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: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Affinity comparison of mAbs derived from chicken immunizations and other sources for 2 unrelated model antigens, PCSK9 and PGRN. (A) Biacore binding curves and global fits of select anti-PCSK9 mAbs from chicken immunization showing a diverse set of kinetic profiles. The colored curves represent the measured binding responses of hPCSK9 when injected at concentrations of 5 nM (red) and 50 nM (green), with the global fit overlaid in black. (B) Isoaffinity plot comparing anti-PCSK9 mAbs generated from chicken immunization (olive green) with those from human phage display libraries (blue). (C) Isoaffinity plot comparing anti-PGRN mAbs generated from immunizations in chicken (olive green) and mouse (purple). The red dotted line indicates the k d limit of 5.70 × 10 −5 (1/s) that was placed on interactions which showed < 5 % signal decay within the allowed dissociation phase of 15 min, also known as the “5 % rule” (see Methods).

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Comparison, Derivative Assay, Binding Assay, Injection, Generated

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Solution affinity determination of 2 high affinity clones obtained from chicken immunization toward their respective serum antigens. (A) Schematic representation of the KinExA assay set-up. The mAb of interest is titrated into human serum and the equilibrated mixtures are injected over beads absorption-coated with a competing mAb to capture the free antigen. The bead-captured antigen is then detected using a Dylight-labeled sandwiching mAb. (B) Global analysis of mAb C34 binding serum PCSK9. (C) Global analysis of mAb C25 binding serum PGRN. In each case, the reported apparent K D value is the best fit and 95 % confidence interval of the fit.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Clone Assay, Injection, Labeling, Binding Assay

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Chicken-mouse merged binning results for PGRN. (A) Heat map showing binning assignments for 32 mAbs generated from chicken immunization (olive green) and 20 mAbs generated from mouse immunization (purple). SPR-derived K D values toward human PGRN are reported, along with their cross-reaction toward mouse (m) PGRN, and their GEP subdomain assignment (just for the human-specific clones; n/d = not determined). (B) Blocking network plot, colored by mAb library, with GEP assignments indicated (determined empirically, or inferred). (C) Dendrogram of the antibody sequence lineages for the chicken mAbs alongside the binning heat map for these clones (drawn from panel A, transposed, and resorted). (D) Blocking network plot for the chicken clones, colored by bin, with GEP assignments indicated. See Table S2.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Generated, Derivative Assay, Clone Assay, Blocking Assay, Sequencing

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Comparison of the epitope coverage observed for anti-PGRN mAbs raised via the immunization of chickens or mouse . *Of the 14 chicken mAbs that showed human/mouse crossreactivity, 8 were mapped to a GEP subdomain (by inferring from the binning data shown in Fig. 4B ) and are included in the A – P tally, and 6 were not assigned to a GEP subdomain.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Comparison

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

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: Mouse serum PGRN levels were measured using a PGRN Mouse EIA kit (Cayman Chemical).

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: Mouse serum PGRN levels were measured using a PGRN Mouse EIA kit (Cayman Chemical).

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: Mouse serum PGRN levels were measured using a PGRN Mouse EIA kit (Cayman Chemical).

Techniques: Western Blot, Infection, Expressing, Fluorescence

Journal: BMC Neuroscience

Article Title: Progranulin is expressed within motor neurons and promotes neuronal cell survival

doi: 10.1186/1471-2202-10-130

Figure Lengend Snippet: PGRN within motor neurons in primary cultures does not colocalize with the nucleus or mitochondria . (A) Motor neuron labeled with antibody to mouse PGRN (left hand image) is attenuated by antigen-competition with 300 ng recombinant mouse PGRN (middle and right hand images). When anti-PRGN was pre-absorbed with 400 ng of mouse recombinant PGRN, no signal was observed in the primary motor neurons (not shown). Shown are confocal images taken at 100×. (B) PGRN is not distributed in nuclei or mitochondria, organelles that are not part of the secretory pathway. Immunolabelling of motor neurons in dissociated spinal cord-DRG cultures with anti-TDP-43 (a) and anti-cytochrome C (b) and anti-PGRN (middle column). Merged images (right column) show no colocalization of TDP-43 or cytochrome C with endogenous mouse PGRN. Confocal images were captured at 63× magnification, hatched boxes represent 3-5× zoom.

Article Snippet: To control for nonspecific binding of the mouse PGRN antibody, antigen-competition was carried out by preadsorbing the antibody at 1:500 dilution with 300 ng/mL and 400 ng/mL recombinant mouse PGRN (Alexis Biochemicals).

Techniques: Labeling, Recombinant

Journal: BMC Neuroscience

Article Title: Progranulin is expressed within motor neurons and promotes neuronal cell survival

doi: 10.1186/1471-2202-10-130

Figure Lengend Snippet: PGRN within motor neurons in primary cultures does not colocalize with the nucleus or mitochondria . (A) Motor neuron labeled with antibody to mouse PGRN (left hand image) is attenuated by antigen-competition with 300 ng recombinant mouse PGRN (middle and right hand images). When anti-PRGN was pre-absorbed with 400 ng of mouse recombinant PGRN, no signal was observed in the primary motor neurons (not shown). Shown are confocal images taken at 100×. (B) PGRN is not distributed in nuclei or mitochondria, organelles that are not part of the secretory pathway. Immunolabelling of motor neurons in dissociated spinal cord-DRG cultures with anti-TDP-43 (a) and anti-cytochrome C (b) and anti-PGRN (middle column). Merged images (right column) show no colocalization of TDP-43 or cytochrome C with endogenous mouse PGRN. Confocal images were captured at 63× magnification, hatched boxes represent 3-5× zoom.

Article Snippet: To control for nonspecific binding of the mouse PGRN antibody, antigen-competition was carried out by preadsorbing the antibody at 1:500 dilution with 300 ng/mL and 400 ng/mL recombinant mouse PGRN (Alexis Biochemicals).

Techniques: Labeling, Recombinant

Journal: PLoS Pathogens

Article Title: Progranulin aggravates lethal Candida albicans sepsis by regulating inflammatory response and antifungal immunity

doi: 10.1371/journal.ppat.1010873

Figure Lengend Snippet: Wild type (WT, n = 5) mice were infected intravenously with 4 × 10 5 colony forming units (CFU) of C . albicans . (A) Organs were removed at the indicated time points, and blood was collected by cardiac puncture. Samples were assayed for PGRN content by specific sandwich enzyme-linked immunosorbent assay (ELISA). (B) PGRN concentrations in the kidney and blood from Toll-like receptor (TLR) 2 knockout (KO), TLR4 KO, TLR7 KO, type I IFN-α/β receptor (IFNAR) KO, and WT mice at the indicated times after intravenous infection with 4 x10 5 CFU of C . albicans . All data were pooled from three independent experiments. The statistical differences were determined by Kruskal-Wallis test followed by Dunn’s multiple comparisons post test, and p values were shown when compared between groups denoted by horizontal lines.

Article Snippet: The protein levels of progranulin (PGRN) were determined by mouse PGRN enzyme linked immunosorbent assay (ELISA) kit (RayBiotech).

Techniques: Infection, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: PLoS Pathogens

Article Title: Progranulin aggravates lethal Candida albicans sepsis by regulating inflammatory response and antifungal immunity

doi: 10.1371/journal.ppat.1010873

Figure Lengend Snippet: (A) Bone-marrow cells from PGRN KO (n = 5) and WT (n = 5) mice were intravenously injected into the irradiated recipient mice separately. Eight weeks later, mice were intravenously infected with 4×10 5 CFU of C . albica n s , and PGRN levels in the kidney, liver and lung were determined by ELISA. The statistical difference was determined by Kruskal-Wallis test followed by Dunn’s multiple comparisons post test, p values were shown when compared between groups denoted by horizontal lines. (B) Bone-marrow cells from PGRN KO (n = 10) and WT (n = 10) mice were intravenously injected into the irradiated recipient mice separately. Eight weeks later, mice were intravenously infected with 4×10 5 CFU of C . albica n s . Survival of these mice was monitored. Kaplan-Meier survival curves were shown, and p value was determined by log-rank survival test. (C) Radiation chimeras were infected with 4×10 5 CFU of C . albicans by intravenous inoculation, and kidney CFU levels at day 9 after C . albicans infection were evaluated. All data were pooled from three independent experiments. The Mann–Whitney U test was used to analyze the difference between groups denoted by horizontal lines, and p values were shown.

Article Snippet: The protein levels of progranulin (PGRN) were determined by mouse PGRN enzyme linked immunosorbent assay (ELISA) kit (RayBiotech).

Techniques: Injection, Irradiation, Infection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Progranulin aggravates lethal Candida albicans sepsis by regulating inflammatory response and antifungal immunity

doi: 10.1371/journal.ppat.1010873

Figure Lengend Snippet: PGRN KO (n = 5) and WT (n = 5) mice were infected intravenously with 4 × 10 5 colony forming units (CFU) of C . albicans , and then inflammatory mediators and leukocytes were quantified at the indicated times. (A) Cytokines and chemokines in the kidneys were quantified by enzyme-linked immunosorbent assay (ELISA) at the indicated times after C . albicans infection. (B) Cytokines and chemokines in the sera were quantified by ELISA at the indicated times after C . albicans infection. (C) Cytokines and chemokines in the livers were quantified by ELISA at the indicated times after C . albicans infection. (D) Cytokines and chemokines in the lungs were quantified by ELISA at the indicated times after C . albicans infection. (E) Cytokines and chemokines in the brains were quantified by ELISA at the indicated times after C . albicans infection. (F) Cytokines and chemokines in the spleens were quantified by ELISA at the indicated times after C . albicans infection. (G) Gene expression levels of ICAM-1 and P-Selectin in the kidneys were quantified by quantitative PCR at the indicated times after C . albicans infection. (H) Kidney cells from C . albicans -infected WT and PGRN KO mice were stained with anti-Ly-6G and anti-F4/80 antibodies, and FACS plots in showed the gating strategy for macrophages (CD11b + F4/80 + ) and neutrophils (CD11b + Ly-6G + ). Graphs showed the absolute numbers of macrophages and neutrophils in homogenized kidneys. All data were pooled from three independent experiments. The Mann–Whitney U test was used to analyze the difference between WT and PGRN KO mice at the same time point after C . albicans infection, and p values were shown when compared between groups denoted by horizontal lines.

Article Snippet: The protein levels of progranulin (PGRN) were determined by mouse PGRN enzyme linked immunosorbent assay (ELISA) kit (RayBiotech).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Progranulin aggravates lethal Candida albicans sepsis by regulating inflammatory response and antifungal immunity

doi: 10.1371/journal.ppat.1010873

Figure Lengend Snippet: WT mice were intravenously injected with 1×10 6 CFU of C . albicans , and PGRN blockade was performed by intraperitoneally administration of 5 μg of anti-PGRN monoclonal antibody on day 0 (same day as C . albicans infection), followed by a booster dose of 2.5 μg 24 hour later. (A) Survival of these mice (n = 10 for each group) was monitored. (B) Mouse Clinical Assessment Score for Sepsis (M-CASS) scores of WT mice (n = 5) treated with or without anti-PGRN antibody after intravenous infection with 1 × 10 6 CFU of C . albicans . (C) Kidney fungal loading was performed in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 10 6 CFU of C . albicans . (D) Kidney IL-6, CXCL1 and CCL2 levels were quantified by ELISA in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 10 6 CFU of C . albicans . (E) The numbers of macrophages (CD11b + F4/80 + ) and neutrophils (CD11b + Ly-6G + ) in the kidneys were quantified by flow cytometry analysis in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 10 6 CFU of C . albicans . (F) The gene expression levels of kidney injury marker-1 (KIM-1) were determined by quantitative PCR in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 10 6 CFU of C . albicans . (G) Serum urea levels were measured in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 10 6 CFU of C . albicans . (H) Serum creatinine levels were measured in WT mice (n = 5) treated with or without anti-PGRN antibody at the indicated times after intravenous infection with 1 × 10 6 CFU of C . albicans . All data were pooled from three independent experiments. Log-rank test was used to analyze the difference between survival curves. The Mann–Whitney U test was used to analyze the difference of other parameters between anti-PGRN-treated and control IgG-treated mice at the same time point after C . albicans infection, and p values were shown when compared between groups denoted by horizontal lines.

Article Snippet: The protein levels of progranulin (PGRN) were determined by mouse PGRN enzyme linked immunosorbent assay (ELISA) kit (RayBiotech).

Techniques: Injection, Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Marker, Real-time Polymerase Chain Reaction, MANN-WHITNEY