Journal: Investigative Ophthalmology & Visual Science

Article Title: Protective Effects of Estradiol on Disease Progression in a Murine Model of Fuchs Endothelial Corneal Dystrophy

doi: 10.1167/iovs.66.15.64

Figure Lengend Snippet: In vivo estradiol (E2) treatment ameliorates the corneal endothelial phenotype in an FECD mouse model. ( A ) Effects of E2 on the corneal endothelial phenotype were investigated in Col8a2 Q455K/Q455K mice, a mouse model of FECD, by supplying E2-supplemented drinking water ad libitum. Representative specular microscopy images of the corneal endothelium from the wild-type, FECD, and FECD + E2 groups. FECD mice exhibited extensive guttae formation and enlarged endothelial cells compared to wild-type mice. E2 treatment reduced guttae formation and maintained smaller endothelial cells with a higher cell density compared with untreated FECD mice. ( B ) Quantification of guttae formation in the wild-type ( n = 9), FECD ( n = 11), and FECD + E2 ( n = 12) groups. The percentage of guttae area was significantly lower in the FECD + E2 group (0.55 ± 0.23%) compared with the FECD group (0.97 ± 0.22%, P < 0.001). ( C ) Quantification of endothelial cell density (ECD). The FECD + E2 group maintained a significantly higher endothelial cell density (2,263 ± 177 cells/mm²) compared with the FECD group (2058 ± 118 cells/mm²; P = 0.004). ( D, E ) A sex-stratified analysis shows that E2 administration significantly reduced the percentage of guttae area in both male mice ( D ; FECD, n = 6; FECD + E2, n = 6; P = 0.002) and female mice ( E ; FECD, n = 5; FECD + E2, n = 6; P = 0.018) compared with their respective untreated controls. ( F, G ) Similarly, the analysis of endothelial cell density, stratified by sex, shows that E2 treatment resulted in a significantly higher density in both male mice ( F ; FECD, n = 6; FECD + E2, n = 6; P = 0.049) and female mice ( G ; FECD, n = 5; FECD + E2, n = 6; P = 0.013). ( H, I ) Quantitative PCR analysis shows that the expression of the ECM-related genes FN1 ( H ) and COL1A1 ( I ), which was elevated in the FECD group, was significantly suppressed by E2 administration. ( J ) Immunofluorescence for fibronectin ( green ) revealed minimal deposition in wild-type corneas, whereas its accumulation was evident in the corneas of FECD model mice. This pathological deposition was markedly suppressed following E2 treatment. Nuclei were counterstained with DAPI ( blue ). Images are representative of four independent experiments. Scale bar = 100 µm.

Article Snippet: The TaqMan primers used were GAPDH , Hs00266705_mL; FN1 , Hs00365052_mL; BGN , Hs00959143_mL; and COL1A1 , Hs00164004_mL (Applied Biosystems).

Techniques: In Vivo, Microscopy, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence

Journal: iScience

Article Title: Gasdermin D promotes hyperuricemia-induced renal tubular injury through RIG-I/caspase-1 pathway

doi: 10.1016/j.isci.2023.108463

Figure Lengend Snippet: Knockout of GSDMD alleviated renal tubule morphological changes in OXO/adenine-induced mice and attenuated fibrosis (A and B) Histological changes in renal tubules after HE (A) and Masson (B) staining in different groups. Scale bar 50 μm and 100μm. (C and D) Western blot showing α-SMA levels in renal tissue from different groups (n = 6 animals per group). (C and E) Western blot showing E-cadherin levels in renal tissue from different groups (n = 5 animals per group). (F and G) RT‒PCR analysis of fibronectin (F) and collagen I (G) levels in renal tissue from different groups (n = 5 animals per group). (H) RT‒PCR analysis of IL-1β levels in renal tissue from different groups (n = 6 animals per group). Data for (D)-(H) are presented as mean ± SEM. One-way ANOVA with Student-Newman-Keuls test for (D–H). WT, wild type. Norm-diet, normal diet. UA, uric acid.

Article Snippet: Anti-Fibronectin antibody , Cell Signaling Technology , Cat# 63779.

Techniques: Knock-Out, Staining, Western Blot

Journal: iScience

Article Title: Gasdermin D promotes hyperuricemia-induced renal tubular injury through RIG-I/caspase-1 pathway

doi: 10.1016/j.isci.2023.108463

Figure Lengend Snippet: The primer sequences used in RT‒PCR

Article Snippet: Anti-Fibronectin antibody , Cell Signaling Technology , Cat# 63779.

Techniques:

Journal: iScience

Article Title: Gasdermin D promotes hyperuricemia-induced renal tubular injury through RIG-I/caspase-1 pathway

doi: 10.1016/j.isci.2023.108463

Figure Lengend Snippet: GSDMD encouraged IL-1β expression and fibrosis in NRK-52E cells in vitro (A–D) HK-2 cells were stimulated with uric acid for 48 h. Western blot showing caspase-1 (A and B), GSDMD (A and C) and RIG-I (A and D) levels in HK-2 cells in the different groups (n = 3 per group). (E–H) NRK-52E cells were stimulated with uric acid for 48 h. Western blot showing cleaved-caspase-1 (E and F), caspase-1 (E and G) and RIG-I (E and H) levels in NRK-52E cells in the different group (n = 6 per group). (I–P) NRK-52E cells were transfected with GSDMD-siRNA or NC-siRNA and treated with UA for 48 h. Western blot showing GSDMD (I and J) and GSDMD-N (I and K) levels after UA treatment with or without GSDMD knockdown (n = 6 per group). (L) IL-1β level in culture medium (n = 6 per group). (M–P) Western blot showing α-SMA (M and N), E-cadherin (M and O) and fibronectin (M and P) levels after UA treatment with or without GSDMD knockdown (n = 6 per group). Data for (B–D), (F–H), (J–L) and (N–P) are presented as mean ± SEM. Student’s t test for (B–D) and (F–H). One-way ANOVA with Student-Newman-Keuls test for (J–L) and (N–P). UA, uric acid.

Article Snippet: Anti-Fibronectin antibody , Cell Signaling Technology , Cat# 63779.

Techniques: Expressing, In Vitro, Western Blot, Transfection, Knockdown

Journal: iScience

Article Title: Gasdermin D promotes hyperuricemia-induced renal tubular injury through RIG-I/caspase-1 pathway

doi: 10.1016/j.isci.2023.108463

Figure Lengend Snippet: RIG-I-siRNA decreased HUA-induced fibrosis and pyroptosis molecules expressions in NRK-52E cells in vitro NRK-52E cells were transfected with RIG-I-siRNA or NC-siRNA and treated with UA for 48 h. (A–D) Western blot showing caspase-1 (A and B), GSDMD (A and C) and RIG-I (A and D) expressions after UA treatment with or without RIG-I knockdown (n = 6 per group). (E–G) Western blot showing cleaved-caspase-1 (E and F) and GSDMD-N (E and G) expressions after UA treatment with or without RIG-I knockdown (n = 6 per group). (H) IL-1β level in culture medium (n = 6 per group). (I–K) Western blot showing fibronectin (I and J) and α-SMA (I and K) expressions after UA treatment with or without RIG-I knockdown (n = 6 per group). Data for (B–D), (F–H) and (J–K) are presented as mean ± SEM. One-way ANOVA with Student-Newman-Keuls test for (B–D), (F–H) and (J–K). UA, uric acid.

Article Snippet: Anti-Fibronectin antibody , Cell Signaling Technology , Cat# 63779.

Techniques: In Vitro, Transfection, Western Blot, Knockdown

Journal: iScience

Article Title: Gasdermin D promotes hyperuricemia-induced renal tubular injury through RIG-I/caspase-1 pathway

doi: 10.1016/j.isci.2023.108463

Figure Lengend Snippet:

Article Snippet: Anti-Fibronectin antibody , Cell Signaling Technology , Cat# 63779.

Techniques: Recombinant, H&E Stain, Staining, Activity Assay, Transfection, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Lysis, Bradford Protein Assay, Bicinchoninic Acid Protein Assay, Negative Control, Software, Fluorescence, Microscopy

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the inhibitory effects of epothilone B on scar formation after spinal cord injury

doi: 10.4103/1673-5374.202921

Figure Lengend Snippet: Effects of EpoB on responsive scar formation at the injury site. (A–D) Masson's trichrome staining of the SCI site at different time points. At both time points, the collagenous staining (blue, arrows) was weaker in the EpoB group (SCI + EpoB) than in the (normal saline) vehicle group at (A, B) 3 days post-SCI and (C, D) 21 days post-SCI. Blue staining indicates collagenous material, and red or dark red staining indicates nervous tissue. (E–H) The FN immunofluorescent staining (red, arrows) was reduced in the EpoB group at both (E, F) 3 days post-SCI and (G, H) 21 days post-SCI. (I–L) GFAP staining (red) did not show an apparent difference. (M–P) The β-tubulin staining (red) was stronger in the EpoB group than in the vehicle group. Nuclei are stained with DAPI (blue). Scale bars: (A–D) 500 μm, (E–H) 100 μm, (I–L) 100 μm, and (M–P) 50 μm. (Q, R) Western blot analysis and quantification (gray value: marker/β-actin) confirmed the histochemical staining results. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are expressed as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett's test). EpoB: Epothilone B; SCI: spinal cord injury; GFAP: glial fibrillary acidic protein; DAPI: 4′,6-diamidino-2-phenylindole; FN: fibronectin.

Article Snippet: The following primary antibodies were used: mouse anti–neuron-glial antigen 2 (anti-NG2) (1:200, ab50009; Abcam, Cambridge, UK), rabbit anti-platelet-derived growth factor receptor β (anti-PDGFRβ) (1:100, ab32570; Abcam), rabbit polyclonal anti-glial fibrillary acidic protein (anti-GFAP, 1:100, ZA-0117; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), rabbit anti-β-tubulin (9F3) (1:200, #2128; Cell Signaling Technology, Inc., Danvers, MA, USA), and rabbit polyclonal anti-fibronectin (1:100, ab2413; Abcam).

Techniques: Staining, Western Blot, Marker

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the inhibitory effects of epothilone B on scar formation after spinal cord injury

doi: 10.4103/1673-5374.202921

Figure Lengend Snippet: Expression of NG2, PDGFRβ in injured spinal cord after treatment with EpoB. (A–H) Immunofluorescence staining of the SCI site at different time points and examination under a confocal microscope. (A–D) Number of NG2 (green)-immunoreactive cells was higher in the vehicle group than in the EpoB group. (E–H) Number of PDGFRβ (red)-immunoreactive cells was higher in the vehicle group (normal saline) than in the EpoB group (SCI + EpoB). Nuclei are stained with DAPI (blue). Scale bars: (A–H) 50 μm. (I, J) The quantification (gray value ratio of marker/β-actin) confirmed the histochemical staining results: the NG2 and PDGFRβ expression levels were decreased in the EpoB group. * P < 0.05, ** P < 0.01. Data are expressed as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett's test). FN: Fibronectin; NG2: neuron-glial antigen 2; PDGFRβ: platelet-derived growth factor receptor β; EpoB: epothilone B; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The following primary antibodies were used: mouse anti–neuron-glial antigen 2 (anti-NG2) (1:200, ab50009; Abcam, Cambridge, UK), rabbit anti-platelet-derived growth factor receptor β (anti-PDGFRβ) (1:100, ab32570; Abcam), rabbit polyclonal anti-glial fibrillary acidic protein (anti-GFAP, 1:100, ZA-0117; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), rabbit anti-β-tubulin (9F3) (1:200, #2128; Cell Signaling Technology, Inc., Danvers, MA, USA), and rabbit polyclonal anti-fibronectin (1:100, ab2413; Abcam).

Techniques: Expressing, Immunofluorescence, Staining, Microscopy, Marker, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the inhibitory effects of epothilone B on scar formation after spinal cord injury

doi: 10.4103/1673-5374.202921

Figure Lengend Snippet: EpoB treatment improved the post-SCI behavior recovery and regulated gene expression. (A) EpoB enhanced the expression of β-tubulin III, suppressed the expression of FN, and minimally affected GFAP (* P < 0.05, ** P < 0.01). (B) EpoB suppressed NG2 and PDGFRβ mRNA expression in pericytes (* P < 0.05, ** P < 0.01, *** P < 0.001). (C) Behavioral test with the BBB score system. EpoB treatment (SCI + EpoB) significantly improved hindlimb performance at 2 weeks post-SCI compared with the vehicle group (normal saline) (* P < 0.05, ** P < 0.01). Data are expressed as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett's test). BBB: Basso, Beattie, and Bresnahan locomotor scale; EpoB: epothilone B; SCI: spinal cord injury; GFAP: glial fibrillary acidic protein; FN: fibronectin; NG2: neuron-glial antigen 2; PDGFRβ: platelet-derived growth factor receptor β.

Article Snippet: The following primary antibodies were used: mouse anti–neuron-glial antigen 2 (anti-NG2) (1:200, ab50009; Abcam, Cambridge, UK), rabbit anti-platelet-derived growth factor receptor β (anti-PDGFRβ) (1:100, ab32570; Abcam), rabbit polyclonal anti-glial fibrillary acidic protein (anti-GFAP, 1:100, ZA-0117; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), rabbit anti-β-tubulin (9F3) (1:200, #2128; Cell Signaling Technology, Inc., Danvers, MA, USA), and rabbit polyclonal anti-fibronectin (1:100, ab2413; Abcam).

Techniques: Expressing, Derivative Assay

Journal: JCI insight

Article Title: Intimomedial tears of the aorta heal by smooth muscle cell-mediated fibrosis without atherosclerosis.

doi: 10.1172/jci.insight.172437

Figure Lengend Snippet: Figure 6. ECM changes in intimomedial tears. Aortas with intimomedial tears were analyzed by immunofluorescence confocal microscopy. (A) Increased type I collagen (green) surrounding SMA+ SMCs in tear neointima versus parallel bundles in distant media with (B) greater signal intensity (integrated den- sity, IntDen). (C) Similarly, increased type III collagen (green) in tear neointima with (D) greater signal intensity. (E) Less elastin (white) in tear neointima with punctate appearance or as short, thin fibers versus parallel thick elastic laminae with extensions of intralaminar elastic fibers in the distant media. (F) Absent elastin in other areas of tear neointima, while elastic laminae are fragmented with fewer intralaminar elastic fibers in some tear bases. (G) Increased elastin precursor tropoelastin (green) in tear neointima compared with distant media. (H) Similar levels of fibronectin (green), although with pericellular pattern in tear neointima versus parallel arrangement in distant media. Scale bars: 25 μm. Data are means, with lines connecting values from individual specimens (n = 9). **P < 0.01 by paired, 2-tailed t test.

Article Snippet: Sections were incubated overnight at 4°C with antibodies against SMA (1-9760-82, Thermo Fisher Scientific or ab5694, Abcam), SMMHC (53-6400-82, Thermo Fisher Scientific), CD31 (ab9498, Abcam), CD45 (LS-B14248300, Lifespan Biosciences), decorin (HPA003315, Atlas Antibodies), PCNA (13110S, Cell Signaling Technology), CD34 (MA5-16924, Thermo Fisher Scientific), calnexin (ab22595, Abcam), LAMP-2 (ab25631, Abcam), PGC-1α (66369-1-Ig, Proteintech), TFAM (22586-1-AP, Proteintech), collagen I (72026, Cell Signaling Technology), collagen III (22734-1-AP, Proteintech), tropoelastin (PR398, Elastin Products Company), fibronectin (15613-1-AP, Proteintech), glycophorin-A (13-9987-82, Thermo Fisher Scientific), albumin (16475-1-AP, Proteintech), fibrin (55169, Cappel), perilipin-1 (9349, Cell Signaling Technology), and perilipin-2 (MAB76341, R&D Systems).

Techniques: Immunofluorescence, Confocal Microscopy