Journal: International immunopharmacology

Article Title: Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury.

doi: 10.1016/j.intimp.2023.110452

Figure Lengend Snippet: Fig. 5. The downstream regulation of AKT1-SKP2 in NTHY cells.

Article Snippet: SKP2 inhibitor (SKP2i, MCE, 5uM) is solubilized by DMSO.

Techniques:

Journal: International immunopharmacology

Article Title: Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury.

doi: 10.1016/j.intimp.2023.110452

Figure Lengend Snippet: Fig. 6. Interaction between SKP2 and PD-L1. (A) DAPI is in blue, SKP2 in red, and PD-L1 in green. In NTHY cells, SKP2 is mainly expressed in the nucleus and PD- L1 in non-nuclear area. (B) NTHY cells are co-transfected with pcDNA3.1-SKP2 plasmid and pcDNA3.1-PDL1 plasmid. The interaction between SKP2 and PD-L1 protein is proved by immunoprecipitation.

Article Snippet: SKP2 inhibitor (SKP2i, MCE, 5uM) is solubilized by DMSO.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation

Journal: International immunopharmacology

Article Title: Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury.

doi: 10.1016/j.intimp.2023.110452

Figure Lengend Snippet: Fig. 8. Hypothesis: NIVO increases the immunosensitivity of NTHY cells by downregulating AKT1-SKP2.

Article Snippet: SKP2 inhibitor (SKP2i, MCE, 5uM) is solubilized by DMSO.

Techniques:

Journal: International immunopharmacology

Article Title: Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury.

doi: 10.1016/j.intimp.2023.110452

Figure Lengend Snippet: Fig. 7. AKT1, SKP2, PD-L1 and IFN-γ in thyroid tissue. (A) The thyroid gland of mice is used for detection by immunohistochemistry. Fewer AKT1, SKP2, PD-L1 and more IFN-γ are observed in the NIVO group than in the CTRL group (10X). (B) Further, levels of various proteins in mouse thyroid are quantified by WB.(C) Human thyroid tissue is used for detection by WB. results revealed that PD-L1 is down-regulated and IFNG is up-regulated in the HT group compared to the non-HT group, however, there is no difference in AKT1 and SKP2.

Article Snippet: SKP2 inhibitor (SKP2i, MCE, 5uM) is solubilized by DMSO.

Techniques: Immunohistochemistry

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 6. SKP2 in oral keratinocytes is upregulated after L-Arginine treatment under high-glucose conditions. TIGK cells were treated with 500 µM Arg in a culture medium having high glucose (48 mM) for 24 and 48 h. Cells were fixed in formalin and permeabilized with Triton X-100 followed by incubation with a mouse anti–human SKP2 antibody and a goat anti-mouse IgG Alexa Fluor 488 secondary antibody. Alexa Fluor 594 Phalloidin was used to stain F-actin as a counterstain. DAPI was used for nuclei staining. The green fluorescence intensity of SKP2 was quantified using ImageJ. Scale bar: 50 µm. # p < 0.01 compared to control.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Incubation, Staining, Control

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 7. SKP2 in oral keratinocytes is upregulated after L-Arginine treatment under high-glucose conditions. TIGK cells were treated with 500 µM Arg in a culture medium having high glucose (48 mM) for 24 and 48 h. Cells were fixed in formalin and permeabilized with Triton X-100 followed by incubation with a rabbit anti-human SKP2 antibody and a goat anti-rabbit IgG Alexa Fluor 488 secondary antibody. Alexa Fluor 594 Phalloidin was used to stain F-actin as a counterstain. DAPI was used for nuclei staining. The green fluorescence intensity of SKP2 was quantified using ImageJ. Scale bar: 50 µm. * p < 0.05 compared to control.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Incubation, Staining, Control

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 8. Knocking down CYP1A1, SKP2, or SRSF5 abolishes enhanced oral keratinocyte proliferation induced by L-Arginine treatment under high-glucose conditions. TIGK cells were transfected with 20 nM siRNAs of CYP1A1, SKP2, or SRSF5 and then treated with 500 µM Arg in a culture medium with high glucose (48 mM). (A) mRNA expression of CYP1A1, SKP2, or SRSF5 48 h after transfection. (B) The proliferation of TIGK cells after knocking down CYP1A1, SKP2, or SRSF5 assessed by cell count at 48 h and MTS assay at 72 and 120 h after transfection. (C) Representative images of cell density at 48 h after treatment. Scale bar: 100 µm. * p < 0.05, # p < 0.01.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Transfection, Expressing, Cell Counting, MTS Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bacteroides Fragilis Transplantation Reverses Reproductive Senescence by Transporting Extracellular Vesicles Through the Gut-Ovary Axis.

doi: 10.1002/advs.202409740

Figure Lengend Snippet: Figure 6. miR-1246 targets SKP2 to regulate the p62/Keap1/Nrf2 pathway. A) The binding site of miR-1246 and SKP2 in the Starbase database. B) The binding relationship between miR-1246 and SKP2 was confirmed using dual-luciferase assay (n = 3, one-way ANOVA). ** p < 0.01. C) After transfection of Control inhibitor, miR-1246 inhibitor, NC mimics, and miR-1246 mimics into GCs, miR-1246 expression was determined using RT-qPCR (n = 3, one- way ANOVA). *** p < 0.001, * p < 0.05. D) SKP2 mRNA expression was determined using RT-qPCR (n = 3, one-way ANOVA). *** p < 0.001, * p < 0.05. E) GCs were transfected with miR-1246 mimic or inhibitor or the corresponding scrambled control. Western blotting was used to detect the protein level of SKP2. F) GCs were transfected with miR-1246 inhibitor or the corresponding scrambled control. Western blotting was used to detect the protein levels of p62, Keap1, and Nrf2. G) GCs were transfected with miR-1246 mimic or the corresponding scrambled control. Western blotting was used to detect the protein levels of p62, Keap1, and Nrf2. H) GCs were co-cultured with EVs (overexpression of SKP2), the SKP2, p62, Keap1, and Nrf2 protein expressions were analyzed by western blotting. I) GCs were co-cultured with EVs (knockdown of SKP2), the SKP2, p62, Keap1, and Nrf2 protein expressions were analyzed by western blotting. The blots represented three independent experiments.

Article Snippet: The following antibody were listed: SKP2 (1:1000; NOVUS; NBP3-16320), p62 (1:500; NOVUS; H00008878-M01), Keap1 (1:1000; Thermo Fisher Scientific; MA5-17106), Nrf2 (1:1000; Thermo Fisher Scientific; PA5-27882), Flag (1:1000; Beyotime; AF5051), Myc (1:1000; Beyotime; AF5054), HA (1:1000; Beyotime; AF5057), and GAPDH (1:1000; Thermo FisherScientific; PA1-987).

Techniques: Binding Assay, Luciferase, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Over Expression, Knockdown

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bacteroides Fragilis Transplantation Reverses Reproductive Senescence by Transporting Extracellular Vesicles Through the Gut-Ovary Axis.

doi: 10.1002/advs.202409740

Figure Lengend Snippet: Figure 8. SKP2 plays a vital role in regulating p62/Keap1/Nrf2 pathway. A) Schematic diagram demonstrating the study design of in vivo knockdown experiments on BF-EVs-relieved ovarian aging. B) The SKP2, p62, and Nrf2 protein expressions were analyzed by western blotting. C) GCs were collected from different groups of mice for protein extraction, and western blotting was performed to detect the protein levels of SKP2, p62, Keap1, and Nrf2. D) Representative images of spindle assembly and chromosomes alignment in different groups. Oocytes were stained with anti-tubulin antibody (red)

Article Snippet: The following antibody were listed: SKP2 (1:1000; NOVUS; NBP3-16320), p62 (1:500; NOVUS; H00008878-M01), Keap1 (1:1000; Thermo Fisher Scientific; MA5-17106), Nrf2 (1:1000; Thermo Fisher Scientific; PA5-27882), Flag (1:1000; Beyotime; AF5051), Myc (1:1000; Beyotime; AF5054), HA (1:1000; Beyotime; AF5057), and GAPDH (1:1000; Thermo FisherScientific; PA1-987).

Techniques: In Vivo, Knockdown, Western Blot, Protein Extraction, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bacteroides Fragilis Transplantation Reverses Reproductive Senescence by Transporting Extracellular Vesicles Through the Gut-Ovary Axis.

doi: 10.1002/advs.202409740

Figure Lengend Snippet: Figure 9. Flowchart of BFT ameliorating ovarian aging by transporting EVs containing miR-1246, which inhibits the expression of SKP2 and promotes p62 accumulation. This process improves reproductive senescence by reducing oxidative stress in the ovaries.

Article Snippet: The following antibody were listed: SKP2 (1:1000; NOVUS; NBP3-16320), p62 (1:500; NOVUS; H00008878-M01), Keap1 (1:1000; Thermo Fisher Scientific; MA5-17106), Nrf2 (1:1000; Thermo Fisher Scientific; PA5-27882), Flag (1:1000; Beyotime; AF5051), Myc (1:1000; Beyotime; AF5054), HA (1:1000; Beyotime; AF5057), and GAPDH (1:1000; Thermo FisherScientific; PA1-987).

Techniques: Expressing

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 3 CLOCK interference promotes GCs proliferation. A The interference efficiency of CLOCK was measured using RT-qPCR. Data are expressed as mean ± SEM (n = 5), **P < 0.01. B Western blotting reveals the expression levels of CLOCK. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 5), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 5), *P < 0.05. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A). GAPDH as a housekeeping protein. H Quantifying the Western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 6 ASB9 interference promotes GCs proliferation. A RT-qPCR detected the interference efficiency of ASB9. Data are expressed as mean ± SEM (n = 6), **P < 0.01. B Western blotting reveals the expression levels of ASB9. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 4), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 16), ****P < 0.0001. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (ASB9, CCNB1, CCNE1, CDK4, and CDKN1A). H Quantifying the western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05, **P < 0.01. I RNA expression of ASB9 in GCs. ZT: zone time

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection, RNA Expression