Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: (A) Experimental scheme of AAV8 injection in cholestatic mice. Retro-orbital injection of AAV8-VEGF-C or AAV8-GFP(control) (1.5×10 11 GC/mouse) was conducted 7 days before BDL surgery. Two weeks after BDL surgery, liver samples were collected for analysis. (B, C) VEGF-C mRNA and protein levels in liver lysates from 2-week BDL mice treated with AAV8-GFP (n=7) or AAV8-VEGF-C (n=7). (D) Liver and spleen weights in mice treated with AAV8-GFP (Control, n=11) or AAV8-VEGF-C (n=9). (E) Immunofluorescence (IF) images of LYVE-1 [Lymphatic vessel (LV); red) and DAPI as well as its quantification (F, G) of LV number and area in livers from 2-week BDL mice treated with AAV8-GFP(n=7) or AAV8-VEGF-C(n=7). (H) Liver lymphatic drainage function in 2-week BDL mice given AAV8-GFP (n=6) or AAV8-VEGF-C (n=7). (I-L) Evaluation of liver fibrosis and damage. Representative Sirius Red staining images (I) and its quantification (J) as well as hydroxyproline level (K), serum ALT levels (L) in livers from 2-week BDL mice treated with AAV8-GFP(n=7) or AAV8-VEGF-C(n=7). (M) Expression of liver fibrotic makers: collagen I (ColI), collagen III (Col III), fibronectin 1 (FN1), and α SMA by western blotting and the quantification (N). AAV8-GFP (n=7) and AAV8-VEGF-C (n=7). *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 μm. PV, portal vein.

Article Snippet: To examine the effect of enhanced lymphangiogenesis by VEGF-C overexpression, mice were randomly divided into two groups, one given Adeno-Associated Virus (AAV)8-mouse VEGF-C (Vector Biolabs, Malvern, PA) and the other given AAV-GFP (control) (Cat#37825-AAV8, Addgene, Watertown, MA).

Techniques: Injection, Control, Immunofluorescence, Staining, Expressing, Western Blot

Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: (A) Representative images of Sirius red staining. (B) Immunofluorescence (IF) images and quantification of hepatic artery (HA) number and portal vein (PV) area in livers from control(n=7) and AAV8-VEGF-C (n=7) treated 2-week BDL mice. (C) IF images and quantification of bile duct (CK19; red) in livers from control(n=7) and AAV8-VEGF-C(n=7)-injected 2-week BDL mice. Scale bars: 100μm.

Article Snippet: To examine the effect of enhanced lymphangiogenesis by VEGF-C overexpression, mice were randomly divided into two groups, one given Adeno-Associated Virus (AAV)8-mouse VEGF-C (Vector Biolabs, Malvern, PA) and the other given AAV-GFP (control) (Cat#37825-AAV8, Addgene, Watertown, MA).

Techniques: Staining, Immunofluorescence, Control, Injection

Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: Immunofluorescence (IF) images and quantification of CD68 (macrophages; red), CD3 (T-cells; red), MPO (neutrophils; red) and CD19 (B cells; red) in livers from control (n=7) and AAV8-VEGF-C (n=7) treated 2-week BDL mice. *p< 0.05. Scale bars: 100 μm.

Article Snippet: To examine the effect of enhanced lymphangiogenesis by VEGF-C overexpression, mice were randomly divided into two groups, one given Adeno-Associated Virus (AAV)8-mouse VEGF-C (Vector Biolabs, Malvern, PA) and the other given AAV-GFP (control) (Cat#37825-AAV8, Addgene, Watertown, MA).

Techniques: Immunofluorescence, Control

Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: (A) Violin plots of gene expression of Flt1 , Kdr , and Flt4 in livers from healthy(n=3) and cirrhotic(n=3) mice (Cdh5-Cre-mTmG +/+ mice) (GSE147581). (B) Immunofluorescence (IF) images and (C) quantification of CD34 (a marker of LSEC capitalization; red) in livers from control(n=7) and AAV8-VEGF-C (n=7) administered 2-week BDL mice. *p< 0.05.

Article Snippet: To examine the effect of enhanced lymphangiogenesis by VEGF-C overexpression, mice were randomly divided into two groups, one given Adeno-Associated Virus (AAV)8-mouse VEGF-C (Vector Biolabs, Malvern, PA) and the other given AAV-GFP (control) (Cat#37825-AAV8, Addgene, Watertown, MA).

Techniques: Expressing, Immunofluorescence, Marker, Control

Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: (A) Experimental scheme of AAV8 injection in rats. Retro-orbital injection was performed for AAV8-VEGF-C or AAV8-GFP(control) (1.5×10 11 GC/rat) 7 days before BDL surgery. Four weeks after BDL surgery, liver samples were collected for analysis. Evaluation of liver fibrosis (B) and hemodynamic characterization performed for Hepatic vascular resistance (C); portal pressure (D); mean arterial pressure (E); systemic vascular resistance (SVR)(F); cardiac index(G); superior mesenteric artery (SMA) resistance(H); splenorenal shunt (SRS) flow(I); and SRS resistance(J), measured 4 weeks after BDL or sham surgery. Sham-operated AAV8-GFP rat (n=9), 4-week BDL rats treated with AAV8-VEGF-C (n=9) or AAV8-GFP(n=9). *P < 0.05, **P < 0.01, and ***P < 0.001. PH, portal hypertension.

Article Snippet: To examine the effect of enhanced lymphangiogenesis by VEGF-C overexpression, mice were randomly divided into two groups, one given Adeno-Associated Virus (AAV)8-mouse VEGF-C (Vector Biolabs, Malvern, PA) and the other given AAV-GFP (control) (Cat#37825-AAV8, Addgene, Watertown, MA).

Techniques: Injection, Control

Journal: Translational Neurodegeneration

Article Title: Critical role of ROCK1 in AD pathogenesis via controlling lysosomal biogenesis and acidification

doi: 10.1186/s40035-024-00442-9

Figure Lengend Snippet: ROCK1 decreases lysosomal biogenesis and impairs lysosomal acid environment. a Schematic illustration of the experimental procedure. Image created with BioRender.com. b Heatmap of mRNA expression of ROCK1 and lysosome-related markers detected with qRT-PCR in HEK-293T cells transfected with control or ROCK1-overexpression plasmid for 48 h. n = 3. Red color, higher expression; blue color, lower expression. c Immunoblotting of lysosome-related markers in HEK-293T cells and quantitation of their protein levels. n = 3. d Transmission electron microscopy of lysosomes in HEK-293T cells. The number of lysosomes was analyzed. n = 10 cells per group. Scale bar, 2 μm. White arrows indicate lysosomes. e Lysotracker Red, Magic Red B and DQ BSA staining in HEK-293T cells. Fluorescence intensities were analyzed. n = 5. Scale bars, 25 μm. f Standard curve of LysoSensor to determine the lysosomal pH values of HEK-293T cells. g Lysosomal pH of HEK-293T cells was determined by LysoSensor. n = 5. h Schematic illustration of the experimental procedure. Image created with BioRender.com. i AAV-Vector and AAV-ROCK1 were successfully microinjected into the hippocampus of WT mice, respectively. GFP (green) was used to visualize viral diffusion. Scale bar, 200 μm. j qRT-PCR analysis of expression levels of indicated mRNAs. n = 3. k Transmission electron microscopy of lysosomes in the hippocampus. The number of lysosomes was analyzed. n = 9 slices from 3 mice. Scale bars, 1 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 versus vector ( b - e , g ) or AAV-Vector group ( j , k ), Student’s t -test

Article Snippet: The AAV carrying either control shRNA with GFP (AAV-sh-Con) (4 µl, 5.8 × 10 12 viral genomes/µl, Sangon Biotech, Shanghai, China) or the ROCK1 shRNA with GFP (AAV-sh-ROCK1) (4 µl, 4.4 × 10 12 viral genomes/µl, Sangon Biotech) was microinjected into the hippocampus of mice using the following microinjection coordinates: anteroposterior − 2.06 mm, lateral ± 1.5 mm, and ventral + 2.0 mm (stereotaxic coordinates from bregma).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Over Expression, Plasmid Preparation, Western Blot, Quantitation Assay, Transmission Assay, Electron Microscopy, Staining, Fluorescence, Diffusion-based Assay

Journal: Translational Neurodegeneration

Article Title: Critical role of ROCK1 in AD pathogenesis via controlling lysosomal biogenesis and acidification

doi: 10.1186/s40035-024-00442-9

Figure Lengend Snippet: Downregulation of ROCK1 promotes TFEB nuclear translocation and lysosomal biogenesis in the brains of APP/PS1 mice. a Timeline of experimental procedure and confocal images of brain sections microinjected with adeno-associated virus carrying control shRNA (AAV-sh-Con) or ROCK1 shRNA (AAV-sh-ROCK1). GFP (green) was used to visualize viral diffusion. Scale bar, 200 μm. b PLA and quantification of ROCK1 and TFEB in the hippocampal CA3 region of APP/PS1 mice microinjected with AAV-sh-Con/AAV-sh-ROCK1. n = 3. Scale bar, 50 μm. c Immunoblotting analysis and quantification of indicated proteins in the hippocampus of APP/PS1 mice microinjected with AAV-sh-Con/AAV-sh-ROCK1. n = 3. d Immunoblotting of cytoplasmic and nuclear TFEB. n = 3. e Transmission electron microscopy of lysosomes in the hippocampus. Number of lysosomes was analyzed. n = 9 slices from 3 mice. Scale bar, 2 μm. White arrows indicate lysosomes. f , g Immunostaining of Aβ (red) and CD68 (green) in the hippocampus of APP/PS1 mice microinjected with AAV-sh-Con/AAV-sh-ROCK1, and co-localization coefficient for Aβ and CD68. n = 3. Scale bar, 50 μm. h qRT-PCR analysis of expression of indicated mRNAs. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001 versus APP/PS1-AAV-sh-Con group, Student’s t -test

Article Snippet: The AAV carrying either control shRNA with GFP (AAV-sh-Con) (4 µl, 5.8 × 10 12 viral genomes/µl, Sangon Biotech, Shanghai, China) or the ROCK1 shRNA with GFP (AAV-sh-ROCK1) (4 µl, 4.4 × 10 12 viral genomes/µl, Sangon Biotech) was microinjected into the hippocampus of mice using the following microinjection coordinates: anteroposterior − 2.06 mm, lateral ± 1.5 mm, and ventral + 2.0 mm (stereotaxic coordinates from bregma).

Techniques: Translocation Assay, Virus, Control, shRNA, Diffusion-based Assay, Western Blot, Transmission Assay, Electron Microscopy, Immunostaining, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 ablation in keratinocytes ameliorates the psoriasiform phenotype. A) Representative images of the dorsal back from mice, and mice PASI scores were depicted (n = 5). B) Representative histological sections of the dorsal back from Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice treated by Vaseline or IMQ for 9 days stained with H&E, and quantification of the epidermal thickness and the infiltrating cells (n = 5). Scale bars = 100 µm. C) Immunoblotting analysis of Cyclin A1, Cyclin D1, and Cyclin E1 protein levels in Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with Vaseline or IMQ for 9 days. β‐Actin was used as a loading control (n = 6). D) Immunofluorescent and quantitative analysis of Ki‐67 positive cells in the skin from Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with Vaseline or IMQ for 9 days. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 100 µm. E) Immunofluorescent and quantitative analysis of K6 in the skin from Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with Vaseline or IMQ for 9 days. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 100 µm. F) qRT‐PCR analysis for IL‐17 , CCL2, CXCL2, S100A8 and IL‐1β mRNA levels in the skin from Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with IMQ for 9 days (n = 5). Error bars show the mean ± SEM. **p < 0.01; ***p < 0.001. &&& p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (A and F) or one‐way ANOVA (B‐E). All numbers (n) are biologically independent experiments.

Article Snippet: AAV expressing GFP under the control of the skin epidermal keratinocyte‐specific Krt14 promoter (Genechem, GOSV0207037_1): AAV9‐ Krt14 GFP (AAV‐ GFP ) or AAV9‐ Krt14 sh‐ p53 (AAV‐sh‐ p53 ) was injected subcutaneously using 35‐gauge needle around the specific region of dorsal skin.

Techniques: Staining, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 promotes proliferation and cell cycle transition of keratinocytes through p53 signaling. A–C) KEGG analysis for the significantly upregulated signaling by the interference of si‐Scr or si‐ Fgf12 in HaCaT cells treated by M5 for 12 h. The Top 15 upregulated GO signal pathways were listed. D) GSEA showing the significant enrichment of p53 signaling in M5 treated HaCaT cells under FGF12 interference. E) Immunoblotting and quantitative analysis of p53 and p21 protein levels in NHEK cells that were treated with si‐Scr or si‐ Fgf12 and stimulated with M5 for 12 h. β‐Actin was used as a loading control (n = 4). F) Immunoblotting and quantitative analysis of p53 and p21 levels in Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with IMQ. β‐Actin was used as a loading control (n = 6). Error bars show the mean ± SEM. **p < 0.01; ***p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (E and F). All numbers (n) are biologically independent experiments.

Article Snippet: AAV expressing GFP under the control of the skin epidermal keratinocyte‐specific Krt14 promoter (Genechem, GOSV0207037_1): AAV9‐ Krt14 GFP (AAV‐ GFP ) or AAV9‐ Krt14 sh‐ p53 (AAV‐sh‐ p53 ) was injected subcutaneously using 35‐gauge needle around the specific region of dorsal skin.

Techniques: Western Blot, Control, Two Tailed Test

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: Loss of p53 abolishes the mitigatory effects of FGF12 knockdown on psoriasis in mice. A) Immunofluorescence images for the skin of mice with knock‐down respective genes were labeled with the indicated antibodies. Nuclei were stained with DAPI (blue). Scar bar = 50 µm. B) Immunoblotting and quantitative analysis of p53 protein level in AAV‐ GFP and AAV‐sh‐ p53 mice. β‐Actin was used as a loading control (n = 5). C) Representative histological sections of the dorsal back from Krt14 +/+ ‐Fgf12 f/f ; AAV‐ GFP , Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐ GFP and Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐sh‐ p53 mice treated by IMQ stained with H&E, and quantification of the epidermal thickness and the infiltrating cells (n = 5). Scale bars = 100 µm. D) Immunofluorescent and quantitative analysis of K6 in the skin from Krt14 +/+ ‐Fgf12 f/f ; AAV‐ GFP , Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐ GFP and Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐sh‐ p53 mice induced by IMQ. Nuclei were stained with DAPI (blue) (n = 5). Scale bars = 50 µm. E) Immunofluorescent and quantitative analysis of Ki‐67 positive cells in the skin from Krt14 +/+ ‐Fgf12 f/f ; AAV‐ GFP , Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐ GFP and Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐sh‐ p53 mice stimulated by IMQ. Nuclei were stained with DAPI (blue) (n = 5). Scale bars = 100 µm. F) Immunoblotting of Cyclin A1, Cyclin D1, and Cyclin E1 protein levels in the skin from Krt14 +/+ ‐Fgf12 f/f ; AAV‐ GFP , Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐ GFP and Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐sh‐ p53 mice treated by IMQ. β‐Actin was used as a loading control (n = 6). Error bars show the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. & p < 0.05; && p < 0.01; &&& p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (B) or one‐way ANOVA (C‐F). All numbers (n) are biologically independent experiments.

Article Snippet: AAV expressing GFP under the control of the skin epidermal keratinocyte‐specific Krt14 promoter (Genechem, GOSV0207037_1): AAV9‐ Krt14 GFP (AAV‐ GFP ) or AAV9‐ Krt14 sh‐ p53 (AAV‐sh‐ p53 ) was injected subcutaneously using 35‐gauge needle around the specific region of dorsal skin.

Techniques: Knockdown, Immunofluorescence, Labeling, Staining, Western Blot, Control, Two Tailed Test