Journal: bioRxiv

Article Title: Systemic inflammation causes microglial dysfunction with a mixed AD-like pathology

doi: 10.1101/2020.07.27.223198

Figure Lengend Snippet: Gene expression changes after the prenatal and early postnatal immune challenge. Volcano plot with log2-fold change (X-axis) and −log10 p-value (Y-axis) at different age groups (A) 3 months (B) 6months (C) 9 months and (D) 16 months in PP versus NN controls. Genes with log2 fold changes >0.5 are shown in green. Significantly regulated genes are shown in red (log2 fold change >0.5, p<0.05, left downregulated, and right upregulated). Genes with insignificant log2 fold changes <0.5 are shown in black [controls (NN) n=3/age, PolyI:C (PP) n=3/age]. (E) Venn diagram indicating the number of uniquely and commonly affected genes in the aging hippocampus following prenatal and early postnatal PolyI:C (PP) treatment. Few common genes along with their Log2 Fold changes either (F) downregulated or (G) upregulated were plotted across staging. Transcript validation analysis for (H) Egr2 and Kcnj2 and (I) Plin4 and Lcn2 . (J) Correlation matrix with highlighted significant association from 9 to 16 months among the differentially expressed cell-type markers ( GFAP, Iba1, and Grin1 ) and Egr2 , Kcnj2 , Plin4 and Lcn2 between PolyI:C and saline controls [controls (NN), n=5/age, PolyI:C (PP) n=4-5/age]. Line plots show values as mean ± SEM. *=p<0.05, **=p<0.01, #=p@0.1, Student’s t-test.

Article Snippet: Following antibodies were used for western blot immunolabeling, immunohistochemistry experiments in this study: Rabbit anti pTau 231 (1:500, ab151559, Abcam, UK), Rabbit anti pTau 205 (1:500, ab181206, Abcam, UK), goat anti-tau (1.25 μg/mL; AF3494, R&D systems, USA), mouse anti-β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), Rabbit anti GFAP (1:500, ab68428, Abcam, UK), Goat anti GFAP (1:500, SAB2500462, Sigma), Goat anti Iba1 (1:500, ab5076, Abcam, UK), Mouse anti-amyloid beta 6E10 clone (1:500, cat:803014; Biolegend, USA), rabbit anti-synaptophysin (1:500; ab14692, Abcam, UK), Rabbit anti-amyloid beta 42 (Aβ-42; 1:500; D54D2 XP, Cell Signaling, USA), Rabbit anti-Lipocalin-2/LCN2 (1:500, 50060-RP02; Sino Biological, China), mouse anti-NF200 (1:500, cat:1178709; Boehringer Mannheim Biochemica, Germany), Nile Red (2 μg/mL, N3013, Sigma, USA), Thioflavin-S (100 mM; CAS 1326-12-1, Santa Cruz Biotechnology, USA) The secondary antibodies used in the study for immunolabeling were: directly conjugated to Cy2, Cy3, or Cy5 in donkey raised secondary antibodies (all 1:1000; Jackson Immuno Europe, UK).

Techniques: Expressing

Journal: bioRxiv

Article Title: Systemic inflammation causes microglial dysfunction with a mixed AD-like pathology

doi: 10.1101/2020.07.27.223198

Figure Lengend Snippet: (A) Representative Lcn2, Aβ 1-42 stained confocal images of saline and PolyI:C treated mice in 9 months and 16 months aged PP mice. Larger aggregates of Lcn2 (insert) visible at the 16 months PolyI:C exposed mice. (B) Quantification of % stained area indicates increased Lcn2 expression in the hippocampal CA3 field (n=4-6 mice for 9- and 16-months groups/treatment). (C) Nile red positive lipid droplet staining (violet) across aging in NN & PP mice. (D) Representative triple fluorescence labeling with Nile Red, Iba1 (teal) and NF200 (grey) shows an overlap between the different marker localized lipid droplets in Iba1 positive cells. Arrows indicating NileRed + /Iba1 + cells. (E) Bar chart representing fold change values in the lipid droplet density, mean size, and % stained area per ROI measured. Data are represented as mean±SEM relative to control (n=3-4 mice per group/treatment). *p<0.05, **p<0.005, Student’s t-test;. Scale bar A= 20μm, C,E= 10μm.

Article Snippet: Following antibodies were used for western blot immunolabeling, immunohistochemistry experiments in this study: Rabbit anti pTau 231 (1:500, ab151559, Abcam, UK), Rabbit anti pTau 205 (1:500, ab181206, Abcam, UK), goat anti-tau (1.25 μg/mL; AF3494, R&D systems, USA), mouse anti-β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), Rabbit anti GFAP (1:500, ab68428, Abcam, UK), Goat anti GFAP (1:500, SAB2500462, Sigma), Goat anti Iba1 (1:500, ab5076, Abcam, UK), Mouse anti-amyloid beta 6E10 clone (1:500, cat:803014; Biolegend, USA), rabbit anti-synaptophysin (1:500; ab14692, Abcam, UK), Rabbit anti-amyloid beta 42 (Aβ-42; 1:500; D54D2 XP, Cell Signaling, USA), Rabbit anti-Lipocalin-2/LCN2 (1:500, 50060-RP02; Sino Biological, China), mouse anti-NF200 (1:500, cat:1178709; Boehringer Mannheim Biochemica, Germany), Nile Red (2 μg/mL, N3013, Sigma, USA), Thioflavin-S (100 mM; CAS 1326-12-1, Santa Cruz Biotechnology, USA) The secondary antibodies used in the study for immunolabeling were: directly conjugated to Cy2, Cy3, or Cy5 in donkey raised secondary antibodies (all 1:1000; Jackson Immuno Europe, UK).

Techniques: Staining, Expressing, Fluorescence, Labeling, Marker

Journal: bioRxiv

Article Title: Systemic inflammation causes microglial dysfunction with a mixed AD-like pathology

doi: 10.1101/2020.07.27.223198

Figure Lengend Snippet: Bar plots with Jitter showing the fold changes (FC) of the DEGs (A-F) in moderate AD and severe AD entorhinal cortices as compared to healthy controls (CTRL) and ( H-N) in hippocampal sections from VaD as compared to healthy controls (CTRL). (A and H) FC of cell-type specific markers for astroglia ( GFAP ), microglia ( Iba1 ) and neurons ( MAP2 ). (B and I) FC of glucose metabolism markers, Glpr2 and Ide . (C and J) FC of plasticity markers, Egr2 and Kcnj2 . (D and K) FC of cell signaling markers, c-fos , c-Jun and Notch1 . (E and L) FC of inflammatory and metabolic markers, Lcn2 and Plin4 . (F and M) . FC of vascular markers, Angptl4 , Cyp1b1 and Klf4 . (G and N) Correlation matrix of the selected biomarkers in the two study cohorts. Data are represented as Geometric mean of fold change± Geometric SEM relative to healthy controls. AD= Alzheimer’s disease and VaD=vascular dementia.

Article Snippet: Following antibodies were used for western blot immunolabeling, immunohistochemistry experiments in this study: Rabbit anti pTau 231 (1:500, ab151559, Abcam, UK), Rabbit anti pTau 205 (1:500, ab181206, Abcam, UK), goat anti-tau (1.25 μg/mL; AF3494, R&D systems, USA), mouse anti-β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), Rabbit anti GFAP (1:500, ab68428, Abcam, UK), Goat anti GFAP (1:500, SAB2500462, Sigma), Goat anti Iba1 (1:500, ab5076, Abcam, UK), Mouse anti-amyloid beta 6E10 clone (1:500, cat:803014; Biolegend, USA), rabbit anti-synaptophysin (1:500; ab14692, Abcam, UK), Rabbit anti-amyloid beta 42 (Aβ-42; 1:500; D54D2 XP, Cell Signaling, USA), Rabbit anti-Lipocalin-2/LCN2 (1:500, 50060-RP02; Sino Biological, China), mouse anti-NF200 (1:500, cat:1178709; Boehringer Mannheim Biochemica, Germany), Nile Red (2 μg/mL, N3013, Sigma, USA), Thioflavin-S (100 mM; CAS 1326-12-1, Santa Cruz Biotechnology, USA) The secondary antibodies used in the study for immunolabeling were: directly conjugated to Cy2, Cy3, or Cy5 in donkey raised secondary antibodies (all 1:1000; Jackson Immuno Europe, UK).

Techniques:

Journal: International Journal of Physiology, Pathophysiology and Pharmacology

Article Title: Tumor associated macrophages deliver iron to tumor cells via Lcn2

doi:

Figure Lengend Snippet: Lcn2 could potentially mediate iron accumulation at tumor metastasis stage. A-D. Western blot analysis of TfR and Lcn2 from tumor tissues, and their statistic. *P<0.05 by two way ANOVA.

Article Snippet: Lcn2 Primary antibody (rabbit anti mouse Lcn2 antibody, Sino biological Inc.) or isotype rabbit IgG (Sino biological Inc.) was applied overnight at 4°C in PBS, followed by three washes in PBS, and then biotinylated secondary antibody, streptavidin-HRP and diaminobenzidine (DAB) peroxidase substrate were added.

Techniques: Western Blot

Journal: International Journal of Physiology, Pathophysiology and Pharmacology

Article Title: Tumor associated macrophages deliver iron to tumor cells via Lcn2

doi:

Figure Lengend Snippet: TAMs colocalized with Lcn2 and secreted Lcn2. A. Immunohistochemical staining of Lcn2 (asterisk marks) in the peripheral region of tumor. B. Isotype IgG was used to address the specificity of Lcn2 primary antibody. Scale bar, 50 μm. C. Co-localization of CD68 positive TAMs and Lcn2. Scale bar, 10 μm. red: Lcn2, green: CD68, blue: DAPI. D. Lcn2 was detected in TAMs culture medium by western blot. Three lanes represented three replicates. E. Tumor weights were evaluated on different days after tumor inoculation. F. Percentage of TAMs in tumors tissue at different growth stages. G. Percentage of TAMs after intraperitoneal injection of iron dextran. H. Percentage of TAMs in in situ breast cancer model. I. immunofluorescense staining of CD68 in tumor tissues from control group and DFO group. J, K. The expression of Lcn2 in control and DFO group detected by western blot and the results were repeated three times. n=3. Data was shown as mean ± SD. *P<0.05, **P<0.01 by Student’s t-test.

Article Snippet: Lcn2 Primary antibody (rabbit anti mouse Lcn2 antibody, Sino biological Inc.) or isotype rabbit IgG (Sino biological Inc.) was applied overnight at 4°C in PBS, followed by three washes in PBS, and then biotinylated secondary antibody, streptavidin-HRP and diaminobenzidine (DAB) peroxidase substrate were added.

Techniques: Immunohistochemical staining, Staining, Western Blot, Injection, In Situ, Expressing

Journal: International Journal of Physiology, Pathophysiology and Pharmacology

Article Title: Tumor associated macrophages deliver iron to tumor cells via Lcn2

doi:

Figure Lengend Snippet: ICP/MS detection of iron concentration. A. Transwell with 0.4 μm pore size of membrane only allows secreted small molecules transported into upper chamber. B. ICP/MS detection of iron concentration in 4T1 cells. n=4. *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test. C. TAMs iron concentration before and after co-culture with 4T1 cells. *P<0.05 by Student’s t-test. n=4. D. The expression of Lcn2 in 67NR cells and 4T1 cells. The western blots were repeated three times. E. Iron concentration of 67NR with and without coculture with TAMs. n=4. Data was shown as mean ± SD. **P<0.01, ***P<0.001 by two way ANOVA.

Article Snippet: Lcn2 Primary antibody (rabbit anti mouse Lcn2 antibody, Sino biological Inc.) or isotype rabbit IgG (Sino biological Inc.) was applied overnight at 4°C in PBS, followed by three washes in PBS, and then biotinylated secondary antibody, streptavidin-HRP and diaminobenzidine (DAB) peroxidase substrate were added.

Techniques: Concentration Assay, Co-Culture Assay, Expressing, Western Blot