Journal: Chemical & Biomedical Imaging

Article Title: Recombinant–Chemosynthetic Biosensors for Probing Cell Surface Signaling of Red Blood Cells and Other Cells

doi: 10.1021/cbmi.4c00067

Figure Lengend Snippet: Figure 2. (A) Optimization of the cell labeling protocol in HeLa cells was performed under three different conditions: (left) cells are in MEM/ DFBS/PS containing HeLa media and purified sortase A and GCaMP6s-LPDTG proteins are in 1% PBS; (middle) cells and purified sortase A and GCaMP6s-LPDTG proteins are in 1% PBS; (right) cells are in 1% PBS and purified sortase A and GCaMP6s-LPDTG proteins are in elution buffer. (B) HeLa cells labeled with ExtraCal showed a reduction in ExtraCal fluorescence upon EDTA addition and its fluorescence intensity was increased upon addition of HBSS containing Ca2+ (n = 25 cells from 3 repeats). The plot shows fluorescence change of ExtraCal upon EDTA followed by HBSS-With- Ca2+ addition over the course of 6 min. The error bars represent SD (standard error of standard deviation). Scale bar = 5 μm. EDTA, ethylenediaminetetraacetic acid; HBSS, Hanks’ balanced salt solution.

Article Snippet: EDTA Disodium salt dihydrate (Fisher Scientific), HBSS-With-Ca2+ (Gibco), Ca2+ free HBSS (Gibco), ATP (Chem-Impex International, Inc.)), Rhod-2 AM (Cayman Chemical), YM-254890 (Cayman Chemical), oxyrase (Oxyrase, Inc.), MgCl2 hexahydrate (SigmaAldrich), PMSF (Research Products International), Lysozyme (Gold Biotechnology), Luciferin (Gold Biotechnology), and EZ-Run Prestained Rec Protein Ladder (Fisher Scientific).

Techniques: Labeling, Purification, Fluorescence, Standard Deviation

Journal: Frontiers in Veterinary Science

Article Title: Analyzing the interactions of mRNAs, miRNAs and lncRNAs to predict ceRNA networks in bovine cystic follicular granulosa cells

doi: 10.3389/fvets.2022.1028867

Figure Lengend Snippet: Analysis of miR-664b sponge HSD17B7 3'-UTR and lncRNA NONBTAT027373.1. (A) : The networks of lncRNA-miRNA- HSD17B7 (B) : The expression of DE lncRNAs in lncRNA-miRNA- HSD17B7 networks. (C) : The expression of HSD17B7 mRNA. (D) : The expression of DE miRNAs in lncRNA-miRNA- HSD17B7 networks. (E) : Interaction between lncRNA NONBTAT027373.1 and miR-664b through Ago2 protein investigated using the RIP assay. lncRNA NONBTAT027373.1 and miR-664b expression quantified by RT-qPCR. (F) : Schematic view of the miR-664b seed region, the motif of HSD17B7 3'-UTR and lncRNA NONBTAT027373.1. (G,H) : Construction of WT and MUT plasmid vectors of HSD17B7 3'-UTR and lncRNA NONBTAT027373.1. (I) : Dual-luciferase reporter gene analysis of the regulatory relationship between HSD17B7 3'-UTR and miR-664b. (J) : Dual-luciferase reporter gene analysis of the regulatory relationship between lncRNA NONBTAT027373.1 and miR-664b. (K) : Transfection efficiency of miR-664b after overexpression and knockdown of miR-664b. (L) : miR-664b regulates the expression of HSD17B7 mRNA. (M) : RT-qPCR analysis of the effect on silence of lncRNA NONBTAT027373.1 expression by siRNA. (N) : Relative expression of HSD17B7 mRNA in groups. NS, Non-significant. Values represent means ± SEM for three individuals. * P < 0.05, ** P < 0.01. Yellow nodes represent HSD17B7 ; red nodes represent miRNAs and blue nodes represent lncRNAs in lncRNA-miRNA- HSD17B7 networks. WT, Wild Type; MUT, Mutant; Mimics, Overexpression of miR-664b; Inhibitor, Knockdown of miR-664b; NC, Negative control.

Article Snippet: Incubated A/G protein beads (MCE, USA) with Ago2 (dilution multiple 1:100) (BOSTER, China)/IgG (dilution multiple 1:100) antibody (ABclonal, China) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Transfection, Over Expression, Mutagenesis, Negative Control