Journal: PLoS ONE

Article Title: Chikungunya virus superinfection exclusion is mediated by a block in viral replication and does not rely on non-structural protein 2

doi: 10.1371/journal.pone.0241592

Figure Lengend Snippet: ( A ) Schematic representation of the tetracycline-inducible construct. PuroR, puromycin resistance. LTR, long terminal repeat; HIV-1 ψ, packaging signal of human immunodeficiency virus type 1; PGK, mouse phosphoglycerate kinase 1; PuroR, puromycin resistance (puromycin N-acetyltransferase); AmpR, ampicillin resistance (β-lactamase); cPPT/CTS, central polypurine tract and central termination sequence of HIV-1; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; RRE, Rev response element of HIV-1; MCS, multiple cloning site, where genes of interest were cloned; IRES, internal ribosome entry site; ori, origin of replication. ( B – D ) 3T3 cells stably co-expressing the transactivator protein (3T3 transactivator) and the tetracycline-inducible construct described in (A) were treated with doxycycline (1 μg/mL) or left untreated for 16 h, then infected with CHIKV-GFP for 8 h at an MOI of 4, and subsequently harvested and analyzed by flow cytometry. Representative flow cytometry plots (B), percentages of cells expressing the transgene (C) and percentage of infected cells (D) and are displayed. SP, structural protein. ( E , F ) 3T3 cells stably expressing the transactivator protein (3T3 transactivator) were transfected with the construct containing nsP2 gene. Twenty-four hours post-transfection, cells were plated and treated or not with 1 μg/mL of doxycycline for 16 h, then challenged with CHIKV-GFP at an MOI of 4. Eight hours post-infection, they were harvested and analyzed by flow cytometry. Representative flow cytometry plot (E) and percentage of infected cells in the mCherry + and mCherry − populations (F) are shown. Bars indicate mean ± SD of biological triplicates and data are representative of two independent experiments. *** p < 0.001 (two-way ANOVA followed by Sidak’s post-test (C) or unpaired t -test (D)).

Article Snippet: 2.2 million 3T3 transactivator cells (Takara #631197) were plated in 10-cm plates.

Techniques: Construct, Virus, Sequencing, Cloning, Clone Assay, Stable Transfection, Expressing, Infection, Flow Cytometry, Transfection

Journal: International Journal of Dentistry

Article Title: Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland

doi: 10.1155/2023/1765317

Figure Lengend Snippet: Mouse parotid acinar cells in primary culture. (a) Cell proliferative capacity of BMP2-added (100 ng/mL) and nonadded (control) groups after 48 hr of culture. The proliferative capacity of BMP2-added cultured cells was significantly increased compared to that in the control group. ∗∗ Indicates significance at P < 0.01, ∗ P < 0.05. Data were shown as mean ± SD. Three independent experiments were performed. (b) Protein expression of E-cadherin (an epithelial marker) and vimentin (a mesenchymal marker) in cultured cells of each group at 48 hr after the addition of BMP2. NIH3T3 (3T3) was used as a positive control for mesenchymal markers. RS: rat serum.

Article Snippet: NIH3T3 whole cell lysate (Novus Biologicals, Centennial, CO, USA) was used as a positive control for mesenchymal markers.

Techniques: Control, Cell Culture, Expressing, Marker, Positive Control

Journal: Cell calcium

Article Title: Ca 2+ -dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics.

doi: 10.1016/j.ceca.2021.102457

Figure Lengend Snippet: Fig. 1. Interaction of S100A6 with cofilin-1 in NIH3T3 fibroblasts. (A) Pull-down assay with the use of protein lysate from NIH3T3 cells and S100A6 affinity resin (upper panel) or empty resin (lower panel). Lanes: 1-input, 2-unbound fraction, 3-last wash, 4-first wash with 0.5 M NaCl, 5-last wash with 0.5 M NaCl, 6-first wash with 1 M NaCl, 7- last wash with 1 M NaCl, 8- elution in buffer containing EGTA. Fractions were analyzed by SDS-PAGE (15% gel) fol lowed by immunoblotting developed with anti- cofilin-1 antibody. (B) Co-immunoprecipitation of S100A6 with cofilin-1 from NIH3T3 cell lysate. 30 μg of protein lysate was used directly for immunoblotting (input; lane 1 in both upper and lower panel) and 2.5 mg of protein lysate was incubated with (upper panel) or without (control, lower panel) anti-S100A6 monoclonal antibody and then with protein A/G agarose. In both panels, lane 2 shows unbound fraction, lane 3-last wash and lane 4-elution. Proteins were identified by immunoblotting using anti- cofilin-1 antibody. (C) Presence of S100A6- cofilin-1 complexes in NIH3T3 cells studied by PLA. Complexes of examined proteins are visualized in red; cell nuclei, stained with DAPI, are in blue. Scale bar is 20 μm.

Article Snippet: For co-immunoprecipitation assays 2.5 mg of protein lysate from NIH3T3 cells, obtained using the Plasma Membrane Protein Extraction Kit (Abcam) according to the manufacturer’s instruction was incubated with protein A/G-Agarose (Santa Cruz Biotechnology) for 1 h at 4◦C, as described by Jurewicz et al. [31].

Techniques: Pull Down Assay, SDS Page, Western Blot, Immunoprecipitation, Incubation, Control, Staining

Journal: bioRxiv

Article Title: Lipid moieties of sonic hedgehog are important for interaction with its inhibitor, WIF1

doi: 10.64898/2026.02.23.707386

Figure Lengend Snippet: Comparison of the signaling activities of rhShh_8908-SH, rhShh_1314-SH and rhShh_1845-SH in the Gli Reporter-NIH3T3 cell line reporter assay. Panel A: rhShh_8908-SH expressed in HEK293 cells was applied at 0–600 nM. EC 50 = 34.22 ± 2.25 nM. Panel B: rhShh_1314-SH expressed in E. coli was applied at 0–2400 nM. EC 50 = 64.81 ± 1.29 nM. Panel C: rhShh_1845-SH expressed in E. coli was applied at 0–2400 nM. EC 50 = 86.09 ± 1.11 nM. In all assays, Gli Reporter-NIH3T3 cells were grown to confluency, the culture medium was removed, and cells were treated with 50 μL aliquots of the indicated Shh concentrations. Data represent one experiment performed in triplicate and are expressed as fold induction relative to luminescence at 0 nM Shh.

Article Snippet: The ONE-StepTM Luciferase Assay System and the Gli Reporter-NIH3T3 cell line were purchased from BPS Bioscience (Inc., San Diego, CA, USA).

Techniques: Comparison, Reporter Assay

Journal: bioRxiv

Article Title: Lipid moieties of sonic hedgehog are important for interaction with its inhibitor, WIF1

doi: 10.64898/2026.02.23.707386

Figure Lengend Snippet: Inhibition of the signaling activities of rhShh_8908-SH, rhShh_1314-SH and rhShh_1845-SH proteins by human WIF1 protein in the Gli Reporter-NIH3T3 cell line reporter assay. Panel A: Inhibition of human rhShh_8908-SH expressed in HEK293 cells (15 nM) by rhWIF1 (0–1000 nM); EC 50 = 3.78 ± 0.13 nM. Panel B: Inhibition of rhShh_1314-SH expressed in E. coli (15 nM) by rhWIF1 (0–1200 nM); EC 50 = 6.83 ± 0.89 nM. Panel C: Inhibition of rhShh_1845-SH expressed in E. coli (15 nM) by rhWIF1 (0–1200 nM); EC 50 = 15.71 ± 1.75 nM. In all assays, Gli Reporter-NIH3T3 cells were grown to confluency, culture medium was removed, and cells were treated with 50 μL aliquots of rhShh preincubated for 5 min with rhWIF1. Data represent representative experiments performed in triplicate (B, C) or quadruplicate (A) and are expressed as fraction of luminescence relative to 0 nM rhWIF1.

Article Snippet: The ONE-StepTM Luciferase Assay System and the Gli Reporter-NIH3T3 cell line were purchased from BPS Bioscience (Inc., San Diego, CA, USA).

Techniques: Inhibition, Reporter Assay