Journal: Cell & Bioscience

Article Title: Aberrant methylation of Serpine1 mediates lung injury in neonatal mice prenatally exposed to intrauterine inflammation

doi: 10.1186/s13578-022-00901-8

Figure Lengend Snippet: Demethylation promoted Rela but inhibited Hdac2 binding to the Serpine1 promoter. A–C Serpine1 expression analysed using RT-qPCR and western blot in Rela-, Hif1a-, and Rest-overexpressing MLE-12 cells. D Transcriptional activity of Rela, Hif1a, and Rest at the Serpine1 promoter analysed using luciferase assay in MLE-12 cells. E The binding ability of Rela and Hdac2 to the biotin-labelled Serpine1 promoter probe analysed via DNA pull-down assay. F–H Serpine1 expression analysed using RT-qPCR and western blot in Hdac2-knockdown MLE-12 cells. I Transcriptional activity of Rela, Hif1a, and Rest at the methylated-Serpine1 promoter analysed using luciferase assay in HEK293T cells. J Transcriptional activity of Rela at the Serpine1 promoter analysed using luciferase assay in Hdac2-knockdown MLE-12 cells. K The enrichment of TFs at the Serpine1 promoter region determined using anti-Rela and anti-Hdac2 antibodies via ChIP-qPCR in 5-Aza-CdR-treated MLE-12 cells. Data show mean ± SEM; Data was analysed using unpaired t -tests

Article Snippet: MLE-12, A549, BEAS-2B and human epithelial kidney 293 T (HEK293T) cell lines were purchased from ATCC.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Pull Down Assay, Methylation

Journal: Nature Communications

Article Title: TRPC channels blockade abolishes endotoxemic cardiac dysfunction by hampering intracellular inflammation and Ca 2+ leakage

doi: 10.1038/s41467-022-35242-0

Figure Lengend Snippet: a The interacted domain of TLR4 (Myc-tagged) with CaM (FLAG-tagged) in co-transfected HEK293T cells measured using Co-IP ( n = 2 biological independent experiments). b Representative immunofluorescent photomicrographs (left panel) and traces of fluorescence intensity spatial profiles (right panel) of TLR4 (Myc-tagged) with CaM (FLAG-tagged) localization in co-transfected HEK293T cells ( n = 6 images from 3 biological independent experiments). c The CD spectroscopies of the synthesized peptides. d The interaction of CaM with the peptides encompassing nonclassical IQ motifs detected by a non-denaturing gel ( n = 2 biological independent experiments). e The dissociation constants of CaM and peptide TLIQ2 measured using MST (mean ± SEM, n = 3 biological independent experiments). f Structural model of signaling complex formed by TLR4 and CaM. Secondary structure elements and position of mutational sites in the human TLR4 sequence (upper panel) and a homologous modeling of TLR4 (TLR2, PDB 1Fyx) and TLIQ2 contacting with CaM (PDB 1QX5) (lower panel) are shown. Source data are provided as a Source Data file.

Article Snippet: Human embryonic kidney 293 T (HEK293T) cell line was obtained from ATCC (CRL-3216).

Techniques: Transfection, Co-Immunoprecipitation Assay, Fluorescence, Synthesized, Sequencing

Journal: Nature Communications

Article Title: The mitochondrial β-oxidation enzyme HADHA restrains hepatic glucagon response by promoting β-hydroxybutyrate production

doi: 10.1038/s41467-022-28044-x

Figure Lengend Snippet: a Luciferase reporter assay of the inhibitory effect of HADHA on Pck1 and G6pc gene promoter co-transfected with HADHA and FOXO1 plasmid in HEK-293T cells. The luciferase activity was normalized with the internal control (Renilla luciferase, n = 6). b Representative confocal images of FOXO1 nuclear translocation in HADHA-overexpressed primary hepatocytes with or without BDH1 siRNA transfection and 100 nM glucagon stimulation for 1 h. Scale bar represents 10 μm. c Confocal images of FOXO1 nuclear translocation in BHB (400 μM, 6 h) treated primary hepatocytes when exposed to glucagon (100 nM, 1 h). Scale bar represents 10 μm. d FOXO1 phosphorylation from the hepatocytes in b ( n = 3). e FOXO1 phosphorylation from the hepatocytes in panel c ( n = 3). f, g FOXO1 acetylation in HADHA-overexpressed or knockdown primary hepatocytes transfected with BDH1 siRNA and 100 nM glucagon stimulation for 1 h. b , c , f and g were repeated 3 times independently with similar results. BHB β-hydroxybutyrate, GLC glucagon, NC normal control. Values represent mean ± SEM. Statistical differences were determined by one-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: HepG2 cell line was obtained from the American Type Culture Collection, and human embryonic kidney 293 T (HEK-293T) cell line was obtained from Stem Cell Bank, Chinese Academy of Sciences.

Techniques: Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Activity Assay, Translocation Assay

Journal: Nature Communications

Article Title: The mitochondrial β-oxidation enzyme HADHA restrains hepatic glucagon response by promoting β-hydroxybutyrate production

doi: 10.1038/s41467-022-28044-x

Figure Lengend Snippet: a The distribution of HDAC4, HDAC5 and HDAC7 in the nucleus and cytoplasm of HADHA-overexpressed hepatocytes transfected with BDH1 siRNA with or without BHB (400 μM, 6 h) and 100 nM glucagon stimulation for 1 h ( n = 3). b Immunoprecipitation analysis of interaction of FOXO1 with HDAC4, HDAC5 or HDAC7 in HADHA-overexpressed hepatocytes with or without BDH1 siRNA transfection. It was repeated 3 times independently with similar results. c Enzyme activity of HDAC4, HDAC5 and HDAC7 in HepG2 cells with 100 nM glucagon stimulation for 1 h after BHB treatment (400 μM, 6 h, n = 5). d Relative mRNA abundance of Pck1 , G6pc and Pgc1a in HADHA-overexpressed hepatocytes transfected with BDH1 siRNA and HDAC7 siRNA ( n = 6). e Immunoprecipitation analysis of FOXO1 acetylation in d . It was repeated 3 times independently with similar results. f Luciferase reporter assay of the effect of HADHA and HDAC7 on Pck1 and G6pc gene promoter. The Pck1 and G6pc luciferase reporters were co-transfected with HADHA, FOXO1 and HDAC7 plasmid in HEK-293T cells. The luciferase activity was normalized with the internal control (Renilla luciferase, n = 6). Values represent mean ± SEM. Statistical differences were determined by one-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: HepG2 cell line was obtained from the American Type Culture Collection, and human embryonic kidney 293 T (HEK-293T) cell line was obtained from Stem Cell Bank, Chinese Academy of Sciences.

Techniques: Transfection, Immunoprecipitation, Activity Assay, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: The mitochondrial β-oxidation enzyme HADHA restrains hepatic glucagon response by promoting β-hydroxybutyrate production

doi: 10.1038/s41467-022-28044-x

Figure Lengend Snippet: a The interaction of BHB with HDACs by surface plasmon resonance. A series of BHB concentrations was injected onto the HDAC4, HDAC5 or HDAC7 biosensor surface. The frequency response and fitting curves were displayed. b CETSA for in-cell HDAC7 target engagement. Representative western blots showed thermostable HDAC7 with indicated heat shocks in the presence or absence of BHB (50 µM, n = 3). c Immunoprecipitation analysis of interaction between biotin-BHB and HDAC7 in primary hepatocytes transfected with HDAC7 plasmid with or without unlabeled BHB treatment. It was repeated 3 times independently with similar results. d Binding modes and sites of BHB with HDAC7 predicted by AutoDock. The protein is shown as cartoon and BHB as sticks. e Immunoprecipitation analysis of interaction between BHB-biotin and HDAC7 in HEK-293T cells transfected with HDAC7 (WT, Mut-His541, Mut-Glu543 or Mut-Asp626) plasmid. It was repeated 3 times independently with similar results. f Enzyme activity of HDAC7 in HepG2 cells transfected with HDAC7 (WT, Mut-His541, Mut-Glu543 or Mut-Asp626) plasmid ( n = 5). g Relative mRNA abundance of Pck1 , G6pc and Pgc1a in primary hepatocytes transfected with HDAC7 (WT, Mut-His541, Mut-Glu543 or Mut-Asp626) plasmid after BHB treatment ( n = 6). Asp aspartic acid, BHB β-hydroxybutyrate, CETSA, cellular thermal shift assay, GLC glucagon, Glu glutamic acid, His histidine, Mut mutant, WT wild type. Values represent mean ± SEM. Statistical differences were determined by one-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: HepG2 cell line was obtained from the American Type Culture Collection, and human embryonic kidney 293 T (HEK-293T) cell line was obtained from Stem Cell Bank, Chinese Academy of Sciences.

Techniques: SPR Assay, Injection, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Binding Assay, Activity Assay, Thermal Shift Assay, Mutagenesis

Journal: Molecular Biology and Evolution

Article Title: Human-Specific Mutations and Positively Selected Sites in MARCO Confer Functional Changes

doi: 10.1093/molbev/msx298

Figure Lengend Snippet: Mutation of sites Q452 and F282 does not affect expression of MARCO. HEK 293 T cells transfected with empty vector, MARCO, MARCOII, Q452A, Q452D, or F282S were examined for total protein expression by western blot (A) and surface expression by flow cytometry (B). V, vector; M, MARCO; II, MARCOII; A, Q452A; D, Q452D; S, F282S.

Article Snippet: Cell Lines HEK 293 T (ATCC #CRL-3216) were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C and 5% CO 2 .

Techniques: Mutagenesis, Expressing, Transfection, Plasmid Preparation, Western Blot, Flow Cytometry

Journal: Molecular Biology and Evolution

Article Title: Human-Specific Mutations and Positively Selected Sites in MARCO Confer Functional Changes

doi: 10.1093/molbev/msx298

Figure Lengend Snippet: Residues at positions 452 and 282 of MARCO influence ligand association and bacterial internalization. (A) Mutation of glutamine residue 452 to alanine (Q452A) reduces ligand-coated microsphere association in HEK 293 T cells, whereas mutation to the ancestral aspartic acid (Q452D) does not. Mutation of residue phenylalanine 282 to the ancestral serine (F282S) also reduces microsphere association. Statistical comparisons relative to MARCO were made using one-way ANOVA with Tukey’s post hoc test. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Experiments were performed a minimum of three times with a minimum of three technical replicates. (B) Immunofluorescence microscopy of microsphere association in HEK 293 T transfected with empty vector, MARCO, MARCOII, or variants Q452A, Q452D, and F282S. Scale bars represent 25 µm. (C) Mutation of residue 452 to alanine (Q452A) or residue 282 to serine (F282S) greatly reduces the phagocytic index of RAW 264.7 macrophages. Phagocytic index was calculated as number of internalized bacteria divided by the number of RAW 264.7 macrophages counted. Statistical comparisons relative to MARCO were made using one-way ANOVA with Tukey’s post hoc test. Error bars indicate mean ± SEM. **P < 0.01.

Article Snippet: Cell Lines HEK 293 T (ATCC #CRL-3216) were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C and 5% CO 2 .

Techniques: Mutagenesis, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation