-tagged granulin A through G were transfected in HEK293T. Lysates were incubated with anti-GFP-conjugated beads. After washes, products were analyzed by Western blot using anti-GFP and anti-Myc antibodies as indicated. C, FLAG-tagged PGRN and GFP-tagged CD68 were transfected in HEK293T cells. Lysates were incubated with anti-GFP-conjugated beads. After washes, products were analyzed by Western blot using anti-GFP and anti-FLAG antibodies as indicated. D, conditioned media containing FLAG-tagged PGRN were incubated with beads bound to GFP or GFP-CD68. After washes, products were analyzed by Western blot using anti-GFP and anti-FLAG antibodies as indicated. E, HEK293T cells were transfected with GFP-tagged CD68 and FLAG-tagged PGRN or PGRN-ΔE. Lysates were incubated with anti-FLAG-conjugated beads. After washes, products were analyzed by Western blot using anti-GFP and anti-FLAG antibodies as indicated. AP, alkaline phosphatase; HEK293T, human embryonic kidney 293T cell line; PGRN, progranulin.

Article Snippet: Blood was spun at 3500g for 15 min two times, and serum was collected and analyzed using mouse PGRN ELISA Kit (R&D Systems; catalog no.: MPGRN0).

Techniques: Transfection, Incubation, Western Blot

Journal: The Journal of biological chemistry

Article Title: Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

doi: 10.1016/j.jbc.2022.102348

Figure Lengend Snippet: Figure 2. CD68 binds PGRN and granulin E via the mucin–proline-rich domain. A, schematic of CD68 domain structure: mucin–praline-rich domain (M + P) or the LAMP-like domain (Lamp-D), and the transmembrane (TM) domain. B, Myc-tagged CD68 constructs containing the M + P or Lamp- D domains were expressed in HEK293T cells with GFP-tagged PGRN, and the lysates were coimmunoprecipitated using anti-Myc-conjugated beads. IP products were analyzed by Western blot and probed with Myc and GFP antibodies as indicated. Full length-CD68 (FL-CD68) is used as a control. C, M + P or the Lamp-D domains of CD68 were fused to the transmembrane domain of PDGFR (pDisplay vector; Invitrogen) and transfected into COS- 7 cells. FL-CD68 is used as a control. Cells were incubated with AP-tagged PGRN and granulin E (100 nM). The scale bar represents 100 μm. AP, alkaline phosphatase; HEK293T, human embryonic kidney 293T cell line; IP, immunoprecipitation; LAMP, lysosomal-associated membrane protein; PDGFR, platelet-derived growth factor receptor; PGRN, progranulin.

Article Snippet: Blood was spun at 3500g for 15 min two times, and serum was collected and analyzed using mouse PGRN ELISA Kit (R&D Systems; catalog no.: MPGRN0).

Techniques: Construct, Western Blot, Control, Plasmid Preparation, Transfection, Incubation, Immunoprecipitation, Membrane, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

doi: 10.1016/j.jbc.2022.102348

Figure Lengend Snippet: Figure 3. CD68 is not required for PGRN lysosomal trafficking. A, WT and Cd68−/−bone marrow–derived macrophages (BMDMs) were stained with anti- cathepsin D (CathD), PGRN, and CD68 antibodies. The scale bar represents 10 μm (inset: 5 μm). B, brain sections from adult WT and Cd68−/−mice were stained with anti-PGRN, LAMP1, and IBA1 antibodies. The scale bar represents 100 μm (inset: 25 μm). Representative images from three mice of each genotype were shown. C, serum PGRN levels in WT and Cd68−/−mice were measured by ELISA. Six mice per genotype were analyzed (n = 6). D, Western blot analysis of PGRN and granulin peptides in spleen lysates of WT and Cd68−/−mice. Six mice per genotype were analyzed (n = 6). PGRN and granulin in- tensities were normalized to GAPDH. LAMP1, lysosomal-associated membrane protein 1; ns, not significant; PGRN, progranulin.

Article Snippet: Blood was spun at 3500g for 15 min two times, and serum was collected and analyzed using mouse PGRN ELISA Kit (R&D Systems; catalog no.: MPGRN0).

Techniques: Derivative Assay, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Membrane

Journal: The Journal of biological chemistry

Article Title: Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

doi: 10.1016/j.jbc.2022.102348

Figure Lengend Snippet: Figure 5. PGRN deficiency leads to decreased molecular weight of CD68. A, Western blot analysis of CD68 in WT and Grn−/−spleen lysates. CD68 levels were quantified and normalized to GAPDH. n = 4, **p < 0.01, unpaired t-test. B, WT and Grn−/−BMDM cells were treated with lysosomal inhibitors, bafilomycin (50 nM), and chloroquine (250 μM) for 8 h at 37 C. Representative blots from two replicates were shown. C, Western blot analysis of CD68 in primary microglia derived from WT and Grn−/−mice. Results were from three sets of microglia cultured independently from mouse pups of corresponding genotypes (n = 3). ns, not significant; unpaired t-test. D, spleen lysates from WT, Grn−/−, and Cd68−/−mice were immunoprecipitated using rat antimouse CD68 antibodies. The beads were then incubated with or without PNGase F. Representative blots from three replicates are shown. Immunoglobulin G (IgG) bands are indicated by arrows. BMDM, bone marrow–derived macrophage; PGRN, progranulin.

Article Snippet: Blood was spun at 3500g for 15 min two times, and serum was collected and analyzed using mouse PGRN ELISA Kit (R&D Systems; catalog no.: MPGRN0).

Techniques: Molecular Weight, Western Blot, Derivative Assay, Cell Culture, Immunoprecipitation, Incubation

Journal: The Journal of biological chemistry

Article Title: Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

doi: 10.1016/j.jbc.2022.102348

Figure Lengend Snippet: Figure 6. PGRN does not affect CD68 localization. A, WT and Grn−/−BMDMs were stained with antibodies against CD68, cathepsin D, and PGRN. The scale bar represents 10 μm (inset: 5 μm). B, deletion of PGRN in RAW264.7 cells leads to the decreased molecular weight of CD68 and increased CD68 levels. CD68 levels were quantified and normalized to GAPDH. n = 3, *p < 0.05, unpaired t-test. C, control and Grn−/−RAW264.7 cells were fixed, permeabilized, and stained with anti-CD68, cathepsin D, and PGRN antibodies. The scale bar represents 10 μm (inset: 2.5 μm). D, live control and Grn−/−RAW264.7 cells were incubated with rat anti-mouse CD68 antibodies on ice followed by washing, fixation, blocking, and staining with secondary antibody and Hoechst. Representative images from three replicates are shown. The scale bar represents 10 μm. E, cell surface and total levels of CD68 were quantified by ImageJ for experiments in C and D. n = 3. BMDM, bone marrow–derived macrophage; ns, not significant; PGRN, progranulin.

Article Snippet: Blood was spun at 3500g for 15 min two times, and serum was collected and analyzed using mouse PGRN ELISA Kit (R&D Systems; catalog no.: MPGRN0).

Techniques: Staining, Molecular Weight, Control, Incubation, Blocking Assay, Derivative Assay

Journal: Journal of neurochemistry

Article Title: The interaction between progranulin and prosaposin is mediated by granulins and the linker region between saposin B and C

doi: 10.1111/jnc.14110

Figure Lengend Snippet: (A) Domain structure of human PGRN (aa 1-593). (B) HEK293T cells were co-transfected with FLAG tagged PGRN truncation constructs and untagged human PSAP as indicated. Cells were lysed two days after transfection and the lysates were immunoprecipitated with anti-FLAG antibodies. The IP products were analyzed by Western blot using anti-FLAG and anti-PSAP antibodies (RRID:AB_2172462). (C) Conditioned medium from HEK293T expressing different AP-fusion proteins were incubated with FLAG beads only or FLAG beads with FLAG-PSAP recombinant proteins. The amount of AP proteins co-immunoprecipitated with FLAG-PSAP was analyzed by Western blot. (D) Conditioned medium from HEK293T expressing different GFP-fusion proteins were mixed with 1μg purified recombinant FLAG-PSAP. Immunoprecipitations were then performed with anti-GFP antibodies and the IP products were analyzed by Western blot.

Article Snippet: The following antibodies were used in this study: mouse anti-FLAG (M2) from Sigma (RRID:AB_439685), rabbit anti-human PSAP antibodies from Proteintech Group (RRID:AB_2172462), goat anti-human PGRN (RRID:AB_2114489), and sheep anti-mouse PGRN (RRID:AB_2114504) from R&D systems, rat anti-mouse LAMP1 (1D4B) from BD Biosciences (RRID:AB_2134499).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Expressing, Incubation, Recombinant, Purification

Journal: Journal of neurochemistry

Article Title: The interaction between progranulin and prosaposin is mediated by granulins and the linker region between saposin B and C

doi: 10.1111/jnc.14110

Figure Lengend Snippet: (A) Conditioned medium containing AP, AP-PGRN, AP-Grn D, or AP-Grn E were incubated with COS-7 cells transfected with PSAP fused to PDGFR. Cells were fixed and AP binding was visualized with AP substrates. Scale bar=100μm. (B) AP–PGRN binding to PSAP-PDGFR expressing COS-7 cells measured as a function of AP–PGRN concentration. (C-E) Scatchard plot of AP-PGRN (C), AP-GRN D (D) or AP-GRN E (E) binding to PSAP-PDGFR expressing COS-7 cells. KD, mean ± sem, n = 4.

Article Snippet: The following antibodies were used in this study: mouse anti-FLAG (M2) from Sigma (RRID:AB_439685), rabbit anti-human PSAP antibodies from Proteintech Group (RRID:AB_2172462), goat anti-human PGRN (RRID:AB_2114489), and sheep anti-mouse PGRN (RRID:AB_2114504) from R&D systems, rat anti-mouse LAMP1 (1D4B) from BD Biosciences (RRID:AB_2134499).

Techniques: Incubation, Transfection, Binding Assay, Expressing, Concentration Assay

Journal: Journal of neurochemistry

Article Title: The interaction between progranulin and prosaposin is mediated by granulins and the linker region between saposin B and C

doi: 10.1111/jnc.14110

Figure Lengend Snippet: (A) HEK293T cells were co-transfected with FLAG tagged PGRN and full length his-PSAP or PSAP truncation mutants as indicated. Cells were lysed two days after transfection and the lysates were immunoprecipitated with anti-FLAG antibodies. The IP products were analyzed by Western blot using anti-FLAG and anti-PSAP antibodies (RRID:AB_2172462). (B) HEK293T cells were co-transfected with untagged PGRN and FLAG tagged PSAP fragments as indicated. Cells were lysed two days after transfection and the lysates were immunoprecipitated with anti-FLAG antibodies. (C) Recombinant Gst- or his-sumo tagged saposins purified from bacteria were incubated with FLAG beads only or FLAG beads with FLAG-PGRN recombinant proteins. The amount of saposin co-immunoprecipitated with FLAG-PGRN was analyzed by Western blot. (D) HEK293T cells were co-transfected with untagged PGRN and FLAG tagged full-length PSAP, full-length LAMP1, saposin B with BC linker fused with transmembrane and cytoplasmic domain of LAMP1 (Tc-LAMP1) and BC linker fused with Tc-LAMP1 as indicated. Cells were lysed two days after transfection and the lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-PGRN and anti-FLAG antibodies.

Article Snippet: The following antibodies were used in this study: mouse anti-FLAG (M2) from Sigma (RRID:AB_439685), rabbit anti-human PSAP antibodies from Proteintech Group (RRID:AB_2172462), goat anti-human PGRN (RRID:AB_2114489), and sheep anti-mouse PGRN (RRID:AB_2114504) from R&D systems, rat anti-mouse LAMP1 (1D4B) from BD Biosciences (RRID:AB_2134499).

Techniques: Transfection, Immunoprecipitation, Western Blot, Recombinant, Purification, Bacteria, Incubation

Journal: Journal of neurochemistry

Article Title: The interaction between progranulin and prosaposin is mediated by granulins and the linker region between saposin B and C

doi: 10.1111/jnc.14110

Figure Lengend Snippet: (A) HEK293T cells were co-transfected with FLAG tagged wild type (WT) or mutant (mut) PSAP and untagged human PGRN as indicated. Media was collected and cells were lysed three days after transfection. The lysates and media were separately immunoprecipitated with anti-FLAG antibodies. (B) Conditioned medium containing AP or AP-PGRN were incubated with COS-7 cells transfected with WT or mutant PSAP fused to PDGFR. Cells were fixed and AP binding were visualized with AP substrates. Scale bar=100μm.

Article Snippet: The following antibodies were used in this study: mouse anti-FLAG (M2) from Sigma (RRID:AB_439685), rabbit anti-human PSAP antibodies from Proteintech Group (RRID:AB_2172462), goat anti-human PGRN (RRID:AB_2114489), and sheep anti-mouse PGRN (RRID:AB_2114504) from R&D systems, rat anti-mouse LAMP1 (1D4B) from BD Biosciences (RRID:AB_2134499).

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Incubation, Binding Assay

Journal: Journal of neurochemistry

Article Title: The interaction between progranulin and prosaposin is mediated by granulins and the linker region between saposin B and C

doi: 10.1111/jnc.14110

Figure Lengend Snippet: (A) PSAP-/- mouse fibroblasts were infected with lentivirus expressing WT or mutant PSAP with the BC linker replaced by CD linker. Cells were fixed and stained with rabbit anti-human saposin B, sheep anti-mouse PGRN and rat anti-LAMP1 antibodies. Scale bar=10 μm. (B) The colocalization of PGRN with lysosomal marker LAMP1 in (A) were quantified using Image J. n=3, ***, p<0.001. (C) Grn-/- cortical neurons were incubated with C-terminally FLAG his tagged human PGRN which does not bind to sortilin (5ug/ml) together with recombinant WT PSAP or mutant PSAP with the BC linker replaced by CD linker (5ug/ml). 12 hours later, cells were fixed and stained with rabbit anti-human saposin B, goat anti-human PGRN and rat anti-LAMP1 antibodies. Scale bar=10 μm. (D, E) The endocytosed PGRN (D) and PSAP (E) for the experiment in (C) were quantified by using Image J. n=3; ns, no significance; ***, p<0.001. One way ANOVA.

Article Snippet: The following antibodies were used in this study: mouse anti-FLAG (M2) from Sigma (RRID:AB_439685), rabbit anti-human PSAP antibodies from Proteintech Group (RRID:AB_2172462), goat anti-human PGRN (RRID:AB_2114489), and sheep anti-mouse PGRN (RRID:AB_2114504) from R&D systems, rat anti-mouse LAMP1 (1D4B) from BD Biosciences (RRID:AB_2134499).

Techniques: Infection, Expressing, Mutagenesis, Staining, Marker, Incubation, Recombinant

Journal: Neurobiology of disease

Article Title: Human progranulin-expressing mice as a novel tool for the development of progranulin-modulating therapeutics.

doi: 10.1016/j.nbd.2021.105314

Figure Lengend Snippet: Fig. 1. Generation and characterization of human progranulin (GRN)-expressing mice. (A) The region included in the targeting BAC shown in the UCSC Genome Browser (accession date January 23, 2020). For both the SegWay and GeneHancer tracks, red bars indicate predicted promotors and yellow bars predicted enhancers. The arc in the predicted GeneHancer promotor (red bar) is indicative of two regions that physically interact. (B) A schematic representing the full targeting construct, which included homology arms for Hprt to allow for homologous recombination in mouse ESCs. (C) Bands representing the PCR products from 7 different reactions used to verify correct insertion of the BAC targeting construct. Primers for each reaction were designed to amplify the following: (1) Wild-type Hprt locus, indicating reconstitution of the Hprt locus; (2) Null Hprt locus, resulting in no band after correct homologous recombination; (3) 3′ junction of the genomic insert to the BAC backbone; (4) 5′ junction of the genomic insert to the BAC backbone; (5) mouse Grn as a positive control; (6) the 5′ end of the genomic insert, a region upstream of the GRN coding start site; and (7) the GRN ATG transcriptional start site, approximately in the middle of the genomic insert. Primer sequences are listed in Table S1. (D) Results of quantitative PCR analysis of the relative copy number of the inserted GRN transgene in DNA derived from the GRN-positive ESC clone used to produce GRNTg mice. Mouse Grn was amplified and the quantity obtained normalized to 4 reference genes; the quantity of Grn DNA was set to 2 (autosomal gene in a diploid genome). Four separate primer pairs were used to amplify human GRN and the quantities obtained again normalized to 4 reference genes. The quantity of GRN DNA was plotted relative to mouse Grn; all 4 primer pairs produced values of approximately 1, suggesting a single copy of GRN DNA is present.

Article Snippet: Proteins were transferred to 0.45 mm nitrocellulose membranes, which were then blocked with 5% milk in PBS, and then blotted for progranulin (AF2420, polyclonal goat anti-progranulin, R&D Systems).

Techniques: Expressing, Construct, Homologous Recombination, Positive Control, Real-time Polymerase Chain Reaction, Derivative Assay, Amplification, Produced

Journal: Neurobiology of disease

Article Title: Human progranulin-expressing mice as a novel tool for the development of progranulin-modulating therapeutics.

doi: 10.1016/j.nbd.2021.105314

Figure Lengend Snippet: Fig. 3. Human progranulin effectively rescues exaggerated lipofuscin deposition in Grn−/−mice. (A) Representative images of lipofuscin accumulation, measured as autofluorescence, in the thalamus of 6-month-old WT, Grn−/−, and GRNTg; Grn−/−mice. (B) Quantification of relative lipofuscin fluorescence in the thalamus from WT, Grn−/−, and GRNTg; Grn−/−mice. Data represent the mean ± SEM measured in arbitrary units (a.u.). N = 4–5 mice per group. *p < 0.05 by Tukey’s post-hoc test after one-way ANOVA. Scale bar = 50 μm.

Article Snippet: Proteins were transferred to 0.45 mm nitrocellulose membranes, which were then blocked with 5% milk in PBS, and then blotted for progranulin (AF2420, polyclonal goat anti-progranulin, R&D Systems).

Techniques: Fluorescence

Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: lncRNA dropout CRISPR-Cas9 screen in MM cell lines. (A) Schematic representation of the CRISPR screening pipeline. (B) Spearman's correlation between pgRNA read count profiles (from DNA collected 30 days after transduction and selection of the library) across screen replicates = 0.85 and 0.88, respectively, for AMO-1 and ABZB, with color bars on top/left indicating cluster membership obtained via hierarchical clustering (complete distance method). (C) Representation of pgRNA abundance log fold changes (logFCs) in DNA collected 30 days after library transduction and selection vs plasmidic amounts for 3 groups of pgRNAs: nontargeting (negative controls [median logFC = 0.27 and 0.35, respectively, for AMO-1 and ABZB]), targeting ribosomal protein genes (control essential genes, median logFC = −0.68 and −0.43, with a logFC ≤−0.5, corresponding to a MAGeCK FDR ≤20%), and lncRNAs, across the 2 screens. Each point represents 1 of the 12 472 pgRNAs in the library with coordinates on the y-axis indicating the median logFC across screen replicates. (D) Gene-wise MAGeCK robust rank aggregation (RRA) scores for significant dependencies identified in the 2 screens at an FDR ≤20%. Top essential control genes, dependencies that are private to each cell line and shared across them (as per the color scheme) are highligted. (E) Number of significantly essential lncRNAs (at an FDR ≤20%) in the 2 screened cell lines and their overlap. MOI, multiplicity of infection.

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: CRISPR, Transduction, Selection, Control, Infection

Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: Functional validation of prioritized oncogenic lncRNA candidates. (A) RP11-350G8.5 and LINC00467 basal expression levels via quantitative real time PCR (qRT-PCR) in MM cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors (values are normalized to the expression of GAPDH). (B) Representative image of genomic PCR products before and after KO of LINC00467 and RP11-350G8.5 in AMO-1 cells, visualized on 1.5% agarose gels. On the right: Sanger sequence of the amplicons encompassing the CRISPR-targeted region. Blue rectangles highlight pgRNA binding sites, whereas colored lines refer to the schematic picture of the KO reported above the gel picture (on the left). (C) Representative image of flow cytometric monitoring of AMO-1 and ABZB cells transduced with a SCRAMBLE-GFP-CRISPR vector (dark gray) or LINC00467/KO-GFP-CRISPR vector (light blue) or RP11-350G8.5/KO- GFP-CRISPR vector (red). Light-gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction) with overlapping colored curves at day 20. (D) Representative images of colony assay of AMO-1 and ABZB GFP-sorted cells, 15 days after plating, were generated using EVOS XL-Core microscope (Invitrogen by Thermo Fisher) (magnification ×10). (E) Number of colonies in 3 independent wells. (F) Dose-response curves 24 hours after treatment with bortezomib (1-10 nM). Percentage of viable cells ± standard deviation are normalized with respect to DMSO-treated cells (vehicle) for each experimental condition. Statistical differences were assessed across all plots via Student t test; ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001.

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Sequencing, CRISPR, Binding Assay, Transduction, Plasmid Preparation, Colony Assay, Generated, Microscopy, Standard Deviation

Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: RP11-350G8.5 putative oncogenic role: in vitro validation and preliminary data from in vivo models. (A) Flow cytometric monitoring of GFP expression in ABZB cells transduced with a SCRAMBLE-GFP-CRISPR negative control vector, an RPL8 /KO-GFP-CRISPR positive control vector (selected from Project Score [37]), and 2 GFP-CRISPR constructs encoding for 2 pgRNAs targeting RP11-350G8.5. Gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction), while colored curves represent the percentage of viable cells at day 20. Bars on the right represent the fold change in percentage of GFP-expressing cells 20 days after target depletion against day 0. (B) Evaluation of IL-6R RNA expression level through quantitative real time PCR (qRT-PCR) on ABZB after transduction with SCRAMBLE vector or KO of RP11-350G8.5 with pgRNA#1 or pgRNA#2 or with a vector overexpressing RP11-350G8.5 (UP). (Data are normalized to the expression of GAPDH.) Statistics were obtained using multiple t -tests, resulting in no significant (ns) differences, as per the reported P values. (C) Flow cytometric monitoring of GFP in JJN.3 and NCI-H929 MM transduced cells, and percentage of GFP-positive cells is reported by overlapping curves referred to day 20 (colored curves) against day 0 (light gray curves). (D) Validation of RP11-350G8.5 KO in nontumoral cells, performed as described for A and C. (E) Representative images of RNA-FISH analysis. Nuclei are counterstained with DAPI (blue signal), whereas C3-fluorescein–conjugated GAPDH (green signal) has been used as cytoplasmic marker. Customly designed Stellaris probes targeting RP11-350G8.5 have been conjugated with 5-carboxytetramethylrhodamine (TAMRA) dye (red signal). Representative pictures acquired with a DMI6000-AF6000 Leica (Wetzlar, Germany) fluorescence microscope at magnification ×63 are reported, followed by specific regions of interest (ROIs), which are represented as enlarged images. (F) Dose-response curves 24 hours after treatment with bortezomib in AMO-1 cells overexpressing RP11-350G8.5 (1-10 nM). Statistics were analyzed using multiple t -tests (cutoff ∗ P < .05, ∗∗ P < .01). (G) In vivo imaging of engrafted ABZB cells. A total of 5 × 10 6 ABZB cells, which previously underwent highly efficient transduction (multiplicity of infection = 1) of RP11-350G8.5 KO-GFP or the SCRAMBLE vectors, were subcutaneously inoculated in mice (n = 2 per group). Images of tumors were acquired when the tumoral masses became palpable (identified as DAY 1), and at the end of the experiment (DAY 16, when tumors reached 2 cm in diameter). Both DAY 1 and DAY 16 were set up by considering SCRAMBLE mice, because SCRAMBLE cells have been faster to generate tumoral masses, due to their higher proliferative rate, and to grow up to 2 cm in diameter, with respect to KO cells. Tumors appear as yellow high-density signals on the right flank of the mice. Pictures were obtained with the IVIS (Perkin Elmer) system. (H) Tumor growth as mean measurement ± standard deviation (SD) across mice groups (n = 2). (I) Photographs of excised tumors were captured by a digital camera. (J) Weights of excised tumors, reported as mean ± SD across mice groups. Statistics were analyzed using multiple t -tests (cutoff: ∗ P < .05).

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: In Vitro, Biomarker Discovery, In Vivo, Expressing, Transduction, CRISPR, Negative Control, Plasmid Preparation, Positive Control, Construct, RNA Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Fluorescence, Microscopy, In Vivo Imaging, Infection, Standard Deviation

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Constructs including coding region, domains, and epitope tags that were packaged into rAAV2/1 (Twin-Strep tag; V5 tag; FLAG tag; SP, signal peptide; paragranulin; granulin-1 [GRN1; G]; GRN2 [F]; GRN3 [B]; GRN4 [A]; GRN5 [C]; GRN6 [D]; GRN7 [E]). (B) Experimental workflow includes intracerebroventricular (i.c.v.) injection of rAAV, mouse aging, sample collection, and sample analysis. (C) ELISA quantification of hPGRN in cortical tissue from rAAV-injected mice as mean ± SD. One-way ANOVA with Tukey’s post hoc correction. n = 6–7 mice/group. * p < 0.05 and ** p < 0.01. (D) Immunoblot of cortical and hippocampal lysates verifying expression of GFP, hGRN2, and hGRN4 (β-tubulin loading control). (E) IHC images of Twin-Strep to visualize expression of GFP, hPGRN, hGRN2, and hGRN4 in coronal section plus magnified images of the cortex, hippocampus, and thalamus. Scale bars: 2 μm (full section) and 200 μm (magnified boxes). (F) Immunofluorescence (IF) images co-staining for hPGRN, hGRN2, and hGRN4 and antibody markers for neurons (Map2) and microglia (Iba1) in the cortex of an hPGRN, hGRN2- Grn −/− , and hGRN4- Grn −/− mouse. Scale bar: 10 μm.

Article Snippet: Human PGRN ELISA , R and D Systems , Cat#DY2420.

Techniques: Construct, Strep-tag, FLAG-tag, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Heatmap of differentially expressed (log 2 Z score-transformed) proteins associated with microglial activation and dysfunction (rows) in all treatment groups in GFP- Grn −/− compared to GFP- Grn +/+ (columns). (B) Abundance of CD45 (PTPRC) across all treatment groups. (C) Abundance of P2RY12 across all treatment groups. (D) Representative CD68 IHC of 12-month-old mouse coronal brain sections across all treatment groups. Scale bar: 2 mm. (E) Quantification of CD68 IHC signal of cortex, hippocampus, and thalamus. (F) Immunoblot of CD68 in cortical and thalamic brain tissue from all injection groups. (G) Quantification of immunoblot of cortical CD68 signal normalized to H3. (H) Quantification of immunoblot of thalamic CD68 signal normalized to H3. (I) Quantification of GPNMB levels in thalamus using ELISA. Data are presented as means ± SD. p values were calculated by one-way or two-way (E) ANOVA with Tukey’s post hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Human PGRN ELISA , R and D Systems , Cat#DY2420.

Techniques: Transformation Assay, Activation Assay, Western Blot, Injection, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Representative images of fluorescent immunohistochemistry of Grn −/− mice injected with AAV-hPGRN, AAV-hGRN2, or AAV-hGRN4 stained for hGRNs (hPGRN, hGRN2, or hGRN4; red), lysosomal protein cathepsin D (CTSD; green), and nucleus (DAPI stain; blue). Images were analyzed with IMARIS software for voxel co-localization (white). Scale bar: 10 μm. (B) Representative images of fluorescent immunocytochemistry of MEF Grn −/− TMEM192 3xHA cells expressing hPGRN, hGRN2, and hGRN4 stained for lysosomal protein CTSD (green), hGRNs (PGRN, GRN2, or GRN4; red), mitochondrial protein heat shock protein 60 (HSP60; gray), and nucleus (DAPI stain; blue). Scale bar: 10 μm. (C) Quantification of Pearson’s correlation coefficients (PCCs) between CTSD and hGRNs vs. HSP60 and hGRNs in MEF Grn −/− TMEM192–3xHA cells expressing hPGRN, hGRN2, or hGRN4. Data are represented as mean ± SD. n = 5 area/group. p values were determined by two-way ANOVA with Tukey’s post hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Human PGRN ELISA , R and D Systems , Cat#DY2420.

Techniques: Immunohistochemistry, Injection, Staining, Software, Immunocytochemistry, Expressing

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Lysosome immunoprecipitation (lyso-IP) workflow using MEF Grn −/− cells co-expressing TMEM192–3xHA and hPGRN, hGRN2, or hGRN4. (B) Immunoblots of cell lysate (cyto), input, and lyso-IP fractions isolated from MEF Grn −/− TMEM192–3xHA cells expressing hPGRN, hGRN2, and hGRN4 probed for lyso-tag (HA), mouse PGRN, hPGRN, hGRN2, hGRN4, lysosome (LAMP1 and CTSZ), mitochondria (HSP60), endoplasmic reticulum (PDI), and cytoskeleton (β-actin).

Article Snippet: Human PGRN ELISA , R and D Systems , Cat#DY2420.

Techniques: Immunoprecipitation, Expressing, Western Blot, Isolation

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human PGRN ELISA , R and D Systems , Cat#DY2420.

Techniques: Recombinant, Plasmid Preparation, Virus, Staining, Blocking Assay, Hydrophilic Interaction Liquid Chromatography, Transfection, Magnetic Beads, Polymer, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: Proteomic screen for PGRN interactors. (A) Schematic illustration of the SILAC experiment searching for PGRN interactors. (B) Volcano plot of SILAC hits. Top hits identified in the heavy fraction are highlighted. The complete list of proteins is summarized in Table S1 .

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Multiplex sample analysis

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: Physical interaction between PGRN and PSAP. (A) PGRN and PSAP interact when overexpressed in HEK293T cells. PSAP-V5 and FLAG-PGRN constructs were transfected into HEK293T cells as indicated. Lysates were prepared 2 d later and immunoprecipitated with anti-FLAG antibodies. (B and C) PSAP and PGRN interact with each other at endogenous levels in fibroblasts. Cell lysates (B) and conditioned medium (C) from wild-type (WT), PSAP −/− (PS−/−), and PGRN −/− (GRN−/−) fibroblasts were immunoprecipitated using rabbit anti-PGRN antibodies. The presence of PGRN and PSAP in the immunoprecipitation was detected using sheep anti–mouse PGRN and rabbit anti–mouse PSAP antibodies, respectively. Asterisk indicates IgG bands. (D) Direct interaction between PGRN and PSAP. Purified recombinant his-PSAP and Flag-PGRN proteins of indicated amounts were mixed in 100 µl PBS + 0.2% Triton X-100 for 1 h before adding anti-FLAG beads. Beads were washed after a 2-h incubation. 0.2 µg of each purified protein was loaded as input. After SDS-PAGE, the gel was stained with Krypton dye to visualize proteins. The binding ratio of PGRN–PSAP was provided based on the densitometric evaluation of PGRN and PSAP band intensities. All the results shown are the representative pictures from at least three independent experiments.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Construct, Transfection, Immunoprecipitation, Purification, Recombinant, Incubation, SDS Page, Staining, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: PSAP is required for PGRN lysosomal targeting in fibroblasts. (A) Mislocalization of PGRN in PSAP −/− fibroblasts. Immunostaining for PGRN, LAMP1, and PSAP in fibroblasts derived from wild-type and PSAP −/− mice using sheep anti– mouse PGRN, rat anti–mouse LAMP1, and rabbit anti–mouse PSAP antibodies. PGRN mislocalization was observed in all the PSAP −/− fibroblasts examined. (B) Quantification of PGRN localization within LAMP1-positive vesicles in wild-type and PSAP −/− fibroblasts using ImageJ. Data are presented as mean ± SEM from three independent experiments. ***, P < 0.001, Student’s t test. (C) Increased PGRN secretion in PSAP −/− fibroblasts. Fibroblasts were cultured in serum-free medium for 48 h before the lysates and conditioned media (CM) were harvested. Proteins from the conditioned media were precipitated with TCA. (D) Quantification of experiments in C. PGRN levels are normalized to transferrin in the conditioned media and Gapdh in the cell lysates. Data are presented as mean ± SEM from four independent experiments. *, P < 0.05, Student’s t test; N.S., no significance. (E) Immunostaining for PGRN, PDI, and cathepsin D in fibroblasts derived from wild-type and PSAP −/− mice. Representative images from three replicated experiments are shown Bars: (main) 20 µm; (inset) 5 μm.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Immunostaining, Derivative Assay, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: PSAP is not required for lysosomal localization of cathepsin D and TMEM106B in fibroblasts. (A) Immunostaining for PGRN, LAMP1, and cathepsin D in fibroblasts derived from wild-type and PSAP −/− mice. (B) Immunostaining for PGRN, LAMP1, and TMEM106B in fibroblasts derived from wild-type and PSAP −/− mice. Representative images from three replicated experiments are shown. Bars: (main) 20 µm; (inset) 5 μm.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Immunostaining, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: PSAP facilitates PGRN lysosomal targeting from the extracellular space . (A) PSAP facilitates PGRN lysosomal targeting from the extracellular space in fibroblasts. GRN −/− fibroblasts were treated with recombinant human his-PSAP and human FLAG-PGRN-his at a concentration of 5 µg/ml in serum-free media for 12 h as indicated. Fixed cells were stained with goat anti–human PGRN, rat anti–mouse LAMP1, and rabbit anti–human saposin B (PSAP) antibodies. Representative images from three replicated experiments are shown. Bars: (main) 20 µm; (inset) 5 μm. (B) Western blot analysis of PGRN and PSAP proteins in the uptake assay. GRN −/− fibroblasts were treated with PGRN and PSAP proteins as in A and the proteins in the lysate after 24-h uptake as well proteins in the medium before and after 24-h uptake are shown. Western blots were detected using goat anti–human PGRN and rabbit anti–human saposin B antibodies. For unknown reasons, PSAP signal is always stronger in the medium when PGRN is added together in the Western blot (although the same amount of PSAP protein was added). (C) Quantification of PGRN–PSAP levels in the medium for experiment in B. Data are presented as mean ± SEM from three independent experiments. ***, P < 0.01, Student’s t test; N.S., no significance.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Recombinant, Concentration Assay, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: Sortilin and PSAP comprise two independent and complementary pathways for PGRN lysosomal targeting. (A) Immunoblot for sortilin with lysates prepared from fibroblasts and N2a cells. Gapdh was used as a loading control. (B) Immunostaining for PGRN, LAMP1, and PSAP in fibroblasts derived from wild-type and Sort1 −/− mice. (C) Ectopic expression of sortilin in PSAP −/− fibroblasts rescues the PGRN trafficking defect. PSAP −/− fibroblasts were transfected with GFP-sortilin. 48 h after transfection, the cells were fixed and stained with sheep anti–mouse PGRN and rabbit anti–Cathepsin D. Representative images from three replicated experiments are shown. Bars: (main) 20 µm; (inset) 5 μm.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Western Blot, Control, Immunostaining, Derivative Assay, Expressing, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: PSAP interacts with M6PR. (A) Volcano plot of the SILAC experiment using recombinant Flag-PSAP protein to identify interactors from cell lysates prepared from fibroblasts grown in SILAC medium. The top hits are highlighted. (B) PSAP interacts with M6PR. Lysates prepared from PSAP −/− fibroblasts were incubated with anti-FLAG antibody-conjugated beads with Flag-PSAP or beads only. After washing, the immunoprecipitation products were analyzed by Western blot using anti-M6PR and anti-Flag antibodies. (C) PSAP bridges the binding between PGRN and M6PR. Lysates prepared from PSAP −/− fibroblasts were incubated with anti-FLAG antibody-conjugated beads with purified FLAG-PGRN or FLAG-PGRN+PSAP or beads only. After washing, the immunoprecipitation products were analyzed by Western blot using anti-M6PR, anti-PSAP, and anti-Flag antibodies. Representative images from three replicated experiments are shown.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Multiplex sample analysis, Recombinant, Incubation, Immunoprecipitation, Western Blot, Binding Assay, Purification

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: M6PR and LRP1 mediate PGRN–PSAP lysosomal trafficking. (A) Both PSAP and PGRN are mislocalized in M6PR-deficient fibroblasts. Fibroblasts infected with lentiviruses harboring guide RNA against M6PR and Cas9 were selected with puromycin and fixed and stained with goat anti–mouse PSAP or sheep anti–PGRN antibodies as indicated together with rat anti-LAMP1 and rabbit anti-M6PR antibodies. Two neighboring cells with (a) or without (b) M6PR expression are shown. (B and C) Quantification of PSAP (B) and PGRN (C) localization within LAMP1-positive vesicles in control and M6PR −/− fibroblasts as shown in A using ImageJ. Data are given as mean ± SEM from three independent experiments: **, P < 0.01; Student’s t test. (D and E) M6PR is required for PSAP-mediated PGRN lysosomal targeting from the extracellular space. Fibroblasts infected with lentiviruses harboring guide RNA against M6PR and Cas9 were selected with puromycin for a week and treated with recombinant human his-PSAP and human FLAG-PGRN-his proteins (5 µg/ml) in serum-free medium for 12 h, and then stained with hPGRN, LAMP1, and M6PR antibodies. Two neighboring cells with (a) or without (b) M6PR expression are shown. Intensities of endocytosed PGRN were quantified using ImageJ. Data are presented as mean ± SEM from three independent experiments: ***, P < 0.001, Student’s t test. (F and G) LRP1 is also critical for PGRN–PSAP uptake in fibroblasts. Fibroblasts infected with lentiviruses harboring guide RNA against LRP1 and Cas9 were selected with puromycin for 1 wk and treated as in D. Data are presented as mean ± SEM from three independent experiments: ***, P < 0.001, Student’s t test. Bars: (main) 20 µm; (inset) 5 μm.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Infection, Staining, Expressing, Control, Recombinant

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: PSAP regulates PGRN trafficking in vivo. (A) PGRN and PSAP colocalize with LAMP1 in cortical neurons of the adult mouse brain. (B) Both sortilin and PSAP are expressed in cortical neurons of the adult mouse brain. (C) Enlarged lysosomes and PGRN mislocalization in cortical neurons of PSAP −/− mice. PSAP −/− and littermate wild-type mice were sacrificed at postnatal day 21 and 15-µm brain sections were stained as indicated. Bars: (main) 20 µm; (inset) 5 μm. (D) Quantification of PGRN signals inside LAMP1-positive vesicles in C using ImageJ. Data are given as mean ± SEM from three pairs of mice: *, P < 0.05, Student’s t test. (E) Serum PGRN levels in newborn wild-type, PSAP +/− , and PSAP −/− littermate mice before the appearance of any apparent lysosomal phenotype in PSAP −/− mice. Data are given as mean ± SEM from four groups of mice. **, P < 0.01; ***, P < 0.001, one-way analysis of variance.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: In Vivo, Staining

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: A model for PGRN lysosomal targeting. PSAP and Sortilin are two independent and complementary pathways for PGRN lysosomal targeting in both biosynthetic and endocytic pathways. M6PR and LRP1 are required for PSAP-mediated PGRN lysosomal trafficking.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: