Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: CU-rich RNA promotes heterochromatin condensate organization during differentiation. ( A ) Nuclei of C2C12 myotubes (MT) treated with 1.5% 1,6-hexanediol (1,6-HD) or 1.5% 2,5-hexanediol (2,5-HD). Left: Representative images of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar, 5 μm. Middle: Time-course quantification of the number of heterochromatin foci per nucleus. Right: Boxplots show foci area (μm 2 , y -axis) at 5, 10, and 15 min posttreatment ( x -axis). n = 55 nuclei, three biological replicates. ( B ) Representative live-cell images of Hoechst 33342-stained MT nuclei before and after 1,6-HD treatment (0 and 15 min, respectively), taken from . Arrows indicate changes in heterochromatin foci intensity (pink, increased; blue, decreased), and the red arrow highlights an alteration in chromocenter integrity. ( C ) Number of heterochromatin foci per nucleus in MT following recovery from 1.5% 1,6-hexanediol (1,6-HD) treatment for 15 min, measured at indicated time points. n = 68 nuclei, three biological replicates. ( D ) Representative images of nuclei of myoblast (MB) and MT with or without 1,6-HD treatment (1.5%, 15 min). Right: Quantification of the number of heterochromatin foci per nucleus. n = 40 (MB), n = 60 (MT), three biological replicates. ( E ) Quantification of colocalization between indicated proteins and DAPI foci in MT with or without 1,6-HD treatment (1.5%, 15 min), by Pearson’s correlation coefficients. n = 60, three biological replicates. ( F ) Boxplot showing the distribution of Z -score of the interchromosomal interaction frequencies in MB and MT. ( G ) RNA FISH using ChRO1 and LacZ biotinylated probes. Biotin signal was detected by Fluorescein-conjugated Avidin DCS and amplified with biotinylated anti-Avidin and additional Fluorescein Avidin DCS. Right: Quantification of colocalization between biotin signal and DAPI foci. n = 50 nuclei. ( H ) Number of heterochromatin foci per nucleus of mouse fibroblast cells (NIH3T3) with or without doxycyline (Dox)-induced ChRO1a expression and/or 1,6-HD treatment (1.5%, 15 min). (EV; empty vector). n = 50, three biological replicates. ( I ) Number of heterochromatin foci per nucleus in MB with or without Dox-induced ChRO1a fragment (1–413, CUR) expression and/or 1,6-HD treatment (1.5%, 15 min). n = 75, three biological replicates. Statistical analyses and data presentation details are described in the “Materials and methods” section.

Article Snippet: C2C12 murine myoblast cells and NIH3T3 mouse fibroblast cells were obtained from the American-type culture collection and grown in a growth medium (GM) consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Staining, Avidin-Biotin Assay, Amplification, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: Osthol ameliorates obesity-associated lipid metabolic disorders by inhibiting ADRA1D-dependent Th17 cell differentiation

doi: 10.1038/s41598-025-20719-x

Figure Lengend Snippet: OST Regulates Adipocyte Lipid Metabolism via ADRA1D. ( A ) Oil red O staining was performed to assess lipid droplet accumulation in 3T3-L1 adipocytes treated with OST. ( B ) Intracellular triglyceride (TG) content was quantified to evaluate lipid accumulation. ( C , D ) RT-qPCR and western blotting were used to measure the mRNA and protein expression levels of FASN, PPARγ, and ADRA1D. (E) Western blot was used to detect the phosphorylation levels of ERK1/2 and PI3K. n = 3. ** P < 0.01, **** P < 0.0001. One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons.

Article Snippet: 3T3-L1 preadipocytes (400,107, Cytion, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle medium (DMEM; 11,885,084, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; A5670401, Thermo Fisher Scientific) at 37 °C until reaching 100% confluence.

Techniques: Staining, Quantitative RT-PCR, Western Blot, Expressing, Phospho-proteomics

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: Infection and Immunity

Article Title: Escherichia coli Cytotoxic Necrotizing Factor and Pasteurella multocida Toxin Induce Focal Adhesion Kinase Autophosphorylation and Src Association

doi: 10.1128/iai.69.9.5931-5935.2001

Figure Lengend Snippet: FIG. 1. Dose- and time-dependent induction of tyrosine 397 autophosphorylation of FAK by CNF1 and PMT. (A) Quiescent Swiss 3T3 cells were treated with purified PMT over a concentration range of 0.01 to 5 ng ml21, with lysates from recombinant E. coli expressing CNF1 over a concentration range of 0.01 to 5 mg ml21, or with lysates from E. coli harboring pBluescript (pBS) for 4 h. The cellular FAK was immunopre- cipitated, and the isolated proteins were separated by SDS-PAGE. A Western blot of the gel was probed with a polyclonal antiserum specific for the Tyr397 autophosphorylation site (FAKPY397). The antibodies were stripped from the membranes and probed with the anti-FAK antibody (FAK). (B) Quiescent Swiss 3T3 cells were treated with lysates from E. coli expressing CNF1 or harboring control pBluescript plasmid at a final concentration of 0.5 mg ml21 or with PMT or the C1165S mutant PMT (PMTc-s) at a final concentration of 5 ng ml21. Cells were harvested before the toxin preparations were added and at 1, 2, 3, 4, 6, and 8 h after addition of the toxins. The cellular FAK was immunoprecipitated, and the isolated proteins were separated by SDS-PAGE. A Western blot of the gel was probed with a polyclonal antiserum specific for the Tyr397

Article Snippet: The stimulation of FAK phosphorylation at Tyr by PMT or CNF1 was investigated, as described previously (41), by exposing quiescent Swiss 3T3 cells to each of the toxins at different concentrations for 4 h. After treatment, the cells were solubilized and FAK was immunoprecipitated from the cleared lysate by using polyclonal rabbit anti-FAK antibody C-20 (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Recombinant, Expressing, Isolation, SDS Page, Western Blot, Control, Plasmid Preparation, Mutagenesis, Immunoprecipitation

Journal: Infection and Immunity

Article Title: Escherichia coli Cytotoxic Necrotizing Factor and Pasteurella multocida Toxin Induce Focal Adhesion Kinase Autophosphorylation and Src Association

doi: 10.1128/iai.69.9.5931-5935.2001

Figure Lengend Snippet: FIG. 2. Induction of FAK-Src association by CNF1 and PMT. (A) Quiescent Swiss 3T3 cells were either left untreated (Unt) or were treated with PMT or the C1165S mutant (PMTc-s) at a final concen- tration of 5 ng ml21 or with lysates from E. coli expressing CNF1 or harboring the control pBluescript plasmid (pBS) at a final concentra- tion of 1 mg ml21. Cells were harvested after 4 h. The cellular Src was immunoprecipitated, and the isolated proteins were separated by SDS- PAGE. A Western blot of the gel was probed with a polyclonal anti- FAK antibody (FAK) to detect FAK that had coimmunoprecipitated with Src. (B) Quiescent Swiss 3T3 cells were treated with bombesin at a final concentration of 10 nM or with lysates from E. coli expressing CNF1 at a final concentration of 1 mg ml21 for 15 min or 4 h, respec- tively, or were left untreated for 4 h. FAK coimmunoprecipitating with Src was detected as described above for panel A.

Article Snippet: The stimulation of FAK phosphorylation at Tyr by PMT or CNF1 was investigated, as described previously (41), by exposing quiescent Swiss 3T3 cells to each of the toxins at different concentrations for 4 h. After treatment, the cells were solubilized and FAK was immunoprecipitated from the cleared lysate by using polyclonal rabbit anti-FAK antibody C-20 (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Expressing, Control, Plasmid Preparation, Immunoprecipitation, Isolation, SDS Page, Western Blot, Concentration Assay

Journal: Infection and Immunity

Article Title: Escherichia coli Cytotoxic Necrotizing Factor and Pasteurella multocida Toxin Induce Focal Adhesion Kinase Autophosphorylation and Src Association

doi: 10.1128/iai.69.9.5931-5935.2001

Figure Lengend Snippet: FIG. 3. Effect of p160/ROCK inhibitors on CNF1- and PMT-induced FAK autophosphorylation and actin stress fiber formation. (A) Quiescent Swiss 3T3 cells were incubated in DMEM with p160/ROCK inhibitors HA1077 (2.0 and 10 mM) and Y-27632 (0.5, 2.0, and 10 mM) for 1 h prior to the addition of a lysate from E. coli expressing CNF1 at a final concentration of 0.5 mg ml21. Cells were harvested after 4 h. The cellular FAK was immunoprecipitated, and the isolated proteins were separated by SDS-PAGE. A Western blot of the gel was probed with a polyclonal antiserum specific for the Tyr397 autophosphorylation site (FAKPY397). The antibodies were stripped from the membranes and reprobed with the anti-FAK antibody (FAK). Cells were similarly pretreated (1) or not pretreated (2) for 4 h with Y-27632 (B) or HA1077 (C) at a final concentration of 10 mM prior to the addition of wild-type PMT or mutant C1165S PMT (PMTc-s) at a final concentration of 5 ng ml21. The basal level of Tyr397 phosphorylation was determined from cells not treated with toxin (Unt). (D) Quiescent Swiss 3T3 cells were incubated in DMEM with (1) or without (2) p160/ROCK inhibitor Y-27632 (10 mM) for 1 h prior to the addition of lysate from E. coli harboring control pBluescript plasmid or expressing CNF1 at a final concentration of 0.5 mg ml21 or wild-type or mutant PMT at a final concentration of 5 ng ml21 or not treated with a toxin. After 16 h, the cells were fixed in paraformaldehyde (3.7%), permeabilized, and stained with phalloidin-rhodamine to demonstrate actin organization. The cells were examined by fluorescence microscopy.

Article Snippet: The stimulation of FAK phosphorylation at Tyr by PMT or CNF1 was investigated, as described previously (41), by exposing quiescent Swiss 3T3 cells to each of the toxins at different concentrations for 4 h. After treatment, the cells were solubilized and FAK was immunoprecipitated from the cleared lysate by using polyclonal rabbit anti-FAK antibody C-20 (Santa Cruz Biotechnology).

Techniques: Incubation, Expressing, Concentration Assay, Immunoprecipitation, Isolation, SDS Page, Western Blot, Mutagenesis, Phospho-proteomics, Control, Plasmid Preparation, Staining, Microscopy