anti serpinf1  (R&D Systems)

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet: In vivo expression of eGFP, PEDF, sFlt-1, and sCD59 following IVT administration of AdCs (A) Representative fundus images of the retina from individual mice following IVT injection of 1.5×10 9 vp and 7.5×10 9 vp AdC68-eGFP. The eGFP signal could be detected from 48 h to 35 days post-injection. The dotted circles represent the edge of mouse retina. (B–D) Assessment of PEDF, sFlt-1, and sCD59 mRNA expression in retina-choroid-sclera complexes isolated from 10 mice. In each mouse, one eye was injected with AdC68-PFC (five mice for 1.5×10 9 vp and five mice for 7.5×10 9 vp) whereas the contralateral, un-injected eye served as control (only five eyes were used for analysis). At 4 days post-injection, RNA was purified from the retina-choroid complexes and real-time qPCR was conducted. Absolute number of mRNA copies were calculated using the standard curve method. (E–H) Images of western blot and quantification of the PEDF, sFlt-1, and sCD59 protein amount expressed in retina-choroid complexes of five mice. In each mouse, one eye was injected with AdC68-PFC (7.5×10 9 vp) whereas the contralateral, un-injected eye served as control. Total protein was obtained from retina-choroid-sclera complexes isolated from AdC68-PFC-treated (7.5×10 9 vp) and un-injected eyes 7 days post-injection. Antibodies against GAPDH were used for the internal control. The relative expression of PEDF, sFlt-1, and sCD59 in the un-injected eyes was set to 1. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA multiple comparisons with Tukey’s method among groups in (B) and Student’s t test (two-tailed) in (C) (∗p < 0.05, ∗∗p < 0.01). PEDF, pigment epithelium-derived factor; sFlt-1, soluble fms-like tyrosine kinase-1; sCD59, soluble forms of CD59; IVT, intravitreal.

Article Snippet: PVDF membranes were blocked with 5% milk in PBST (PBS+Tween-20) for 2 h at RT and then target proteins were detected by specific primary antibodies including PEDF (1:500, 11104-RP02, Sino Biological, China), VEGFR1 (1:500, AF7748, Affinity, US), sCD59 (1:500, 12474-RP02, Sino Biological, China), Erk1/2 p44/42 (1:1000, 9102, Cell Signaling Technology, US), Erk1/2 phospho-p44/42 (T202/Y204) (1:1000, 9106, Cell Signaling Technology, US), p38 MAPK(1:1000, 8690, Cell Signaling Technology, US), Phospho-p38 MAPK (Thr180/Tyr182) (1:1000, 4631, Cell Signaling Technology, US), ICAM-1 (1:1000, 10831-1-AP, Proteintech, US), VCAM-1 (1:200, sc-13160 , Santa Cruz biotechnology, US).

Techniques: In Vivo, Expressing, Injection, Isolation, Control, Purification, Western Blot, Two Tailed Test, Derivative Assay

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet:

Article Snippet: PVDF membranes were blocked with 5% milk in PBST (PBS+Tween-20) for 2 h at RT and then target proteins were detected by specific primary antibodies including PEDF (1:500, 11104-RP02, Sino Biological, China), VEGFR1 (1:500, AF7748, Affinity, US), sCD59 (1:500, 12474-RP02, Sino Biological, China), Erk1/2 p44/42 (1:1000, 9102, Cell Signaling Technology, US), Erk1/2 phospho-p44/42 (T202/Y204) (1:1000, 9106, Cell Signaling Technology, US), p38 MAPK(1:1000, 8690, Cell Signaling Technology, US), Phospho-p38 MAPK (Thr180/Tyr182) (1:1000, 4631, Cell Signaling Technology, US), ICAM-1 (1:1000, 10831-1-AP, Proteintech, US), VCAM-1 (1:200, sc-13160 , Santa Cruz biotechnology, US).

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, Gel Extraction, Software

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-PEDF , Sino Biological , Cat#11104-RP02.

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, Gel Extraction, Software

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

doi: 10.1016/j.biopha.2021.111951

Figure Lengend Snippet: Fig. 1. Increased expression of PEDF in expanded human and mouse epidermis. A. Immunofluorescence staining for PEDF (green) and DAPI (blue) in the epithelial cells of human expanded skin (n = 9) and control skin (n = 6, ×40 magnification); B. Quantification of PEDF-positive cells per field in human skin (n = 6, mean ± SEM; **P < 0.01); C. Immunofluorescence staining for PEDF (green) and DAPI (blue) in the epithelial cells in mouse expanded skin and control skin (n = 6, ×40 magnification); D. Quantification of PEDF-positive cells per field in mouse skin (n = 6, mean ± SEM; ***P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After deparaffinized and rehydrated in alcohol series, skin sections were kept in a sodium citrate solution and heated in a microwave oven to 95 °C for 20 min before being incubated with a rabbit anti-mouse PEDF primary polyclonal antibody at a 1:30 dilution (Boster Bio-Engineering, Hubei Province, China) at 4 °C overnight.

Techniques: Expressing, Immunofluorescence, Staining, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

doi: 10.1016/j.biopha.2021.111951

Figure Lengend Snippet: Fig. 3. PEDF accumulated in the subcutaneous exudates of a rat skin expansion model. The quantity of PEDF protein in the subcutaneous exudates obtained at indicated times in expanded and control rats were determined by ELISA assays (n = 6, mean ± SEM; ***P < 0.001).

Article Snippet: After deparaffinized and rehydrated in alcohol series, skin sections were kept in a sodium citrate solution and heated in a microwave oven to 95 °C for 20 min before being incubated with a rabbit anti-mouse PEDF primary polyclonal antibody at a 1:30 dilution (Boster Bio-Engineering, Hubei Province, China) at 4 °C overnight.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

doi: 10.1016/j.biopha.2021.111951

Figure Lengend Snippet: Fig. 2. Skin expansion resulted in dermal thinning and PEDF up-regulation in a mouse skin expansion model. A. H&E staining at the 3rd week after expansion, showing thinner dermis in the expanded skin (×40 magnification); B. Quantification of dermal thickness between expanded and control skin (n = 6, mean ± SEM; **P < 0.01); C. The mRNA expression levels of PEDF in skin obtained at the 3rd week compared with the control skin (n = 6, mean

Article Snippet: After deparaffinized and rehydrated in alcohol series, skin sections were kept in a sodium citrate solution and heated in a microwave oven to 95 °C for 20 min before being incubated with a rabbit anti-mouse PEDF primary polyclonal antibody at a 1:30 dilution (Boster Bio-Engineering, Hubei Province, China) at 4 °C overnight.

Techniques: Staining, Control, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

doi: 10.1016/j.biopha.2021.111951

Figure Lengend Snippet: Fig. 5. In vivo injection of PEDF promoted M1 macrophage polarization. A. A larger number of CD68+/iNOS+ double-positive macrophages were detected in the PEDF injection group compared with PBS treatment. In contrast to PEDF injection, LR antibody injection decreased the number of CD68+/iNOS+ double-positive macrophages; B. Quantification of M1 macrophages in differently treated skin (mean ± SEM; **P < 0.01，*P < 0.05).

Article Snippet: After deparaffinized and rehydrated in alcohol series, skin sections were kept in a sodium citrate solution and heated in a microwave oven to 95 °C for 20 min before being incubated with a rabbit anti-mouse PEDF primary polyclonal antibody at a 1:30 dilution (Boster Bio-Engineering, Hubei Province, China) at 4 °C overnight.

Techniques: In Vivo, Injection

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

doi: 10.1016/j.biopha.2021.111951

Figure Lengend Snippet: Fig. 4. Blockage of PEDF receptor rescued dermal thinning in vivo. A. HE staining results showed that recombinant PEDF protein injection decreased the thickness of expanded dermis, while LR blockage rescued dermal thinning of expanded skin; B. Quantification of dermal thickness between differently treated skin (n = 6, mean ± SEM; ***P < 0.001，*P < 0.05).

Article Snippet: After deparaffinized and rehydrated in alcohol series, skin sections were kept in a sodium citrate solution and heated in a microwave oven to 95 °C for 20 min before being incubated with a rabbit anti-mouse PEDF primary polyclonal antibody at a 1:30 dilution (Boster Bio-Engineering, Hubei Province, China) at 4 °C overnight.

Techniques: In Vivo, Staining, Recombinant, Injection

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

doi: 10.1016/j.biopha.2021.111951

Figure Lengend Snippet: Fig. 7. Hypoxia induced PEDF expression in HaCaT cells. The mRNA expression levels of PEDF in HaCaT cells under normoxic and hypoxic condi tions were determined by RT-qPCR (mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: After deparaffinized and rehydrated in alcohol series, skin sections were kept in a sodium citrate solution and heated in a microwave oven to 95 °C for 20 min before being incubated with a rabbit anti-mouse PEDF primary polyclonal antibody at a 1:30 dilution (Boster Bio-Engineering, Hubei Province, China) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

doi: 10.1016/j.biopha.2021.111951

Figure Lengend Snippet: Fig. 8. PEDF promoted macrophages to polarize towards the M1 subtype under hypoxic conditions. A. FACs analysis of Raw264.7 single-cell sus pensions prepared after treating with PEDF under hypoxic conditions. CD11c+ cells indicate M1 macrophages. B. Quantification of CD11c+ cells under hypoxia (mean ± SEM; **P < 0.01). C. FACS analysis of Raw264.7 single-cell suspensions prepared after treating with PEDF under hypoxic conditions. CD206+ cells indicate M2 macro phages. D. Quantification of CD206+ cells under hypoxic conditions. The mRNA expression of M1 marker genes (E. iNOS, F. TNF-α) and M2 marker genes (G. Arg-1, H. Ym-1) determined by RT-qPCR and normalized to GAPDH mRNA expression. (mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: After deparaffinized and rehydrated in alcohol series, skin sections were kept in a sodium citrate solution and heated in a microwave oven to 95 °C for 20 min before being incubated with a rabbit anti-mouse PEDF primary polyclonal antibody at a 1:30 dilution (Boster Bio-Engineering, Hubei Province, China) at 4 °C overnight.

Techniques: Expressing, Marker, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

doi: 10.1016/j.biopha.2021.111951

Figure Lengend Snippet: Fig. 9. PEDF hindered collagen syn thesis in a macrophage-mediated manner under hypoxic conditions. A, B. Relative mRNA expression of COLI (A) and COLIII (B) in fibroblasts after co-culture with PEDF treated macro phages determined by RT-qPCR (mean

Article Snippet: After deparaffinized and rehydrated in alcohol series, skin sections were kept in a sodium citrate solution and heated in a microwave oven to 95 °C for 20 min before being incubated with a rabbit anti-mouse PEDF primary polyclonal antibody at a 1:30 dilution (Boster Bio-Engineering, Hubei Province, China) at 4 °C overnight.

Techniques: Expressing, Co-Culture Assay, Quantitative RT-PCR