Journal: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet: Muscle fibre cross-sectional area in oxidative ( A ) and non-oxidative ( B ) fibres from irradiated mice at 16 months of age. Frequencies of p21-positive myonuclei in oxidative ( C ) and glycolytic ( D ) fibres. ( E ) Telomere-associated foci (TAF) frequencies in myonuclei. Data are from at least five animals/group.

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques: Irradiation

Journal: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet: Immunostaining and blotting methods.

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques: Immunostaining, Plasmid Preparation, Avidin-Biotin Assay, Immunohistochemistry-IF

Journal: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet: List of metal conjugated antibodies for stress response pathway analysis by CyTOF.

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques:

Journal: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet:

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques: Recombinant, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fra-1 Inhibits Cell Growth and the Warburg Effect in Cervical Cancer Cells via STAT1 Regulation of the p53 Signaling Pathway

doi: 10.3389/fcell.2020.579629

Figure Lengend Snippet: List of the human-specific primer sequences used for qPCR analyses.

Article Snippet: Then 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene difluoride membrane (HyClone Laboratories, Logan, UT, United States), which was sealed with 5% non-fat milk diluted in PBS-Tween20 for ∼2 h. Primary antibody solution was then added for incubation at 4°C for 12 h. The primary antibodies used were: anti-sirtuin 3 (SIRT3, dilution ratio: 1:500), anti-Mn superoxide dismutase 2 (MnSOD2, dilution ratio: 1:1,000), anti-isocitrate dehydrogenase 2 (IDH2, dilution ratio: 1:500), anti-liver kinase B1 (LKB1, dilution ratio: 1:500), anti-p-AMP-activated protein kinase (AMPK, dilution ratio: 1:1,000), anti-pyruvate kinase muscle isozyme 2 (PKM2, dilution ratio: 1:1,000), anti-phosphofructose kinase 1 (PFK1, dilution ratio: 1:1,000), anti-pyruvate dehydrogenase (PDH, dilution ratio: 1:1,000), anti-FOS like 1, AP-1 transcription factor subunit (FOSL1, Fra-1, Abcam, Cambridge, MA, United States, dilution ratio: 1:500), anti-signal transducer and activator of transcription 1 (STAT1, dilution ratio: 1:500), anti-c-Rel (c-Rel, dilution ratio: 1:1,000), anti-lactate dehydrogenase A (LDHA, dilution ratio: 1:1,000), anti-sirtuin 1 (SIRT1, dilution ratio: 1:500), anti- B-cell lymphoma-2 (Bcl-2, dilution ratio: 1:1,000), anti-NF kappa-B (NF-κB/p65, dilution ratio: 1:1,000, Immunoway Technology, Newark, DE, United States), anti-TP53 (p53, dilution ratio: 1:1,000), anti-cyclin-dependent kinase 4 (CDK4, dilution ratio: 1:1,000), anti-cyclin D1 (dilution ratio: 1:1,000), anti-p21 (dilution ratio: 1:1,000), anti-MAPK14/p38 (dilution ratio: 1:1,000), and anti-MDM2 (dilution ratio: 1:1,000) anti-p16 (dilution ratio: 1:1,000, Boster Technology, Wuhan, China).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fra-1 Inhibits Cell Growth and the Warburg Effect in Cervical Cancer Cells via STAT1 Regulation of the p53 Signaling Pathway

doi: 10.3389/fcell.2020.579629

Figure Lengend Snippet: Fra-1 regulated p53 signaling pathway activity in cervical cancer cells. (A) mRNA chip analysis in HeLa/vector and HeLa/Fra-1 cells revealed differences in mRNA expression between the two groups. Differentially active signaling pathways were identified by gene ontology analysis. (B) Western blotting analysis of signal transducer and activator of transcription 1 (STAT1), p53, Bcl-2, p38, p21, MDM2, CDK4, and cyclinD1 protein levels in HeLa/vector and HeLa/Fra-1 cells. (C) Real-time polymerase chain reaction (PCR) analysis the mRNA expression levels of STAT1, p53, Bcl-2, p38, p21, MDM2, CDK4, and cyclinD1 in HeLa/vector and HeLa/Fra-1 cells. * P < 0.05; ** P < 0.01; *** P < 0.001, n = 3.

Article Snippet: Then 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene difluoride membrane (HyClone Laboratories, Logan, UT, United States), which was sealed with 5% non-fat milk diluted in PBS-Tween20 for ∼2 h. Primary antibody solution was then added for incubation at 4°C for 12 h. The primary antibodies used were: anti-sirtuin 3 (SIRT3, dilution ratio: 1:500), anti-Mn superoxide dismutase 2 (MnSOD2, dilution ratio: 1:1,000), anti-isocitrate dehydrogenase 2 (IDH2, dilution ratio: 1:500), anti-liver kinase B1 (LKB1, dilution ratio: 1:500), anti-p-AMP-activated protein kinase (AMPK, dilution ratio: 1:1,000), anti-pyruvate kinase muscle isozyme 2 (PKM2, dilution ratio: 1:1,000), anti-phosphofructose kinase 1 (PFK1, dilution ratio: 1:1,000), anti-pyruvate dehydrogenase (PDH, dilution ratio: 1:1,000), anti-FOS like 1, AP-1 transcription factor subunit (FOSL1, Fra-1, Abcam, Cambridge, MA, United States, dilution ratio: 1:500), anti-signal transducer and activator of transcription 1 (STAT1, dilution ratio: 1:500), anti-c-Rel (c-Rel, dilution ratio: 1:1,000), anti-lactate dehydrogenase A (LDHA, dilution ratio: 1:1,000), anti-sirtuin 1 (SIRT1, dilution ratio: 1:500), anti- B-cell lymphoma-2 (Bcl-2, dilution ratio: 1:1,000), anti-NF kappa-B (NF-κB/p65, dilution ratio: 1:1,000, Immunoway Technology, Newark, DE, United States), anti-TP53 (p53, dilution ratio: 1:1,000), anti-cyclin-dependent kinase 4 (CDK4, dilution ratio: 1:1,000), anti-cyclin D1 (dilution ratio: 1:1,000), anti-p21 (dilution ratio: 1:1,000), anti-MAPK14/p38 (dilution ratio: 1:1,000), and anti-MDM2 (dilution ratio: 1:1,000) anti-p16 (dilution ratio: 1:1,000, Boster Technology, Wuhan, China).

Techniques: Activity Assay, Plasmid Preparation, Expressing, Protein-Protein interactions, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fra-1 Inhibits Cell Growth and the Warburg Effect in Cervical Cancer Cells via STAT1 Regulation of the p53 Signaling Pathway

doi: 10.3389/fcell.2020.579629

Figure Lengend Snippet: Silencing of signal transducer and activator of transcription 1(STAT1) by small interfering RNA promoted proliferation of cervical cancer cells via activation of the p53 signal pathway. (A) Real-time polymerase chain reaction (PCR) analysis of the silencing effects of three siRNAs for STAT1 in HeLa/Fra-1 cells. (B) Cell viability of HeLa/Fra-1 and HeLa/Fra-1/siSTAT1 cells according to CCK8 assay. (C) Colony forming ability of HeLa/Fra-1 and HeLa/Fra-1/siSTAT1 cells. (D) In vivo analysis of subcutaneous implanted tumor growth to compare the tumorigenic abilities of HeLa/vector, HeLa/Fra-1, and HeLa/Fra-1/siSTAT1 cells in mice. (E) Western blot analysis of protein expression of STAT1, CDK4, cyclin A, cyclin D1, p53, Bcl-2, c-REL, p38, p21, and MDM2 in HeLa/Fra-1 and HeLa/Fra-1/siSTAT1 cells. (F) Real-time PCR analysis of mRNA expression of STAT1, CDK4, cyclin D1, p53, Bcl-2, p38, p21, and MDM2 in HeLa/Fra-1 and HeLa/Fra-1/siSTAT1 cells. * P < 0.05; ** P < 0.01; *** P < 0.001, n = 3.

Article Snippet: Then 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene difluoride membrane (HyClone Laboratories, Logan, UT, United States), which was sealed with 5% non-fat milk diluted in PBS-Tween20 for ∼2 h. Primary antibody solution was then added for incubation at 4°C for 12 h. The primary antibodies used were: anti-sirtuin 3 (SIRT3, dilution ratio: 1:500), anti-Mn superoxide dismutase 2 (MnSOD2, dilution ratio: 1:1,000), anti-isocitrate dehydrogenase 2 (IDH2, dilution ratio: 1:500), anti-liver kinase B1 (LKB1, dilution ratio: 1:500), anti-p-AMP-activated protein kinase (AMPK, dilution ratio: 1:1,000), anti-pyruvate kinase muscle isozyme 2 (PKM2, dilution ratio: 1:1,000), anti-phosphofructose kinase 1 (PFK1, dilution ratio: 1:1,000), anti-pyruvate dehydrogenase (PDH, dilution ratio: 1:1,000), anti-FOS like 1, AP-1 transcription factor subunit (FOSL1, Fra-1, Abcam, Cambridge, MA, United States, dilution ratio: 1:500), anti-signal transducer and activator of transcription 1 (STAT1, dilution ratio: 1:500), anti-c-Rel (c-Rel, dilution ratio: 1:1,000), anti-lactate dehydrogenase A (LDHA, dilution ratio: 1:1,000), anti-sirtuin 1 (SIRT1, dilution ratio: 1:500), anti- B-cell lymphoma-2 (Bcl-2, dilution ratio: 1:1,000), anti-NF kappa-B (NF-κB/p65, dilution ratio: 1:1,000, Immunoway Technology, Newark, DE, United States), anti-TP53 (p53, dilution ratio: 1:1,000), anti-cyclin-dependent kinase 4 (CDK4, dilution ratio: 1:1,000), anti-cyclin D1 (dilution ratio: 1:1,000), anti-p21 (dilution ratio: 1:1,000), anti-MAPK14/p38 (dilution ratio: 1:1,000), and anti-MDM2 (dilution ratio: 1:1,000) anti-p16 (dilution ratio: 1:1,000, Boster Technology, Wuhan, China).

Techniques: Small Interfering RNA, Activation Assay, Real-time Polymerase Chain Reaction, CCK-8 Assay, In Vivo, Plasmid Preparation, Western Blot, Expressing

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: HDBECs (2×10 5 ) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2-AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Groups of cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to the addition of TF. The cells were harvested after 24 h, into separate aliquots (1×10 5 cells). Total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 mRNA (n=5), and (B) the relative amounts of p21 CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21 CIP1/WAF1 protein, (using a mouse anti-human antibody) and against GAPDH. TF, tissue factor; PAR2, protease-activated receptor 2.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Incubation, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were cultured in 25 cm 2 flasks and supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on the indicated wks, total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 mRNA (n=3), and (B) the relative amounts of p21 CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21 CIP1/WAF1 protein (using a mouse anti-human antibody) and against GAPDH. TF, tissue factor; Wk, week.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Reverse Transcription, Expressing, Cell Culture, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Proposed model for the mechanism by which the level of TF on the cell surface may have differential outcomes on G1/S checkpoint regulation. The presence of TF on the cell surface differentially upregulates the expression of Inhibitor of CDK p16 INKa , CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 and Alternative reading frame p14 ARF , which is dependent on the concentration of TF and the ability of the cell to dissipate excess TF. Therefore, alterations in p21 CIP1/WAF1 are highly effective in the regulation of the cellular response to acute stress. The interplay between these proteins modulates the signal permitting passage through the cell cycle, or alternatively its arrest. Consequently, the concentration of TF may be an ideal gauge for determining the level of cellular damage. However, the adaptive loss of p16 INK4a function may be promoted by prolonged inflammation leading to permissive transition through the G1/S checkpoint, even in the absence of mutational loss of p16 INKa . TF, tissue factor; PAR2, protease-activated receptor 2, Rb, retinoblastoma protein; E2F, early region 2 binding factor; p21 CIP1/WAF1 , CDK interacting protein/wildtype p53-activated fragment; p16 INK4a , inhibitor of CDK; p14 ARF , alternative reading frame.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Expressing, Concentration Assay, Binding Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage

doi: 10.1155/2022/4155565

Figure Lengend Snippet: Ionizing radiation causes cardiomyocyte senescence. (a) Senescence-associated β -galactosidase (SA- β gal) staining of sham-irradiated and irradiated H9C2 cells. (b)–(d) Western blotting and quantification analyses of p21 and p16 protein expression in H9C2 cells in the control group and after 6h, 12h, and 24h of irradiation ( ∗ p < 0.05, ∗∗ p < 0.01). (e) q-PCR analysis of the mRNA levels of inflammation- and fibrosis-associated factors in H9C2 cells of the control, 6h, 12h, and 24h irradiation groups ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Bar: 100 μ m; h: hour.

Article Snippet: Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [ ] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti- α -tubulin monoclonal antibody (1 : 1000; Boster).

Techniques: Staining, Irradiation, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage

doi: 10.1155/2022/4155565

Figure Lengend Snippet: Atp5f1c is hyperacetylated at the 55th lysine site, which leads to metabolic disorders and senescence. (a) The expression of anti-His antibody after His-tagged Atp5f1c WT, K55R, and K55Q point mutation plasmids transfection in H9C2 cells. (b) Atp5f1c acetylation level verified by Co-IP after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells. (c, d) Senescence-associated β -galactosidase (SABG) staining and statistical analysis after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; NS: not significant). (e) q-PCR analysis of the mRNA levels of inflammation- and fibrosis-associated factors after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (f)–(i) Western blotting and quantification analyses of p21 and p16 protein expression after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells; relative protein levels were normalized to GAPDH or actin expression ( ∗ p < 0.05). (j, k) ATP synthase activity and ATP production after transfection of Atp5f1c WT, K55R, and K55Q plasmids into H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; NS: not significant). Co-IP: Coimmunoprecipitation; WB: western blotting; WT: wild type.

Article Snippet: Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [ ] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti- α -tubulin monoclonal antibody (1 : 1000; Boster).

Techniques: Expressing, Mutagenesis, Transfection, Co-Immunoprecipitation Assay, Staining, Western Blot, Activity Assay

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