Journal: Frontiers in cell and developmental biology

Article Title: Patient-derived and gene-edited pluripotent stem cells lacking NPHP1 recapitulate juvenile nephronophthisis in abnormalities of primary cilia and renal cyst formation.

doi: 10.3389/fcell.2024.1370723

Figure Lengend Snippet: FIGURE 1 Generation of NPHP1-overexpressing hiPSCs derived from patient-derived hiPSCs with whole NPHP1 deletion. (A) Schematic construct of pPB- CAG-NPHP1-PGK-HygR in DNA transposon. pPB: piggybac transposon plasmid, CAG: Cytomegalovirus (CMV) early enhancer element, the promoter, the first exon and the first intron of chicken beta-Actin gene, and the splice acceptor of the rabbit beta-Globin gene, PGK: phosphoglycerate kinase, HygR: hygromycin resistant gene. (B,C) Phase contrast images of patient-derived hiPSCs and their transfected cells treated with hygromycin at 50, 100, and 200 μg/mL for 5 days. Scale bars = 500 µm. (D) Relative mRNA expression of NPHP1 in healthy donor hiPSCs (1383D6 line), patient-derived hiPSC lines, HPS0447 and HPS0450, transfected hiPSC lines. HPS0447 NPHP1-OE and HPS0450 NPHP1-OE. Each value was normalized with GAPDH value from the same sample. Data are shown as the mean ± SE (n = 3). (E) Representative images of an automatic capillary Western blot on nephrocystin-1 protein in healthy donor hiPSCs (1383D6 line), patient-derived hiPSC lines, HPS0447 and HPS0450, transfected hiPSC lines, HPS0447 NPHP1-OE and HPS0450 NPHP1-OE. (F) Relative protein expression of nephrocystin-1 calculated from the data of automatic capillary Western blot. Each value was normalized with GAPDH value from the same sample. Data are shown as the mean ± SE (n = 3). (G,H) Immunocytochemistry of NPHP1 in patient-derived hiPSC line, HPS0447 and HPS0450, transfected hiPSC lines, HPS0447 NPHP1-OE and HPS0450 NPHP1-OE. Nuclei are stained with DAPI. Scale bars = 100 µm.

Article Snippet: Western blot was performed with a capillary automatic Western blot device (Simple Western, Wes; ProteinSimple) following the manufacturer’s instructions.

Techniques: Derivative Assay, Construct, Plasmid Preparation, Transfection, Expressing, Western Blot, Immunocytochemistry, Staining

Journal: Biosafety and Health

Article Title: Rapid evaluation of heterologous chimeric RBD-dimer mRNA vaccine for currently-epidemic Omicron sub-variants as booster shot after inactivated vaccine

doi: 10.1016/j.bsheal.2023.02.002

Figure Lengend Snippet: In vitro characterization of seven mRNA vaccines with dimeric receptor-binding domains (RBDs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A) In vitro expression of the seven mRNAs encoding dimeric RBDs. Each mRNA was transfected into HEK293T cells. The expression of dimeric RBDs in the supernatant was analyzed by western blot. B) Cryo-electron microscopy image of the lipid nanoparticles (LNPs) of seven mRNA vaccines and empty LNP. Scale bar, 100 nm. C) Particle size distributions of LNPs characterized by dynamic light scattering (DLS). The number in the legend indicates z-average or polydispersity index (PDI). D) Zeta potential for LNPs at pH 4.0 and 7.4. For C and D, one representative result from two independent experiments is shown.

Article Snippet: Diluted LNPs were added into folded capillary zeta cells and loaded into Zetasizer Pro (Malvern Panalytical).

Techniques: In Vitro, Vaccines, Binding Assay, Expressing, Transfection, Western Blot, Cryo-Electron Microscopy, Zeta Potential Analyzer

Journal: Journal of Nanobiotechnology

Article Title: Biomimetic nanoparticles blocking autophagy for enhanced chemotherapy and metastasis inhibition via reversing focal adhesion disassembly

doi: 10.1186/s12951-021-01189-5

Figure Lengend Snippet: Preparation of tumor-targeting nanoformulation TH-NP for the co-delivery of OXA and HCQ. A Schemes showing the fabrication of TH-NP and usage as an HCC targeted co-delivery system, which was synthesized through TRAIL expressing HUVECs membrane coating on PLGA NPs loading OXA and HCQ. B DLS measurements (hydrodynamic size and zeta potential ζ) of TH-NP. C TEM of the targeted co-delivery NPs TH-NPs, showing the intact membrane coating onto PLGA NPs and the uniform size distribution. Scale bar:100 nm. D Western blotting results showing TRAIL protein was integrally preserved on the outer membrane of TH-NPs without damaging its activity. E Release profiles of HCQ from the TH-NPs. F Release profiles of OXA from the TH-NPs. G Confocal microscopy displayed the tumor targeting of Dil-conjugated various membrane coated NPs in the HCCLM3 cells. TH-NPs showed the most potent HCC targeting compared to H-NPs, HEK-NPS or THEK-NPs. (Dil, red; Hoechst, blue). Scale bar: 20 μm. H Comparison of relative mean fluorescence intensity (MFI) of different nanoparticles of HCC targeting. Data were displayed as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: The human umbilical vein endothelial cell (HUVEC) from ATCC was cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin.

Techniques: Synthesized, Expressing, Membrane, Zeta Potential Analyzer, Western Blot, Activity Assay, Confocal Microscopy, Comparison, Fluorescence

Journal: Journal of medicinal chemistry

Article Title: Discovery of a Novel Chemotype of Histone Lysine Methyltransferase EHMT1/2 (GLP/G9a) Inhibitors: Rational Design, Synthesis, Biological Evaluation, and Co-crystal Structure

doi: 10.1021/acs.jmedchem.8b02008

Figure Lengend Snippet: Direct binding measurements of the interaction between G9a and compounds 7 (left) and 12a (right). (a, b) MST binding affinity assays were performed as described in Experimental Section. Representative dose–response curves of 7 (a) and 12a (b) binding to G9a are shown; Fnorm, normalized fluorescence. Experiments were repeated independently three times. Reported KD is the mean ± SD of three independent experiments. (c, d) Sensorgrams obtained from the SPR interaction analysis of compounds 7 (c) and 12a (d) binding to immobilized G9a. Each compound was injected at eight different concentrations (1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 μM). The equilibrium dissociation constants (KD) were derived from the ratio between kinetic dissociation (koff) and association (kon) constants.

Article Snippet: Pretests using standard-treated and premium-coated MST capillaries (NanoTemper Technologies) were performed to test for adsorption of NT647-G9a to capillary walls by analyzing capillary scans recorded by the Monolith NT.115pico prior to MST experiments.

Techniques: Binding Assay, Fluorescence, Injection, Derivative Assay