transfection ac16 human cardiomyocytes  (Procell Inc)

Journal: Molecular metabolism

Article Title: Super-enhancer-driven LncRNA PPARα-seRNA exacerbates glucolipid metabolism and diabetic cardiomyopathy via recruiting KDM4B.

doi: 10.1016/j.molmet.2024.101978

Figure Lengend Snippet: Figure 1: Identification of PPARa-seRNA as a key element participating in glucolipid metabolism disorder. (A) Selection strategy of DbCM-associated seRNAs from the Encyclopedia of DNA Elements (ENCODE) datasets in different human tissues. (B) Rank Ordering of Super-Enhancers (ROSE) algorithm to identify super enhancer and its related gene in heart. (C) Identification of potential super-enhancer-related long noncoding RNAs (lncRNAs) expressed in different metabolic tissues and heart by Venn diagram. (D) Expression of super-enhancer-related lncRNAs in AC16 treated with the BRD4 inhibitor JQ1 at 1 mmol/L for 24 h. (E) Expression of super-enhancer-related lncRNAs according to qRT-PCR in AC16 treated with low (5 mmol/L) or high (33.3 mmol/L) glucose and palmitate (200 mmol/l) for 24 h. (F) ChIP-seq data from WashU website showing peaks of different histone markers. Four constituent enhancers are indicated with short blue lines. (G) Relative expression of PPARa-seRNA in human cardiomyocyte-like cells (AC16), human cardiac fibroblasts (HCF), and human umbilical vein endothelial cells (HUVECs). (HeI) Subcellular localization of PPARa-seRNA detected by qRT-PCR in cytosol and nuclear fractions from AC16 cells (H) and FISH (I, scale bar ¼ 10 mm). U6 RNA was used as a nucleus RNA marker, while Gapdh were used as cytosol RNA marker. (J) Activity of four constituent enhancers (E1, E2, E3, and E4) constructed with pGL3-promoter reporter vector, evaluated by luciferase assay. (K) Diagram of activated and deactivated enhancer using the CRISPR/dCAS9-p300 or CRISPR/dCAS9-KRAB system with single guide RNA (sgRNA). (L) Expression of PPARa-seRNA with super-enhancer deactivated by sgRNA-dCAS9-KRAB. (M) Expression of PPARa-seRNA with super-enhancer activated by sgRNA-dCAS9-p300. One-way ANOVA with Tukey’s multiple comparisons test was used in (G) and (J); student’s two-tailed t test was used otherwise. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant. All data are illustrated as mean SD.

Article Snippet: Cell culture and transfection AC16 human cardiomyocyte-like cells, murine HL-1 cardiomyocytelike cells, and HEK293T cell lines from ATCC (Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 5% glucose) supplemented with 10% Fetal Bovine Serum (FBS) and 1% of penicillin/streptomycin mixture, and cultured at 37 C in a humidified atmosphere of 5% CO2.

Techniques: Selection, Expressing, Quantitative RT-PCR, ChIP-sequencing, Marker, Activity Assay, Construct, Plasmid Preparation, Luciferase, CRISPR, Two Tailed Test

Journal: Molecular metabolism

Article Title: Super-enhancer-driven LncRNA PPARα-seRNA exacerbates glucolipid metabolism and diabetic cardiomyopathy via recruiting KDM4B.

doi: 10.1016/j.molmet.2024.101978

Figure Lengend Snippet: Figure 2: PPARa-seRNA aggravates cardiomyocytes metabolic disorders. (A&F) qRT-PCR assay used to detect the effect overexpression (A) and silencing (F) of PPARa- seRNA in the AC16 cell line. (B&G) Oil O Red (upper panel) and BODIPY staining (lower panel) showing the lipid droplet content after PPARa-seRNA overexpression (B) or silencing (G) with high glucose (33.3 mmol/L) for 24 h and palmitate (200 mmol/L) treatment for 6 h. Scale bar ¼ 25 mm. (C&H) In Vitro Glucose Uptake Assay demonstrating that PPARa- seRNA overexpression reduces (C), and PPARa-seRNA silencing increases (H) the efficiency of insulin-mediated 2D-glucose uptake. (D&I) Total ATP levels declined in response to PPARa-seRNA overexpression (D) or decreased when was PPARa-seRNA silenced (I) with high glucose (33.3 mmol/L) for 24 h and palmitate (200 mmol/L) treatment for 6 h (E&J) PPARa-seRNA overexpression reduced (E), and PPARa-seRNA silencing increased (J) the NADþ/NADH ratio with high glucose (33.3 mmol/L) for 24 h and palmitate (200 mmol/L) treatment for 6 h. Statistical analysis was performed with student’s two-tailed t test in (A) and (F). Two-way ANOVA with multiple comparisons tests otherwise. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are depicted as mean SD.

Article Snippet: Cell culture and transfection AC16 human cardiomyocyte-like cells, murine HL-1 cardiomyocytelike cells, and HEK293T cell lines from ATCC (Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 5% glucose) supplemented with 10% Fetal Bovine Serum (FBS) and 1% of penicillin/streptomycin mixture, and cultured at 37 C in a humidified atmosphere of 5% CO2.

Techniques: Quantitative RT-PCR, Over Expression, Staining, In Vitro, Two Tailed Test

Journal: Molecular metabolism

Article Title: Super-enhancer-driven LncRNA PPARα-seRNA exacerbates glucolipid metabolism and diabetic cardiomyopathy via recruiting KDM4B.

doi: 10.1016/j.molmet.2024.101978

Figure Lengend Snippet: Figure 5: PPARa-seRNA interacted with KDM4B. (A) Gene Ontogeny (GO) analysis of PPARa-seRNA binding proteins identified by mass spectrometry. (B) The detected proteins associated with chromatin remodeling. (C) RNA pull-down to detect the binding intensity of KDM4B or RBBP4 with PPARa-seRNA. (D) RNA immunoprecipitation with an antibody against KDM4B indicating significant enrichment of PPARa-seRNA. (E) The localization of PPARa-seRNA and KDM4B in AC16 cells. Scale bar ¼ 10 mm. (F) Binding ability of PPARa-seRNA and KDM4B predicted by the catRAPID tool. (G) Full length and three domains of KDM4B were inserted into the pc3.1-DNA vector with 3 flag tag, respectively. (H) RNA pull-down verifying the binding intensity of the domains to PPARa-seRNA. (I) Relative PPARa-seRNA levels detected by three domains-RIP in AC16 cells. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons test for D; student’s two-tailed t test for I. *P < 0.05, **P < 0.01. Data are depicted as mean SD.

Article Snippet: Cell culture and transfection AC16 human cardiomyocyte-like cells, murine HL-1 cardiomyocytelike cells, and HEK293T cell lines from ATCC (Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 5% glucose) supplemented with 10% Fetal Bovine Serum (FBS) and 1% of penicillin/streptomycin mixture, and cultured at 37 C in a humidified atmosphere of 5% CO2.

Techniques: Binding Assay, Mass Spectrometry, RNA Immunoprecipitation, Plasmid Preparation, Two Tailed Test