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: Scientific Reports

Article Title: Protein delivery to living cells by thermal stimulation for biophysical investigation

doi: 10.1038/s41598-022-21103-9

Figure Lengend Snippet: ( A ) Schematic representation of the developed thermal stimulation delivery method (HS). ( B ) HSP70s WB assay conducted on the HEK 293 T, HeLa and Jurkat T cells after the thermal stimulation delivery. ( C ) PDB structures of the proteins used in this work (Ub: 1UBQ.pdb GFP: 1GFL.pdb). ( D ) Trypan blue Viability assay for control and treated cells with the three tested thermal stimulation schemes in absence of external protein. The raw western blot membrane is shown if the Supplementary Information, Fig. .

Article Snippet: HEK 293 T cells (ATCC CRL-3216) and HeLa cells (obtained from Swiss Institute for Experimental Cancer Research) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco) supplemented with l-glutamine, antibiotics (penicillin and streptomycin) and 10% fetal bovine serum (FBS; Gibco) in uncoated 75 cm2 plastic flasks.

Techniques: Viability Assay, Western Blot

Journal: Scientific Reports

Article Title: Protein delivery to living cells by thermal stimulation for biophysical investigation

doi: 10.1038/s41598-022-21103-9

Figure Lengend Snippet: ( A ) Viability assay conducted using the 5-5 HS delivery setup in presence of variable external Ubiquitin protein concentrations. ( B ) grey-cw-EPR spectra acquired at RT on HeLa, HEK 293 T and Jurkat T cells where ubiquitin A28C ma-Px-spin labelled was delivered using the 5-5 delivery scheme and an external protein concentration of 500 μM. Blue-cw-EPR spectra acquired for an in vitro reference sample of ubiquitin A28C ma-Px-spin labelled protein at 100 μM. ( C ) In-gel fluorescence PAGE and Western blot assay conducted on Ub A28C-atto488 and GFP delivered cells using the 5-5 delivery setup. In each assay references at known concentrations were used (Supplementary Information, Experimental Methods, Figs. ). All gels reported in this figure have been selected from the raw gels reported in the .

Article Snippet: HEK 293 T cells (ATCC CRL-3216) and HeLa cells (obtained from Swiss Institute for Experimental Cancer Research) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco) supplemented with l-glutamine, antibiotics (penicillin and streptomycin) and 10% fetal bovine serum (FBS; Gibco) in uncoated 75 cm2 plastic flasks.

Techniques: Viability Assay, Protein Concentration, In Vitro, Fluorescence, Western Blot

Journal: Scientific Reports

Article Title: Protein delivery to living cells by thermal stimulation for biophysical investigation

doi: 10.1038/s41598-022-21103-9

Figure Lengend Snippet: ( A ) Model structure of human ubiquitin protein labelled with doubly Maleimide DOTA Gadolinium tag in the S20C and G35C position. ( B ) Derived bulk intracellular concentration for the Ub-ma-DOTA-Gd 3+ delivered cells. The concentration was estimated interpolating the Integral value of the in-cell spectra Gd 3+ transition with a calibration curve. ( C ) Acquired EDFS spectra for the HEK 293 T and HeLa cells over the center of the resonator dip under overcoupling conditions. ( D ) DEER spectra acquired for the in vitro (blue traces) and the relative in-cell sample (red traces).

Article Snippet: HEK 293 T cells (ATCC CRL-3216) and HeLa cells (obtained from Swiss Institute for Experimental Cancer Research) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco) supplemented with l-glutamine, antibiotics (penicillin and streptomycin) and 10% fetal bovine serum (FBS; Gibco) in uncoated 75 cm2 plastic flasks.

Techniques: Derivative Assay, Concentration Assay, In Vitro

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