Journal: Investigative ophthalmology & visual science

Article Title: Untargeted Metabolomics Reveals the Role of Lipocalin-2 in the Pathological Changes of Lens and Retina in Diabetic Mice.

doi: 10.1167/iovs.65.14.19

Figure Lengend Snippet: FIGURE 2. Pathological changes in STZ-induced diabetic mice. (A) Changes in blood glucose and body weight of WT (WT-STZ) and LCN2−/−(LCN2−/−-STZ) mice induced by intraperitoneal injections of STZ. The control group received sodium citrate solution (WT-SC and LCN2−/−-SC). The observation period spanned 12 weeks after STZ injection. ns (blue), compared to the WT-SC; ns (green), compared to the WT-STZ. ****P < 0.0001 compared to the WT-SC (n = 24). (B) Representative images of lenses in four groups of mice. Scale bar: 1 mm. (C) Representative fluorescence signal images of flatmounted retinas after injection of Evans blue dye. Scale bar: 100 μm (n = 4). (D) Representative images of retinal OCT in four groups of mice (n = 3). (E) Representative retinal H&E staining images for the four groups. Scale bar: 50 μm (n = 3).(F, G) Quantitative analysis of total retinal and inner plexiform layer thickness (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Mice in the STZ groups were intraperitoneally injected with STZ solution (50 mg/kg for 5 days, MP Biomedicals, Santa Ana, CA, USA).

Techniques: Control, Injection, Fluorescence, Staining

Journal: Investigative ophthalmology & visual science

Article Title: Untargeted Metabolomics Reveals the Role of Lipocalin-2 in the Pathological Changes of Lens and Retina in Diabetic Mice.

doi: 10.1167/iovs.65.14.19

Figure Lengend Snippet: FIGURE 3. Quality control of untargeted metabolomics data. In each image of Fig. 3, A–D represent the LCN2−/−-STZ group, WT-STZ group, LCN2−/−-SC group, and WT-SC group of the lenses and E–H represent the LCN2−/−-STZ group, WT-STZ group, LCN2−/−-SC group, and WT-SC group of the retinas. (A) Principal component analysis of the lens and retinal groups in cationic mode. (B) Principal component analysis of lens and retinal groups in anion mode. (C, D) Score plots and permutation analysis plot of OPLS-DA among the four lens groups from STZ-induced WT mice and LCN2−/−mice in cationic mode. (E, F) Score plots and permutation analysis plot of OPLS-DA among the four retina groups from STZ-induced WT mice and LCN2−/−mice in cationic mode.

Article Snippet: Mice in the STZ groups were intraperitoneally injected with STZ solution (50 mg/kg for 5 days, MP Biomedicals, Santa Ana, CA, USA).

Techniques: Control

Journal: Investigative ophthalmology & visual science

Article Title: Untargeted Metabolomics Reveals the Role of Lipocalin-2 in the Pathological Changes of Lens and Retina in Diabetic Mice.

doi: 10.1167/iovs.65.14.19

Figure Lengend Snippet: FIGURE 4. Metabolic changes in mice lenses by STZ induction. In each image of Fig. 4, (B) and (D) represent the WT-STZ group (n = 9) and WT-SC group (n = 8) of the lenses. (A) Volcano plot of 136 differential metabolites. Compared to the WT-SC group, metabolites that were significantly upregulated in the WT-STZ group are marked in red, with VIP > 1, P < 0.05, and fold change (FC) > 1.5. Metabolites that were remarkedly downregulated in the WT-STZ group are marked in blue. Metabolites with VIP > 1, P < 0.05, and FC < 1.5 are indicated in yellow. Non-significant metabolites are represented in gray. The size of the dots corresponds to the magnitude of the VIP values. (B) Heatmap of 136 differential metabolites. Red represents significantly upregulated metabolites in the WT-STZ group compared to the WT-SC group, and blue represents downregulated metabolites. (C) Classification of 136 differential metabolites. (D) The top 20 differential abundance score with P < 0.05 based on KEGG enrichment analysis. DA-score = (number of upregulated metabolites −number of downregulated metabolites)/(total number of differential metabolites in the pathway). (E) Chord plots of the top 10 KEGG enrichment terms with P < 0.05. (F) Circle plots of the top 10 KEGG enrichment terms with P < 0.05. Red represents metabolites with elevated expression, and blue represents metabolites with decreased expression.

Article Snippet: Mice in the STZ groups were intraperitoneally injected with STZ solution (50 mg/kg for 5 days, MP Biomedicals, Santa Ana, CA, USA).

Techniques: Expressing

Journal: Investigative ophthalmology & visual science

Article Title: Untargeted Metabolomics Reveals the Role of Lipocalin-2 in the Pathological Changes of Lens and Retina in Diabetic Mice.

doi: 10.1167/iovs.65.14.19

Figure Lengend Snippet: FIGURE 5. Metabolic alterations in lenses of STZ-induced WT and LCN2−/−mice. In each image of Fig. 5, A and B represent the LCN2−/−- STZ group (n = 8) and WT-STZ group (n = 9) of the lenses. (A) Volcano plot of 54 differential metabolites. Compared to the WT-STZ group, metabolites that were significantly upregulated in the LCN2−/−-STZ group are marked in red, with VIP > 1, P < 0.05, and FC > 1.5. Metabolites that were remarkedly downregulated in the LCN2−/−-STZ group are marked in blue. Metabolites with VIP > 1, P < 0.05, and FC < 1.5 are indicated in yellow. Non-significant metabolites are shown in gray. The size of the dots corresponds to the magnitude of the VIP values. (B) Heatmap of 54 differential metabolites. Red represents significantly upregulated metabolites in the LCN2−/−-STZ group compared to the WT-STZ group, and blue represents downregulated metabolites. (C) Chord plots of the top 10 KEGG enrichment terms with P < 0.05. (D) The top 20 differential abundance score with P < 0.05 based on KEGG enrichment analysis. (E) The relative expression levels of differential metabolites in the top three KEGG pathways between the two groups. *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: Mice in the STZ groups were intraperitoneally injected with STZ solution (50 mg/kg for 5 days, MP Biomedicals, Santa Ana, CA, USA).

Techniques: Expressing

Journal: Investigative ophthalmology & visual science

Article Title: Untargeted Metabolomics Reveals the Role of Lipocalin-2 in the Pathological Changes of Lens and Retina in Diabetic Mice.

doi: 10.1167/iovs.65.14.19

Figure Lengend Snippet: FIGURE 6. Metabolic changes in mice retinas by STZ induction. In each image of Fig. 6, F and H represent the WT-STZ group (n = 8) and WT-SC group (n = 8) of the retinas. (A) Volcano plot of 218 differential metabolites. Compared to the WT-SC group, metabolites that were significantly upregulated in the WT-STZ group are marked in red, with VIP > 1, P < 0.05, and FC > 1.5. Metabolites that were remarkedly downregulated in the WT-STZ group are indicated in blue. Metabolites with VIP > 1, P < 0.05, and FC < 1.5 are indicated in yellow. Non-significant metabolites are represented in gray. The size of the dots corresponds to the magnitude of the VIP values. (B) Heatmap of 218 differential metabolites. Red represents significantly upregulated metabolites in the WT-STZ group compared to the WT-SC group, and blue represents downregulated metabolites. (C) Classification of 218 differential metabolites. (D) The top 20 DA-scores with P < 0.05 based on KEGG enrichment analysis. (E) Chord plots of the top 10 KEGG enrichment terms with P < 0.05. (F) Circle plots of the top 10 KEGG enrichment terms with P < 0.05. Red represents metabolites with elevated expression, and blue represents metabolites with decreased expression.

Article Snippet: Mice in the STZ groups were intraperitoneally injected with STZ solution (50 mg/kg for 5 days, MP Biomedicals, Santa Ana, CA, USA).

Techniques: Expressing

Journal: Investigative ophthalmology & visual science

Article Title: Untargeted Metabolomics Reveals the Role of Lipocalin-2 in the Pathological Changes of Lens and Retina in Diabetic Mice.

doi: 10.1167/iovs.65.14.19

Figure Lengend Snippet: FIGURE 7. Metabolic alterations in retinas of STZ-induced WT and LCN2−/−mice. In each image of Fig. 7, E and F represent the LCN2−/−- STZ group (n = 8) and WT-STZ group (n = 9) of the retinas. (A) Volcano plot of 35 differential metabolites. Compared with the WT-STZ group, metabolites that were significantly upregulated in the LCN2−/−-STZ group are marked in red, with VIP > 1, P < 0.05, and FC > 1.5. Metabolites that were remarkedly downregulated in the LCN2−/−-STZ group are marked in blue. Metabolites with VIP > 1, P < 0.05, and FC < 1.5 are indicated in yellow. Non-significant metabolites are represented in gray. The size of the dots corresponds to the magnitude of the VIP values. (B) Heatmap of 35 differential metabolites. Red represents significantly upregulated metabolites in the LCN2−/−-STZ group compared to the WT-STZ group, and blue represents downregulated metabolites. (C) Chord plots of the top 10 KEGG enrichment terms with P < 0.05. (D) The top 20 differential abundance score with P < 0.05 based on KEGG enrichment analysis. (E) Metabolites involving multiple enriched metabolic pathways. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Mice in the STZ groups were intraperitoneally injected with STZ solution (50 mg/kg for 5 days, MP Biomedicals, Santa Ana, CA, USA).

Techniques:

Journal: Investigative ophthalmology & visual science

Article Title: Untargeted Metabolomics Reveals the Role of Lipocalin-2 in the Pathological Changes of Lens and Retina in Diabetic Mice.

doi: 10.1167/iovs.65.14.19

Figure Lengend Snippet: FIGURE 8. Lens and retinas change metabolites in STZ-induced WT mice. In each image of Fig. 8, B and D represent the WT-STZ (n = 9) group and WT-SC group (n = 8) of the lenses, and F and H represent the WT-STZ group (n = 8) and WT-SC group (n = 8) of the retinas. (A) Overlap of differential metabolites in different groups. (B) Relative expression of differential metabolites in different groups. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001.

Article Snippet: Mice in the STZ groups were intraperitoneally injected with STZ solution (50 mg/kg for 5 days, MP Biomedicals, Santa Ana, CA, USA).

Techniques: Expressing

Journal: Investigative ophthalmology & visual science

Article Title: Untargeted Metabolomics Reveals the Role of Lipocalin-2 in the Pathological Changes of Lens and Retina in Diabetic Mice.

doi: 10.1167/iovs.65.14.19

Figure Lengend Snippet: FIGURE 9. Lens and retinas change metabolites in STZ-induced WT mice compared with LCN2−/−mice. In each image of Fig. 9, A and B represent the LCN2−/−-STZ group (n = 8) and WT-STZ group (n = 9) of the lenses, and E and F represent the LCN2−/−-STZ group (n = 8) and WT-STZ group (n = 9) of the retinas. (A) Overlap of differential metabolites in different groups. (B) Relative expression of differential metabolites in different groups. *P < 0.05, **P < 0.01, ##P < 0.01, ###P < 0.001.

Article Snippet: Mice in the STZ groups were intraperitoneally injected with STZ solution (50 mg/kg for 5 days, MP Biomedicals, Santa Ana, CA, USA).

Techniques: Expressing

Journal: eLife

Article Title: RANK + TLR2 + myeloid subpopulation converts autoimmune to joint destruction in rheumatoid arthritis

doi: 10.7554/eLife.85553

Figure Lengend Snippet: ( A ) Dimensional reduction projection of monocyte and macrophage cells expressing RANK, TRAP, and/or TLR2 from all sample groups onto two dimensions using principal components, depicting values of Slingshot pseudotime followed by gene expression levels of St3gal4, Tlr2, Tnfrsf11a, and Acp5 . ( B ) A heat map of dynamic expression of St3gal4, Tlr2, Tnfrsf11a, and Acp5 in monocyte and macrophage cells expressing RANK, TRAP, and/or TLR2 from all sample groups. Pseudotime values and annotated cell types are depicted in the color-coded bars atop the heat map. ( C ) Representative images of TRAP staining and immunofluorescence co-staining for RANK (red), TLR2 (green), and DAPI (blue) staining for nuclei for bone marrow macrophages cultured with macrophage colony stimulating factor (M-CSF) ( + ng/ml) and RANKL (200 ng/ml) for 0, 1, 3, and 5 days. Scale bar, 0.1 mm. ( D ) Quantitative analysis of the percentage of TRAP + cells in bone marrow macrophages at different time points (n=4 or 5, one-way ANOVA with Tukey’s multiple comparisons test). ( E–G ) Quantitative analysis of the mean intensity of RANK and TLR2 positive fluorescence, and the percentage of RANK + TLR2 + positive cells in bone marrow macrophages at different time points (n=5 or 6, one-way ANOVA with Tukey’s multiple comparisons test). All data are means ± SD. N.S=No Significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The sections were incubated with primary antibodies against RANK (1:100, ab13918, Abcam), TLR2 (1:100, mAb12276, Cell Signaling), Maackia Amurensis Lectin (1:100, B-1265–1, Vector Laboratories), ST3GAL4 (1:100, 13546–1-AP, Proteintech), c-Fos (1:100, mAb2250, Cell Signaling), or ST3GAL1 (1:50, PA5-21721, Thermo Fisher Scientific) overnight at 4 °C.

Techniques: Expressing, Gene Expression, Staining, Immunofluorescence, Cell Culture, Fluorescence

Journal: eLife

Article Title: RANK + TLR2 + myeloid subpopulation converts autoimmune to joint destruction in rheumatoid arthritis

doi: 10.7554/eLife.85553

Figure Lengend Snippet: ( A ) Representative histograms of sialic acid α(2,3)-modified cells in the bone marrow of DBA control mice and CIA mice 2 months after the first immunization. ( B ) Quantitative analysis of the frequency of sialic acid α(2,3)-positive cells in DBA mice and CIA mice (n=4, t -test). ( C ) Representative images of the biotin fluorescence staining for sialic acid α(2,3) (green) and of DAPI staining (blue) of the knee joint sections from DBA mice and CIA mice 2 months after the first immunization. Scale bar, 0.5 mm. ( D ) Quantitative analysis of the mean intensity of sialic acid α(2,3)-positive fluorescence of the images represented in ( C ) (n=6, t -test). ( E ) Heat maps of dynamic expression of marker genes Tlr2, Tnfrsf11a, Acp5, sialyltransferases, and Fos in the RANK + TLR2 − myeloid cells (top) and RANK + TLR2 + myeloid cells (bottom). ( F ) Representative images of the immunofluorescence staining for c-Fos (green) of the knee joint section from DBA mice and CIA mice 2 months after the first immunization. Scale bar, 0.5 mm. ( G ) Quantitative analysis of the mean intensity of c-Fos-positive fluorescence in ( F ) (n=5, t -test). ( H–I ) The mRNA expression changes of c-Fos in the knee joint tissue of CIA and DBA mice ( H ) or in the RANK + TLR2 − and RANK + TLR2 + monocytes ( I ) (n=3, t -test). ( J ) Representative images of the immunofluorescence co-staining for RANK (red) and ST3GAL4 (green) of the knee joint sections from DBA mice and CIA mice 2 months after the first immunization. ( K ) Quantitative analysis of the RANK + ST3GAL4 + cells in ( J ) (n=5, t -test). ( L ) The electrophoresis of CHIP assay for the binding sites of c-Fos in the promoter sequence of the St3gal4 gene. ( M ) The diagram of different locations and sequences of the putative binding sites for c-Fos in the promoter of St3gal4 gene. *p<0.05, ***p<0.001, ****p<0.0001. Figure 6—source data 1. Gel blot of CHIP assay of St3gal4 gene expression.

Article Snippet: The sections were incubated with primary antibodies against RANK (1:100, ab13918, Abcam), TLR2 (1:100, mAb12276, Cell Signaling), Maackia Amurensis Lectin (1:100, B-1265–1, Vector Laboratories), ST3GAL4 (1:100, 13546–1-AP, Proteintech), c-Fos (1:100, mAb2250, Cell Signaling), or ST3GAL1 (1:50, PA5-21721, Thermo Fisher Scientific) overnight at 4 °C.

Techniques: Modification, Control, Fluorescence, Staining, Expressing, Marker, Immunofluorescence, Electrophoresis, Binding Assay, Sequencing, Western Blot, Gene Expression