Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 1. PGRN levels are elevated in clinical and experimental influenza/S. pneumoniae coinfection. (A) ELISA detection of PGRN levels in the sera collected from pediatric patients with IAV infection (n = 47), S. pneumoniae infection (n = 53), and influenza/S. pneumoniae coinfection (n = 38). Healthy subjects served as controls (n = 30). (B) Groups of C57BL/6 WT mice were infected intranasally with A/Puerto Rico/8/1934 H1N1 (PR8; 400 PFU) alone, S. pneumoniae (19F; 1 3 108 CFU) alone, or PR8+19F coinfection. Secondary 19F infection was performed 3 d after influenza infection. Mice inoculated with PBS served as controls. The sera, lung homogenates, and sections of mice were collected 3 d after PR8 infection and 1 d after 19F primary infection or coinfection. (C and D) PGRN levels in the lung homogenates and sera were measured by ELISA kits (n = 4–5). (E) The expression and distribution of PGRN in lung tissues were detected by immunohistochemical staining with mouse monoclonal PGRN Ab. Each dot represents one patient or mouse. PGRN quantification in (A), (C), and (D); these experiments were performed once. Experiments in (E) were performed twice with representative data presented. **p , 0.01, ***p , 0.001 by Mann–Whitney U test, compared between groups.

Article Snippet: To demonstrate the protective role of PGRN in coinfection, each PGRN2/2 and WT mouse was administrated i.p. with 20 mg recombinant PGRN (rPGRN; R&D Systems), and the survival and weight loss of rPGRN-treated mice were determined.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Expressing, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 3. PGRN deficiency impairs bacterial clearance of mice in influenza/S. pneumoniae coinfection. Lungs and nasal washes of mice were collected 3 d after PR8 infection and 1 d after 19F primary infection or coinfection. (A and B) Viral loads in lungs were determined by PR8 matrix protein gene copy detected with quantitative PCR (n = 3). (C–F) Lung homogenates and nasal washes were inoculated on blood agar plates, and bacterial loads were de- termined by CFU analysis (n = 4–5). The label CFU/lung or CFU/nasal wash indicates total number of CFU in each lung homogenate or nasal wash sample. Data are representative of at least three independent experiments. *p , 0.05 based on Mann–Whitney U test. ns, not significant.

Article Snippet: To demonstrate the protective role of PGRN in coinfection, each PGRN2/2 and WT mouse was administrated i.p. with 20 mg recombinant PGRN (rPGRN; R&D Systems), and the survival and weight loss of rPGRN-treated mice were determined.

Techniques: Infection, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 2. PGRN deficiency increases mortality of mice in influenza/S. pneumoniae coinfection. WT and PGRN2/2 mice were infected with PR8 alone, 19F alone, or PR8+19F coinfection. Mice were monitored for 2 wk after primary PR8 or 19F infection. (A) Kaplan–Meier survival curves were assessed by log-rank (Mantel–Cox) test for significance (n = 8 for single infection groups and n = 18 for coinfection groups). *p , 0.05. (B–D) Body weight loss of mice was recorded daily and analyzed by two-way ANOVA test for significance. ***p , 0.001. (E and F) Coinfected WT and PGRN2/2 mice were i.p. injected with 20 mg rPGRN at 3 and 5 d after influenza (0 and 2 d after 19F coinfection); the survival (E) and weights (F) were monitored for 2 wk after influenza. Statistical analyses of survival and weight loss were performed in the same way (n = 10 per group, compared between groups). *p , 0.05. Survival and weight loss curves are shown by combining two independent experiments.

Article Snippet: To demonstrate the protective role of PGRN in coinfection, each PGRN2/2 and WT mouse was administrated i.p. with 20 mg recombinant PGRN (rPGRN; R&D Systems), and the survival and weight loss of rPGRN-treated mice were determined.

Techniques: Infection, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 4. PGRN deficiency aggravates lung inflammatory damage in influenza/S. pneumoniae coinfection. Lungs and BALF samples from mice were collected 3 d after PR8 infection and 1 d after 19F primary infection or coinfection. (A) Lung sections were stained with H&E for histopathology assay. (B and C) The LDH activity and total protein levels were tested by LDH and BCA protein assay kits, respectively (n = 4–5). (D) The wet/dry ratios of lungs were determined to evaluate lung edema of mice in different infection scenarios (n = 4–5). (E) Levels of proinflammatory cytokines IL-6, TNF-a, and IL-1b in lungs were detected by ELISA kits (n = 4–5). Data are representative of at least three independent experiments. *p , 0.05, **p , 0.01 based on unpaired Student t test. ns, not significant.

Article Snippet: To demonstrate the protective role of PGRN in coinfection, each PGRN2/2 and WT mouse was administrated i.p. with 20 mg recombinant PGRN (rPGRN; R&D Systems), and the survival and weight loss of rPGRN-treated mice were determined.

Techniques: Infection, Staining, Histopathology, Activity Assay, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 5. PGRN deficiency increases immune cell infiltration in lungs during influenza/S. pneumoniae coinfection. Cells from BALF were counted, purified, and identified by flow cytometry. Neutrophils were determined by gating on CD11b+ Ly6G+ cells, whereas macrophages were determined by gating on CD11b+ F4/80+ cells. (A) Percentages of neutrophils and macrophages. (B–D) The absolute numbers of total cells, neutrophils, and macrophages (n = 5–7). *p , 0.05, **p , 0.01, ***p , 0.001 based on Mann–Whitney U test. (E) Levels of chemokines CXCL1, CCL2, and CXCL10 in lungs were measured by ELISA kits (n = 5). Data are representative of at least three independent experiments. *p , 0.05, based on unpaired Student t test. ns, not significant.

Article Snippet: To demonstrate the protective role of PGRN in coinfection, each PGRN2/2 and WT mouse was administrated i.p. with 20 mg recombinant PGRN (rPGRN; R&D Systems), and the survival and weight loss of rPGRN-treated mice were determined.

Techniques: Cytometry, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 6. PGRN deficiency increases ERS-mediated apoptosis in mice during influenza and coinfection. Lungs and BALF from mice were collected 3 d after PR8 infection and 1 d after 19F primary infection or coinfection. (A) Apoptosis of cells collected from BALF was analyzed by flow cytometry with the staining of annexin V and propidium iodide. Annexin V–positive cells were identified as apoptotic and necrotic cells. Apoptotic cell numbers were calculated from total cell numbers by multiplying their respective apoptosis rates (n = 5–6). **p , 0.01, ***p , 0.001 based on Mann–Whitney U test. ns, not significant. (B) TUNEL staining of lung sections was performed with the use of In Situ Cell Death Detection Kit according to manufacturer instructions (original magnification, 3200). (C) Immunohistochemical assay of cleaved caspase-3 expression in lung tissues. (D) The expressions of XBP1s, CHOP, phospho-JNK, and cleaved caspase-3 in lungs were detected by Western blotting. Data are representative of at least three independent experiments.

Article Snippet: To demonstrate the protective role of PGRN in coinfection, each PGRN2/2 and WT mouse was administrated i.p. with 20 mg recombinant PGRN (rPGRN; R&D Systems), and the survival and weight loss of rPGRN-treated mice were determined.

Techniques: Infection, Cytometry, Staining, MANN-WHITNEY, TUNEL Assay, In Situ, Immunohistochemical staining, Expressing, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Progranulin deficiency causes impairment of autophagy and TDP-43 accumulation

doi: 10.1084/jem.20160999

Figure Lengend Snippet: Impaired defense against Listeria monocytogenes in mice lacking PGRN. (A) 14-d survival curve of GRN +/+ (solid blue line), GRN +/− (dashed green line), and GRN −/− (dotted red line) mice after intravenous L. monocytogenes infection. n = 12 mice per group from one experiment. ****, P < 0.0001 GRN +/+ versus GRN −/− using a log-rank test. (B) L. monocytogenes burden in GRN +/+ (blue circles) and GRN −/− (red triangles) mice 2 d after infection. n = 5 mice per group from one experiment. *, P < 0.05 and **, P < 0.01 versus GRN +/+ using an unpaired t test with Welch’s correction. (C) Serum PGRN levels in response to L. monocytogenes infection. n = 5 mice per group from one experiment. ***, P < 0.001 versus pre-infected using a paired t test. (D) Serum IL-6, IL-10, and MCP-1 levels in GRN +/+ (circles) and GRN −/− (triangles) mice after intravenous L. moncytogenes infection. n = 5 mice per group from one experiment. *, P < 0.05 versus GRN +/+ using an unpaired t test. (E) Serum IL-6, IL-10, and MCP-1 levels in GRN +/+ (circles) and GRN −/− (triangles) mice after treatment with LPS. n = 5 mice per group from one experiment. *, P < 0.05 and **, P < 0.01 using an unpaired t test. Error bars represent mean ± SEM.

Article Snippet: Recombinant mouse PGRN (2557-PG; R&D Systems) and bafilomycin A1 (B1793; Sigma) were used.

Techniques: Infection

Journal: The Journal of Experimental Medicine

Article Title: Progranulin deficiency causes impairment of autophagy and TDP-43 accumulation

doi: 10.1084/jem.20160999

Figure Lengend Snippet: Stimulation of autophagy signaling by PGRN. (A) Representative Western blot from n = 3 independent experiments of cell lysates from GRN −/− cortical neurons untreated or with 10 µg/ml recombinant mouse PGRN or vehicle and probed with the indicated antibodies. (B) Quantification of phosphorylated/total protein signals for the indicated proteins from experiments as shown in A. n = 3 independent experiments. *, P < 0.05 and **, P < 0.01 versus GRN +/+ using an unpaired t test. (C) Dose–response relationship for PGRN stimulation of AMPKα phosphorylation. Representative Western blot from n = 3 independent experiments of GRN −/− cortical cultures treated with the indicated concentrations of recombinant mPGRN and probed with the indicated antibodies. (A and C) Molecular mass is indicated in kilodaltons. (D) Quantification of phospho-AMPKα /total AMPKα signal from experiments as shown in C. n = 3 independent experiments. *, P < 0.05 and ***, P < 0.001 versus untreated using one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent mean ± SEM.

Article Snippet: Recombinant mouse PGRN (2557-PG; R&D Systems) and bafilomycin A1 (B1793; Sigma) were used.

Techniques: Western Blot, Recombinant, Phospho-proteomics

Journal: The Journal of Experimental Medicine

Article Title: Progranulin deficiency causes impairment of autophagy and TDP-43 accumulation

doi: 10.1084/jem.20160999

Figure Lengend Snippet: Accelerated accumulation of pathogenic TDP-43 in GRN −/− neurons. (A) Representative Western blot from n = 4 independent experiments of GRN +/+ and GRN −/− cortical cultures infected with AAV expressing mCherry along with either full-length TDP43-GFP (left) or TDP43CT-GFP (right) and probed with the indicated antibodies. Molecular mass is indicated in kilodaltons. (B) Quantification of GFP/mCherry signal from experiments as shown in A. n = 4 independent experiments. ***, P < 0.001 versus GRN +/+ using an unpaired t test. (C) Representative images of GRN +/+ and GRN −/− hippocampal neurons cotransfected with mCherry (top row) and TDP43CT-GFP (bottom) and treated with vehicle (DMSO; left), 10 µM PGRN (middle), or 100 nM bafilomycin A1 (right). n = 158, 173, 37, 69, 147, and 96 cells per genotype and condition from eight, eight, three, three, eight, and eight independent experiments for the groups listed left to right. Bars, 25 µm. (D) Quantification of total GFP fluorescence per cell from experiments as shown in A. Data were normalized to the mean of GRN +/+ vehicle-treated cells. n = 158, 173, 37, 69, 147, and 96 cells per genotype and condition from eight, eight, three, three, eight, and eight independent experiments for the groups listed left to right. *, P < 0.05 and **, P < 0.01 versus vehicle-treated GRN +/+ using Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons test. Error bars represent mean ± SEM. ns, not significant.

Article Snippet: Recombinant mouse PGRN (2557-PG; R&D Systems) and bafilomycin A1 (B1793; Sigma) were used.

Techniques: Western Blot, Infection, Expressing, Fluorescence

Journal: Npj Dementia

Article Title: Progranulin deficiency does not exacerbate TDP-43 pathology in TDP-43 transgenic mouse models

doi: 10.1038/s44400-025-00020-4

Figure Lengend Snippet: A Immunostaining of TDP-43 and NeuN in brain sections from 16-month-old mice WT, TDP-43 Q331K/Q331K (Q331K), Grn −/− , TDP-43 Q331K/Q331K Grn −/− (Q331K Grn −/− ) mice. Representative images from the cortex were shown. Scale bar, 10 µm. B Analysis of TDP-43, phosphorylated TDP-43 (pS409/410), and PGRN levels in cortical lysates from 16-month-old mice of the indicated genotypes. C TDP-43 levels in RIPA- and urea-soluble fractions were quantified. Data are presented as mean ± SEM from 3 mice per group ( n = 3). One-way ANOVA tests with Bonferroni’s multiple comparisons. D PGRN levels in RIPA soluble fractions were quantified and normalized to GAPDH. Data are presented as mean ± SEM from 3 mice per group ( n = 3). E Total RNAs were extracted from the cortex of 10-month-old WT, TDP-43 Q331K/Q331K , Grn −/− , TDP-43 Q331K/Q331K Grn −/− male mice, and the RT-qPCR was performed to analyze the splicing changes in Sort1 exon 17b (left) and Mapt exons 2 and 3 (right). The relative mRNA levels of transcripts including or excluding exons 2 and 3 represent the inclusion of Mapt exons 2 and 3. Data are presented as mean ± SEM ( n = 4 mice per genotype). p -values were determined using one-way ANOVA tests with Bonferroni’s multiple comparisons. F Expression levels of Tardbp in WT and Q331K mice. Total RNAs were extracted from the cortex of 10-month-old WT and Q331K male mice, and the RNA-seq was performed to analyze gene expression changes. Normalized read counts are shown. Data are presented as mean ± SEM ( n = 5-6 mice per genotype). * p < 0.05, unpaired two-tailed Student's t-test.

Article Snippet: The following antibodies were used in this study: rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-CD68 (Bio-Rad, MCA1957), mouse anti-GFAP (Cell signaling, 3670S), rabbit anti-TDP43 (Proteintech Group, 12892-1-AP (C-terminal) and 10782-2-AP (N-terminal)), rabbit anti-phospho-TDP-43 (Ser409/410) (Proteintech group, 80007-1-RR), mouse anti-PLP (Millipore, MAB388), mouse anti-MBP (Millipore, SMI-99), rabbit anti-MAG (Proteintech Group, 14386-1-AP), sheep anti-mouse PGRN (R&D systems, AF2557), and mouse anti-GAPDH (Proteintech Group, 60004-1-Ig).

Techniques: Immunostaining, Quantitative RT-PCR, Expressing, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: Npj Dementia

Article Title: Progranulin deficiency does not exacerbate TDP-43 pathology in TDP-43 transgenic mouse models

doi: 10.1038/s44400-025-00020-4

Figure Lengend Snippet: A Immunostaining of TDP-43 and NeuN using rabbit anti-TDP-43 CTD antibodies and mouse anti-NeuN antibodies, respectively (left panel) or immunostaining of human TDP-43 (right panel) using mouse anti-human TDP-43 in brain sections from 21-day-old WT, hTDP-43 Tg/Tg (Tg/Tg), Grn −/− , and hTDP-43 Tg/Tg Grn −/− mice. Representative images from the cortex were shown. Scale bar, 10 µm. B Analysis of TDP-43 and phosphorylated TDP-43 (pS409/410) and PGRN levels in brain lysates from 21-day-old mice. Antibodies recognizing the C-terminal or N-terminal domain of TDP-43 were used to detect total TDP-43 levels. C TDP-43 and pTDP-43 levels in RIPA- and urea-soluble fractions were quantified. Data are presented as mean ± SEM from 3 mice per group ( n = 3). One-way ANOVA tests with Bonferroni’s multiple comparisons. ** p < 0.01. D PGRN levels in RIPA soluble fractions were quantified and normalized to GAPDH. Data are presented as mean ± SEM from 3 mice per group ( n = 3).

Article Snippet: The following antibodies were used in this study: rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-CD68 (Bio-Rad, MCA1957), mouse anti-GFAP (Cell signaling, 3670S), rabbit anti-TDP43 (Proteintech Group, 12892-1-AP (C-terminal) and 10782-2-AP (N-terminal)), rabbit anti-phospho-TDP-43 (Ser409/410) (Proteintech group, 80007-1-RR), mouse anti-PLP (Millipore, MAB388), mouse anti-MBP (Millipore, SMI-99), rabbit anti-MAG (Proteintech Group, 14386-1-AP), sheep anti-mouse PGRN (R&D systems, AF2557), and mouse anti-GAPDH (Proteintech Group, 60004-1-Ig).

Techniques: Immunostaining

Journal: Cell death & disease

Article Title: Progranulin attenuates liver fibrosis by downregulating the inflammatory response.

doi: 10.1038/s41419-019-1994-2

Figure Lengend Snippet: Fig. 1 PGRN attenuates CCl4-induced fibrosis in mice. a Schematic diagram of the study. Liver fibrosis was induced by CCl4 injection for 4 weeks. Ad.CON, control adenovirus + CCl4; Ad.PGRN, PGRN adenovirus + CCl4; TA, tibialis anterior. b Serum PGRN levels. c Final body, liver, and spleen weights of Ad.CON and Ad.PGRN mice. d Serum levels of ALT and AST. e Liver morphology and staining with Sirius Red. Scale bar: 200 μm. f Quantification of the Sirius Red-positive area per high-powered field. Graphs show the mean ± SEM. *p < 0.05 and **p < 0.01 versus the corresponding control, as shown

Article Snippet: The serum concentration of mPGRN was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using a mouse PGRN ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Injection, Control, Staining

Journal: Cell death & disease

Article Title: Progranulin attenuates liver fibrosis by downregulating the inflammatory response.

doi: 10.1038/s41419-019-1994-2

Figure Lengend Snippet: Fig. 2 Effect of PGRN on liver fibrosis-related protein expression in CCl4-treated mice. a Immunohistochemical staining of liver sections for α- SMA, collagen 1a1, F4/80 antigen, and apoptotic cells (TUNEL). The mean number of positively stained cells per high-powered field was quantified. Scale bar: 200 μm. b, c Whole liver tissues were subjected to gene expression analysis by real-time qPCR. d Liver tissues were subjected to western blot analysis. Graphs show the mean ± SEM. *p < 0.05 and **p < 0.01 versus the corresponding control, as shown

Article Snippet: The serum concentration of mPGRN was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using a mouse PGRN ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, TUNEL Assay, Gene Expression, Western Blot, Control

Journal: Cell death & disease

Article Title: Progranulin attenuates liver fibrosis by downregulating the inflammatory response.

doi: 10.1038/s41419-019-1994-2

Figure Lengend Snippet: Fig. 3 The effect of PGRN on the macrophage inflammatory response to LPS. a, b Raw 264.7 cells and primary macrophages were pre-treated with PGRN for 30 min. They were then treated with LPS for an additional hour, and the expression of inflammatory genes was analyzed by real-time qPCR. c Human primary stellate cells were pre-treated with PGRN for 30 min. They were then treated with TGF-β1 for an additional 24 h, and gene expression was analyzed. d HepG2 and Huh7 cells were pre-treated with PGRN for 30 min and then stimulated with TNF-α for an additional 24 h and subjected to real-time qPCR analysis. e Raw 264.7 cells were treated with conditioned media from HepG2 or Huh7 cells treated with TNF-α for 24 h with or without 30 min of PGRN pre-treatment. After 1 h, the Raw 264.7 cells were subjected to real-time qPCR analysis. Graphs show the mean ± SEM. *p < 0.05 and **p < 0.01 versus the corresponding control, as shown

Article Snippet: The serum concentration of mPGRN was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using a mouse PGRN ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Gene Expression, Control

Journal: Cell death & disease

Article Title: Progranulin attenuates liver fibrosis by downregulating the inflammatory response.

doi: 10.1038/s41419-019-1994-2

Figure Lengend Snippet: Fig. 4 PGRN attenuates steatohepatitis induced by a MCD diet in mice. a Schematic diagram of the animal study. NASH was induced with a MCD diet for 8 weeks. Ad.CON, control adenovirus + MCD diet; Ad.PGRN, PGRN-expressing adenovirus + MCD diet; TA, tibialis anterior. b Serum PGRN levels. c Final body, liver, and spleen weights of Ad.CON and Ad.PGRN mice. d Serum ALT and AST levels. e Liver sections were stained with H&E and Sirius Red. Scale bar: 200 μm. f Quantification of steatosis, inflammatory foci, and Sirius Red staining. Graphs show the mean ± SEM. *p < 0.05 and **p < 0.01 versus the corresponding control, as shown

Article Snippet: The serum concentration of mPGRN was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using a mouse PGRN ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Control, Expressing, Staining

Journal: Cell death & disease

Article Title: Progranulin attenuates liver fibrosis by downregulating the inflammatory response.

doi: 10.1038/s41419-019-1994-2

Figure Lengend Snippet: Fig. 5 PGRN treatment decreases MCD diet-induced hepatic steatosis and fibrosis in mice. a Oil Red O staining and liver TG content. Scale bar: 200 μm. b Liver expression of genes involved in lipid metabolism was determined by real-time qPCR. c Liver tissues were subjected to western blot analysis. d Immunohistochemical staining for α-SMA, collagen 1a1, and F4/80 antigen. Scale bar: 200 μm. e Whole liver tissues were subjected to gene expression analysis by real-time qPCR. Graphs show the mean ± SEM. *p < 0.05 and **p < 0.01 versus the corresponding controls, as shown

Article Snippet: The serum concentration of mPGRN was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using a mouse PGRN ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Staining, Expressing, Western Blot, Immunohistochemical staining, Gene Expression

Journal: Cell death & disease

Article Title: Progranulin attenuates liver fibrosis by downregulating the inflammatory response.

doi: 10.1038/s41419-019-1994-2

Figure Lengend Snippet: Fig. 6 The effect of PGRN on the macrophage response to palmitate. a, b Raw 264.7 cells and primary macrophages were pre-treated with PGRN for 30 min. They were then treated with palmitate for an additional hour, and expression of inflammatory genes was analyzed by real-time qPCR. c Raw 264.7 cells were treated with conditioned media from HepG2 or Huh7 cells treated with palmitate for 6 h with or without 30 min of PGRN pre- treatment. Raw 264.7 cells were exposed to the conditioned media for 1 h and then subjected to real-time qPCR analysis. Graphs show the mean ± SEM. *p < 0.05 and **p < 0.01 versus the corresponding control, as shown

Article Snippet: The serum concentration of mPGRN was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using a mouse PGRN ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Control

Journal: eNeuro

Article Title: Effects of Exercise on Progranulin Levels and Gliosis in Progranulin-Insufficient Mice 1 , 2 , 3

doi: 10.1523/ENEURO.0061-14.2015

Figure Lengend Snippet: Exercise (3-4 weeks) produces a small increase in hippocampal progranulin in young adult wild-type mice. Two- to 3-month-old wild-type mice were randomized to sedentary or exercise groups for three to four weeks ( A ). B , Exercise produced a robust increase in BDNF (**** p < 0.0001). C , Representative BDNF and α-tubulin blots. D , Exercise did not significantly increase frontal cortex progranulin protein. E , Exercise produced a small but statistically significant increase in hippocampal progranulin (* p = 0.038). F , Exercise did not affect plasma progranulin. n = 8–16 mice per group. Values in B – F are expressed relative to the sedentary group.

Article Snippet: After electrophoresis, transfer, and blocking as described above, the samples were incubated overnight in a sheep polyclonal anti-progranulin antibody (1:5000, R&D Systems AF2557).

Techniques: Produced, Clinical Proteomics

Journal: eNeuro

Article Title: Effects of Exercise on Progranulin Levels and Gliosis in Progranulin-Insufficient Mice 1 , 2 , 3

doi: 10.1523/ENEURO.0061-14.2015

Figure Lengend Snippet: Exercise (4 weeks) does not increase progranulin protein levels in multiple brain regions of Grn +/− mice. Four- to 8-month-old Grn +/− mice were randomized to sedentary or exercise groups for 4 weeks ( A ). Exercise did not increase progranulin protein levels in frontal cortex ( B ), thalamus ( C ), or hippocampus ( D ), despite producing the expected increase in the number of doublecortin-positive neurons in the dentate gyrus ( E , ** p < 0.01). Values in B – D are expressed relative to sedentary Grn +/+ mice, with sedentary Grn +/− mice set at 0.5 to maintain a consistent scale with Figure 3. n = 11–12 per group.

Article Snippet: After electrophoresis, transfer, and blocking as described above, the samples were incubated overnight in a sheep polyclonal anti-progranulin antibody (1:5000, R&D Systems AF2557).

Techniques:

Journal: eNeuro

Article Title: Effects of Exercise on Progranulin Levels and Gliosis in Progranulin-Insufficient Mice 1 , 2 , 3

doi: 10.1523/ENEURO.0061-14.2015

Figure Lengend Snippet: Measures of progranulin levels

Article Snippet: After electrophoresis, transfer, and blocking as described above, the samples were incubated overnight in a sheep polyclonal anti-progranulin antibody (1:5000, R&D Systems AF2557).

Techniques: Clinical Proteomics

Journal: eNeuro

Article Title: Effects of Exercise on Progranulin Levels and Gliosis in Progranulin-Insufficient Mice 1 , 2 , 3

doi: 10.1523/ENEURO.0061-14.2015

Figure Lengend Snippet: Group housing does not potentiate the effects of exercise on progranulin. Six weeks of wheel running ( A ) failed to increase frontal cortex progranulin mRNA ( B ) or hippocampal progranulin protein ( C ) in either solo- or group-housed wild-type mice aged 3–6 months. D-F , Hippocampal BDNF was increased by exercise ( D ; ANOVA effect of exercise, p = 0.041). F , Representative BDNF and α-tubulin blots for each group. n = 8–14 mice per group. Values in B – E are expressed relative to the solo-housed sedentary group.

Article Snippet: After electrophoresis, transfer, and blocking as described above, the samples were incubated overnight in a sheep polyclonal anti-progranulin antibody (1:5000, R&D Systems AF2557).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Anti-sortilin1 Antibody Up-Regulates Progranulin via Sortilin1 Down-Regulation

doi: 10.3389/fnins.2020.586107

Figure Lengend Snippet: Binding profiles of anti-SORT1 mAbs. (A) Epitope binning of anti-SORT1 mAbs. The competitive binding was measured in a sandwich ELISA with 29 anti-SORT1 mAbs. Each column and row in the matrix represent an unlabeled and biotinylated anti-SORT1 mAb. The competitive binding of the 29 anti-SORT1 mAbs is shown as a heat map analyzed by Ward’s hierarchical clustering. The color scale from 0 to 100 shows the competitive binding of the 2 mAbs. (B) Design of chimeric proteins; nABcde (human SORT1 A, B and puffer fish SORT1 N-terminal C, D, E regions), NabCDE (human SORT1 N-terminal, C, D, E and puffer fish SORT1 A, B regions), NAbcDE (human SORT1 N-terminal, A, D, E and puffer fish SORT1 B, C regions), and naBCdE (human SORT1 B, C, E and puffer fish SORT1 N-terminal, A, D regions). The white bar indicates the SORT1 domain derived from human and the black bar indicates the SORT1 domain derived from puffer fish. (C–H) Epitope mapping of anti-SORT1 mAbs. Epitope mapping was analyzed by the binding pattern of anti-SORT1 mAb to human/puffer fish chimeric SORT1 proteins in BLI. The BLI analysis was performed with anti-SORT1 mAb K1-19 (C) , K1-12 (D) , K1-15 (E) , K1-40 (F) , K1-67 (G) , and K1-05 (H) as ligands and chimeric SORT1 protein as an analyte. The vertical axis indicates the BLI signal response (nm). The horizontal axis indicates the time after analyte loading. (I) Epitope mapping results of anti-SORT1 mAbs tested are shown in the table.

Article Snippet: PGRN levels in collected culture media were determined with Mouse Progranulin DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Binding Assay, Sandwich ELISA, Derivative Assay

Journal: Frontiers in Neuroscience

Article Title: Anti-sortilin1 Antibody Up-Regulates Progranulin via Sortilin1 Down-Regulation

doi: 10.3389/fnins.2020.586107

Figure Lengend Snippet: Detailed characterization of anti-SORT1 mAb K1-67. (A) PGRN up-regulation in response to K1-67 in U251 human glioblastoma cells. Cells were treated with various concentrations of K1-67 for 72 h. Human PGRN in U251 culture supernatant was determined by ELISA and is shown as a fold-change relative to PBS-treated cells. Data are mean ± SEM from 3 independent experiments. (B) PGRN up-regulation in response to K1-67 in mouse primary neurons. Cells were treated with various concentrations of K1-67 for 7 days. Mouse PGRN in mouse primary neuron culture supernatant was determined by ELISA and is shown as a fold-change relative to PBS-treated cells. Data are mean ± SEM from 3 independent experiments. (C,D) Affinity of K1-67 to SORT1. BLI was used to determine the affinity of K1-67 toward human and mouse SORT1 with K1-67 as a ligand and with SORT1-His protein as an analyte. The vertical axis indicates the BLI signal response (nm), and the horizontal line indicates the time after analyte loading. Kinetic parameters were analyzed using a 1:1 Langmuir fitting model. Association ( K a ) and dissociation ( K d ) constants were calculated and used to determine the K D value ( K d / K a ).

Article Snippet: PGRN levels in collected culture media were determined with Mouse Progranulin DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Neuroscience

Article Title: Anti-sortilin1 Antibody Up-Regulates Progranulin via Sortilin1 Down-Regulation

doi: 10.3389/fnins.2020.586107

Figure Lengend Snippet: Up-regulation of plasma or CSF PGRN levels in K1-67-treated mice. (A,B) Plasma or CSF PGRN levels in response to K1-67. Mouse plasma or CSF was collected 1- or 3-days after the treatment of K1-67 at 100 mg/kg, i.v., and subjected to ELISA. Pooled CSF from 4 mouse was used due to the small volume of CSF from individual mouse. The vertical axis indicates PGRN concentration. Data are mean ± SD from 12 or 3–4 independent experiments for plasma and CSF, respectively. ∗∗∗ p < 0.001 vs. PBS-treated group by Aspin–Welch t -test. (C) K1-67 concentration in plasma. Mouse plasma was collected 1 or 3 days after the treatment of K1-67 at 100 mg/kg, i.v., and subjected to liquid chromatography-mass spectrometry. Data are mean ± SD from 3 independent experiments.

Article Snippet: PGRN levels in collected culture media were determined with Mouse Progranulin DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Concentration Assay, Liquid Chromatography, Mass Spectrometry

Journal: Frontiers in Neuroscience

Article Title: Anti-sortilin1 Antibody Up-Regulates Progranulin via Sortilin1 Down-Regulation

doi: 10.3389/fnins.2020.586107

Figure Lengend Snippet: ISF PGRN levels in response to K1-67. Mouse ISF was collected from a probe in hippocampal microdialysis over 24 h after the treatment of K1-67 at 50 mg/kg or 100 mg/kg, i.v. PGRN levels were determined by ELISA. The vertical axis indicates PGRN concentration. The horizontal axis indicates the hours after K1-67 injection. Data are mean ± SD from 10 independent experiments. *** p < 0.001, **** p < 0.0001 vs. PBS by Two-way RM ANOVA analysis with Sidak’s multiple comparisons.

Article Snippet: PGRN levels in collected culture media were determined with Mouse Progranulin DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Injection

Journal: The American journal of pathology

Article Title: Accelerated lipofuscinosis and ubiquitination in granulin knockout mice suggest a role for progranulin in successful aging.

doi: 10.2353/ajpath.2010.090915

Figure Lengend Snippet: Figure 1. PGRN immunoreactivity is reflective of genotypes and increases in older GRN/ and GRN/ mice. As shown in 12-month-old mice, GRN/ mice (A) have higher PGRN immunoreactivity than GRN/ mice (B), whereas GRN/ mice (C) have no detectable PGRN expression. Higher magnification insets correspond to the areas identified by black squares. D: In a subset of GRN/ (green) and GRN/ (orange) mice, PGRN immuno- reactivity was quantified by image analysis. Consistent with genotypes, GRN/ mice had significantly reduced PGRN levels compared with GRN/

Article Snippet: Membranes were blocked in 5% milk in Tris-buffered saline-0.1% Triton X-100 and incubated overnight in anti-mouse PGRN (1:500; R&D Systems, Minneapolis, MN) or anti-glyceraldehyde-3-phosphate dehydrogenase (1:10,000; BioSource International, Camarillo, CA) at 4°C.

Techniques: Expressing

Journal: The American journal of pathology

Article Title: Accelerated lipofuscinosis and ubiquitination in granulin knockout mice suggest a role for progranulin in successful aging.

doi: 10.2353/ajpath.2010.090915

Figure Lengend Snippet: Figure 7. Hypothetical model of neurodegeneration based on the chrono- logical appearance of pathology in GRN/ mice. 1 month: young neurons are free of any pathology and microglia are in a ramified or resting state; seven months: through an unknown mechanism, the lack of PGRN results in intraneuronal accumulation of lipofuscin pigment in aging neurons resulting in cellular stress which is detected by nearby microglia; 12 months: neuronal cytoplasm is full of lipofuscin pigment which adversely affects cellular func- tion and long-term neuronal survival, signaling the activation of microglia; 23 months: slow and progressive loss of neurons, resulting in expulsion of neuronal lipofuscin into extracellular space, most of which is phagocytosed by activated microglia with some lipofuscin accumulating in the extracellular space. The lack of PGRN may also affect the normal function of activated microglia resulting in accumulation of endogenous microglial lipofuscin. A severe, sustained, and/or an inappropriate microglia (inflammatory) re- sponse (attributable to absence of microglial PGRN) may further enhance neuronal loss.

Article Snippet: Membranes were blocked in 5% milk in Tris-buffered saline-0.1% Triton X-100 and incubated overnight in anti-mouse PGRN (1:500; R&D Systems, Minneapolis, MN) or anti-glyceraldehyde-3-phosphate dehydrogenase (1:10,000; BioSource International, Camarillo, CA) at 4°C.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

doi: 10.1371/journal.pone.0065542

Figure Lengend Snippet: (A) Schematic diagram of DNA injection. 6- to 8-week old female BALB/c mice were injected subcutaneously with ALD-DNA (50 µg/mice) plus CFA at week 0, followed by two booster injections of ALD-DNA (50 µg/mice) emulsified with IFA at week 2 and week 4 after initial injection. (B) Serum anti-dsDNA IgG levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (C) 8 weeks after initial injection, glomerular immune deposition were detected by direct immunofluorescence for IgG in frozen kidney section from ALD-DNA-injected SLE mice or control mice. Representative images (magnification×200) of 8 mice are shown for each group. (D) Nephritic pathology was evaluated by H&E staining of renal tissues. Images (magnification×200) are representative of 8 mice in each group. (E) The kidney score was assessed using paraffin sections stained with H&E in (D). n = 8. (F) Urine protein levels of the mice were assessed by BCA Protein Assay Kit every 2 weeks. Data are means ± SD from 8 mice in each group. (G) Serum GRN levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (H) The correlation between serum GRN level and kidney score in lupus model. Correlation analysis was performed by Pearson correlation analysis. Each symbol indicates an individual mouse (n = 21). (I) The correlation between serum GRN level and urine protein level in lupus model. Pearson correlation analysis was used to carry out the correlation study. Each symbol indicates an individual mouse (n = 21). *, P <0.05.

Article Snippet: To assess protein levels of GRN in murine serum, ELISA assays were performed with commercial mouse GRN ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Control, Staining, Bicinchoninic Acid Protein Assay

Journal: PLoS ONE

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

doi: 10.1371/journal.pone.0065542

Figure Lengend Snippet: BALB/c mice were administrated intramuscularly with 100 µg/mouse pGRN or pcDNA3.1 to overexpress GRN, intravenously injected with LV-shGRN or LV-shNC (2×10 8 molecules/mouse) to down-regulate GRN expression. And 72h later, mice were then injected subcutaneously with ALD-DNA (50 µg/mouse) for total 3 times in 4 weeks. (A) The dynamics of serum GRN levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (B) Nephritic pathology was evaluated by H&E staining of renal tissues. Images (magnification×200) are representative of at least 8 mice in each group. (C) The kidney score was assessed using paraffin sections stained with H&E in (B). n = 8. (D) Urine protein levels of the mice were assessed by BCA Protein Assay Kit every 2 weeks. Data are means ± SD from 8 mice in each group. *, P <0.05.

Article Snippet: To assess protein levels of GRN in murine serum, ELISA assays were performed with commercial mouse GRN ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Bicinchoninic Acid Protein Assay

Journal: PLoS ONE

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

doi: 10.1371/journal.pone.0065542

Figure Lengend Snippet: (A–B) Primary macrophages were stimulated with increasing amounts of ALD-DNA for 24 h. mRNA levels of GRN in macrophages were analyzed by real time PCR analysis (A), and protein levels of GRN in the supernatants of macrophages were analyzed by western blot (B). Above, quantitative results of western blots, the band intensity was measured by Image J; Below, representative western blots. Similar results were obtained in three independent experiments. Data are representative of results obtained in three independent experiments. Primary peritoneal macrophages were stimulated by ALD-DNA (50 µg/mL) with GRN (5 µg/mL) for 24 h (C and F), or were pretreated with elastase inhibitor (100 µM) or DMSO (0.1%) for 12 h (D and G), and then were exposed to ALD-DNA, UnALD-DNA, or PBS for another 24 h. Macrophages were transfected with control siRNA (200 nM) or GRN siRNA (siGRN, 200 nM). 36 h posttransfection, macrophages were stimulated with PBS, UnALD-DNA or ALD-DNA (50 µg/mL) (E and H). (C–E) ELISA assay was used to analyze the levels of TNF-α, IL-1β, IL-6, IL-10, IL-12, and MCP-1 in the culture supernatants of macrophages. Data are means ± SD of three independent experiments. (F–H) Western blot analysis was used to analyze the protein levels of iNOS in macrophages. Data are representative of three separate experiments. Similar results were obtained in three independent experiments. Band intensity was measured by Image J and the ratios of iNOS to β-actin were calculated. *, P <0.05.

Article Snippet: To assess protein levels of GRN in murine serum, ELISA assays were performed with commercial mouse GRN ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

doi: 10.1371/journal.pone.0065542

Figure Lengend Snippet: (A) Primary macrophages were stimulated with 50 µg/mL ALD-DNA together with 5 µg/mL purified GRN for the indicated time. Phospho-Akt, ERK, JNK, and p38 were detected by immunoblotting. Data are representative of three separate experiments. Similar results were obtained in three independent experiments. (B) Primary macrophages were pretreated with elastase inhibitor (100 µM) for 12 h, and then were stimulated with 50 µg/mL ALD-DNA for the indicated time. Phospho-Akt, ERK, JNK, and p38 were detected by Western blot. Data are representative of three separate experiments. Similar results were obtained in three independent experiments. Left, representative western blots; Right, quantitative results, the band intensity was measured by Image J and the ratios of phospho-Akt, phospho-ERK, phospho-JNK and phospho-p38 to β-actin were calculated. Primary peritoneal macrophages were pretreated with U0126 (10 µM) (C), SP600125 (10 µM) (D), SB203580 (10 µM) (E) for 30 min, and then were stimulated with 50 µg/mL ALD-DNA together with 5 µg/mL purified GRN for 24 h. Cytokine expression levels of TNF-α, IL-1β and IL-10 in the supernatants of macrophages were determined by ELISA assay. Data are means ± SD of three independent experiments. *, P <0.05.

Article Snippet: To assess protein levels of GRN in murine serum, ELISA assays were performed with commercial mouse GRN ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Purification, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay