cells  (MedChemExpress)

Journal: Molecular Therapy. Nucleic Acids

Article Title: MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

doi: 10.1016/j.omtn.2022.12.020

Figure Lengend Snippet: MDM2 antagonists promote CRISPR-Cas9-mediated precise genome editing in SFFs (A) Schematic of the targeting strategy for knockin T2A-EGFP with 1,000 bp HA to the sheep FGF5 locus. (B) Detection of the relative mRNA expression levels of the p21 and Cdc25C genes in the Cas9/MSTN sgRNA/HDR/MDM2 antagonist-treated cells at 24 h post transfection. (C and E) FACS analysis of EGFP-positive cells at 48 h after transfection of the pX330-MSTN/FGF5 plasmid and MSTN/FGF5-T2A-EGFP repair template with 1,000 bp HA into SFFs. Representative FACS results of samples treated with 0.1% DMSO, 10 μM pifithrin-β, 10 nM RITA, 10 μM Nutlin3, 1.5 μM CTX1 alone, or the MDM2 antagonist mixture are shown. (D and F) Effects of p53 inhibitor and MDM2 antagonists on HDR efficiency at the MSTN and FGF5 loci. DMSO-treated transfected cells were used as a control. n = 3 biological replicates. Error bars represent SD. Significance was calculated using Student’s t test: ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: For MSTN/FGF5-T2A-EGFP knockin assays, co-transfected cells were treated with 0.1% DMSO (Sigma); 4, 20, and 40 μM nicotinamide (Beyotime); 0.4, 1.4, and 4.2 mM Ca 2+ (Sigma); 1, 5, and 10 ng/mL IL-10 (PeproTech); 0.5, 2, and 10 μM pifithrin-β (MCE); 10, 50, and 100 μM SCR7 (Selleck); 0.05, 0.2, and 0.5 μM SF2523 (Selleck); 0.05, 0.2, and 0.5 μM CC-115 (Selleck); 0.01, 0.05, and 0.1 μM PIK-75 (Selleck); 10 nM RITA (MCE); 10 μM Nutlin3 (MCE); or 1.5 μM CTX1 (MCE) for 48 h, followed by determining the HDR efficiency of each treatment through flow cytometry.

Techniques: CRISPR, Knock-In, Expressing, Transfection, Plasmid Preparation, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

doi: 10.1016/j.omtn.2022.12.020

Figure Lengend Snippet: Effect of cell-cycle stage on HDR efficiency in SFFs (A and B) Cell-cycle distribution of co-transfected SFFs after treatment with DMSO, 10 μM pifithrin-β, 10 nM RITA, 10 μM Nutlin3, and 1.5 μM CTX1 for 24 h. Samples were stained by PI and analyzed by FACS. (C and D) Analysis of SFFs with different cell-cycle blocks by PI staining and flow cytometry. (E) FACS analysis of the HDR efficiency upon transfection with the pX330-MSTN plasmid and MSTN-T2A-EGFP with the 1,000 bp HA repair template into SFFs in different phases of the cell cycle. (F) Effects of different cell-cycle blocks on HDR efficiency. DMSO-treated cells served as a control. n = 3 biological replicates. Error bars represent SD. Significance was calculated using Student’s t test: ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: For MSTN/FGF5-T2A-EGFP knockin assays, co-transfected cells were treated with 0.1% DMSO (Sigma); 4, 20, and 40 μM nicotinamide (Beyotime); 0.4, 1.4, and 4.2 mM Ca 2+ (Sigma); 1, 5, and 10 ng/mL IL-10 (PeproTech); 0.5, 2, and 10 μM pifithrin-β (MCE); 10, 50, and 100 μM SCR7 (Selleck); 0.05, 0.2, and 0.5 μM SF2523 (Selleck); 0.05, 0.2, and 0.5 μM CC-115 (Selleck); 0.01, 0.05, and 0.1 μM PIK-75 (Selleck); 10 nM RITA (MCE); 10 μM Nutlin3 (MCE); or 1.5 μM CTX1 (MCE) for 48 h, followed by determining the HDR efficiency of each treatment through flow cytometry.

Techniques: Transfection, Staining, Flow Cytometry, Plasmid Preparation, Control

Journal: EBioMedicine

Article Title: Exercise downregulates HIPK2 and HIPK2 inhibition protects against myocardial infarction.

doi: 10.1016/j.ebiom.2021.103713

Figure Lengend Snippet: Fig. 2. Suppression of HIPK2 prevents apoptosis in NRCMs. (a) Immuno-histochemical staining for sarcomeric a-actinin and EdU followed by quantification of cardiomyocyte area in NRCMs infected with lentiviral vectors expressing scrambled (lenti-scrambled) or short-hairpin (sh) RNA against mouse HIPK2 (lenti-sh-HIPK2), or lentiviral vectors expressing Fugw (lenti-Fugw) or mouse HIPK2 (lenti-HIPK2, HIPK2 OE), and HIPK2 had no effect on NRCMs proliferation and hypertrophy (n=5 in sh-Scramble, 5 in sh-HIPK2, 5 in Fugw, and 4 in HIPK2 OE); (b and c) Lenti-sh-HIPK2 reduced the percentage of TUNEL staining positive cardiomyocytes (n=6 per group), Bax/Bcl2, cleaved Caspase3/Caspase3 (n=3 per group) in NRCMs under oxygen glucose deprivation/reperfusion (OGD/R); (d and e) HIPK2 inhibitor (PKI1H) decreased the percentage of TUNEL staining positive cardiomyocytes (n=6 per

Article Snippet: and Recombinant Proteins DPBQ MedChemExpress HY-U00441 CTX1 MedChemExpress HY-U00442 PKI1H (Protein kinase inhibitors 1 hydrochloride) MedChemExpress HY-U00439A WGA(wheat germ agglutinin) Sigma L4895

Techniques: Staining, Infection, Expressing, TUNEL Assay

Journal: EBioMedicine

Article Title: Exercise downregulates HIPK2 and HIPK2 inhibition protects against myocardial infarction.

doi: 10.1016/j.ebiom.2021.103713

Figure Lengend Snippet: Fig. 3. HIPK2 inhibition protects acute MI. (a and b) HIPK2 inhibitor (PKI1H) reduced the infarct sizes (IS) and Bax/Bcl2, cleaved Caspase3/Caspase3 in heart of acute MI mice as deter- mined by TTC staining (n=8 per group) and western blotting (n=3 per group); (c and d) HIPK2/ mice reduced IS and Bax/Bcl2, cleaved Caspase3/Caspase3 in heart of acute MI mice as determined by TTC staining (n=8 in WT+AMI, 7 in HIPK2/+AMI) and western blotting (n=3 per group). Data are represented as mean § SD. Significant differences were assessed by two-tailed student t-test in a and c, or two-way ANOVA followed by Bonferroni's multiple comparisons test in b and d. *: p<0.05, ***: p<0.001 versus respective control.

Article Snippet: and Recombinant Proteins DPBQ MedChemExpress HY-U00441 CTX1 MedChemExpress HY-U00442 PKI1H (Protein kinase inhibitors 1 hydrochloride) MedChemExpress HY-U00439A WGA(wheat germ agglutinin) Sigma L4895

Techniques: Inhibition, Staining, Western Blot, Two Tailed Test, Control

Journal: EBioMedicine

Article Title: Exercise downregulates HIPK2 and HIPK2 inhibition protects against myocardial infarction.

doi: 10.1016/j.ebiom.2021.103713

Figure Lengend Snippet: Fig. 4. HIPK2 inhibitor protects against cardiac dysfunction after MI. (a) The graphical description for the experimental setup. Mice were subjected to either sham or MI and treated with or without HIPK2 inhibitor (PKI1H) for 3

Article Snippet: and Recombinant Proteins DPBQ MedChemExpress HY-U00441 CTX1 MedChemExpress HY-U00442 PKI1H (Protein kinase inhibitors 1 hydrochloride) MedChemExpress HY-U00439A WGA(wheat germ agglutinin) Sigma L4895

Techniques:

Journal: EBioMedicine

Article Title: Exercise downregulates HIPK2 and HIPK2 inhibition protects against myocardial infarction.

doi: 10.1016/j.ebiom.2021.103713

Figure Lengend Snippet: Fig. 5. HIPK2 inhibition mediates cardiac protection through downregulating P-P53. (a) P-P53 (S15)/P53 were reduced in the heart of swimming mice (n=3 per group); (b) Cardiac P-P53 (S15)/P53 were increased in post-MI remodeling for 9 weeks and 8-week-running training decreased cardiac P-P53 (S15)/P53 during post-MI remodeling (n=3 per group); (c) Cardiac P- P53 (S15)/P53 were increased in post-MI remodeling for 3 weeks and HIPK2/ mice decreased cardiac P-P53 (S15)/P53 during post-MI remodeling (n=3 per group); (d) Cardiac P-P53 (S15)/P53 were increased in post-MI remodeling for 3 weeks and PKI1H decreased cardiac P-P53 (S15)/P53 during post-MI remodeling (n=3 per group); (e) Immunoprecipitation (IP) and immunoblotting were performed using the antibodies indicated and showed that HIPK2 interacted with P53; (f and g) P53 activators (CTX1 and DPBQ) increased TUNEL staining in NRCMs under OGD/R infected with lenti-sh-HIPK2 (n=6 per group in f and n=4 per group in g); (h and i) CTX1 and DPBQ increased Bax/Bcl2, cleaved Caspase3/Caspase3 in NRCMs under OGD/R

Article Snippet: and Recombinant Proteins DPBQ MedChemExpress HY-U00441 CTX1 MedChemExpress HY-U00442 PKI1H (Protein kinase inhibitors 1 hydrochloride) MedChemExpress HY-U00439A WGA(wheat germ agglutinin) Sigma L4895

Techniques: Inhibition, Immunoprecipitation, Western Blot, TUNEL Assay, Staining, Infection

Journal: EBioMedicine

Article Title: Exercise downregulates HIPK2 and HIPK2 inhibition protects against myocardial infarction

doi: 10.1016/j.ebiom.2021.103713

Figure Lengend Snippet:

Article Snippet: CTX1 , MedChemExpress , HY-U00442 , .

Techniques: Recombinant, Western Blot, Lysis, Bicinchoninic Acid Protein Assay, Staining, TUNEL Assay, Software