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: 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: Molecular Cancer

Article Title: NF-κB/STAT3/PI3K signaling crosstalk in iMyc Eμ B lymphoma

doi: 10.1186/1476-4598-9-97

Figure Lengend Snippet: AKT is constitutively phosphorylated, in a PTEN independent-manner, in a majority of LBLs and iMyc Eμ -1 cells . (A) Western blot analysis of the activating-phosphorylation status of key proteins of the PI3K (AKT, P-AKT S473 and T308), MAPK (ERK 1/2, P-ERK1/2, total p 38, P-p 38) and mTOR (p70S6K, P-p70S6K) signaling pathways. Positive controls for P-ERK1/2, P-p38 and P-p70S6K were from extracts of UV-treated HeLa cells, NIH 3T3 cells and insulin-treated MCF-7 cells, respectively. (B and C) Levels of PTEN protein (B) and mRNA (C) in LBLs and iMyc Eμ -1. α-tubulin and β-actin served as loading controls, respectively. "C" denotes control BL6 splenic B cells.

Article Snippet: Total cell extracts from UV-treated HeLa and NIH 3T3 cells were used as positive controls for P-ERK1/2 (sc-2221) and P-p38 (sc-2210), respectively (Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Protein-Protein interactions, Control

Journal: Cell Death and Differentiation

Article Title: BRCA1 and BRCA2 gene expression: p53- and cell cycle-dependent repression requires RB and DREAM

doi: 10.1038/s41418-025-01566-9

Figure Lengend Snippet: Wild-type (WT), Lin37 -/- (Lin37-KO), Rb -/- (Rb-KO) or Rb -/- /Lin37 -/- (DKO) NIH3T3 cells were used. Brca1 ( A ) and Brca2 ( B ) mRNA expression was measured from proliferating, density-arrested, or serum-starved cells. A , B Mean ± SD; n = 4, two-way ANOVA; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Analogous experiments were performed with serum-starved cells predominantly in G 0 and cells serum-restimulated to re-enter the cell cycle. From G 0 and restimulated cells, mRNA levels of Brca1 ( C ) and Brca2 ( D ) were analyzed. Maximal fold-changes were calculated as the ratio of the highest to lowest expression levels. Averages from two technical replicates each from four independent cell lines for each cell variant are shown. Expression was normalized to WT proliferating ( A , B ) or to maximum expression of WT ( C , D ).

Article Snippet: RPE-1, NIH3T3, HFF, and T98G cells (DSMZ, Braunschweig, Germany) as well as HCT116 wild-type, p53 -/- and p21 -/- cells [ ] were grown in DMEM (Lonza) supplemented with 10% FCS and penicillin/streptomycin and maintained at 37 °C and 10% CO 2 .

Techniques: Expressing, Variant Assay

Journal: Cell Death and Differentiation

Article Title: BRCA1 and BRCA2 gene expression: p53- and cell cycle-dependent repression requires RB and DREAM

doi: 10.1038/s41418-025-01566-9

Figure Lengend Snippet: NIH3T3 cells were transfected with luciferase reporter constructs of the wild-type human promoters (wt) and mutant promoters for potential transcription factor binding sites (E2F-A, E2F-B, and A/B) of ( A ) BRCA1 , with A/B representing a mutant of E2F-A and E2F-B or ( B ) wt and mutant promoter construct of BRCA2 together with a Renilla luciferase control reporter plasmid. Cells were synchronized in G 0 by serum starvation, stimulated to re-enter the cell cycle be serum addition and collected after 24 h. Relative luciferase activity is given (Mean ± SEM, n = 3–4; two-tailed unpaired t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: RPE-1, NIH3T3, HFF, and T98G cells (DSMZ, Braunschweig, Germany) as well as HCT116 wild-type, p53 -/- and p21 -/- cells [ ] were grown in DMEM (Lonza) supplemented with 10% FCS and penicillin/streptomycin and maintained at 37 °C and 10% CO 2 .

Techniques: Transfection, Luciferase, Construct, Mutagenesis, Binding Assay, Control, Plasmid Preparation, Activity Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: BRCA1 and BRCA2 gene expression: p53- and cell cycle-dependent repression requires RB and DREAM

doi: 10.1038/s41418-025-01566-9

Figure Lengend Snippet: A Chromatin immunoprecipitations (ChIPs) were performed with cross-linked chromatin from serum-starved (0 h) or restimulated (22 h) T98G cells. Antibodies targeted E2F4, E2F1, E2F3, or B-MYB. A non-targeting antibody (IgG) and the promoter of the GAPDHS gene served as a negative control. The BRCA1 and BRCA2 promoters were detected by real-time qPCR. All signals are given relative to the input DNA signal. B ChIPs were performed with cross-linked chromatin from starved (0 h) or restimulated (22 h) RPE-1 cells. Antibodies targeted E2F4, LIN9, LIN37, A-MYB, or B-MYB. A non-targeting antibody (IgG) and the promoter of the GAPDHS gene served as a negative control. The BRCA1 and BRCA2 promoters were detected by real-time qPCR. All signals are given relative to the input DNA signal. A , B Mean ± SD; two-way ANOVA; n = 3; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. C Nuclear extracts of density-arrested RPE-1 cells and cells restimulated for 20 h were employed for DNA affinity purification using biotinylated wt and mutant (E2F-A, E2F-B, and A/B) BRCA1 promoter probes and a promoter probe of the late cell cycle gene Cyclin B2 as well as a mouse promoter probe of the early cell cycle gene Dhfr . As a negative control, a fragment of the mouse Gapdhs promoter (neg. Ctrl.) was used. D DREAM components (p130, E2f4, Lin37, Lin54, and Lin9) were purified from nuclear extracts of density-arrested NIH3T3 mouse cells by DNA affinity purification and detected by Western blot. Binding to the human BRCA2 wild-type promoter probe (wt) was compared with binding to mutant probes (E2F-A, E2F-B, and A/B). Background protein binding was determined with a probe of the mouse cyclin B2 CHR mutant promoter (neg. Ctrl.). E p130, RB, E2F4, and LIN37 were purified from nuclear extracts of proliferating HCT116 human cells by DNA affinity purification and detected by Western blot. Binding to the wild-type promoter probe (wt) was compared with binding to E2F site mutant probes (E2F-A, E2F-B, and A/B) as well as binding to the promoter of the late cell cycle gene Cyclin B2 and the mouse promoter of the early cell cycle gene Dhfr . As a negative control, binding to a fragment of the mouse Gapdhs promoter was tested (neg. Ctrl.).

Article Snippet: RPE-1, NIH3T3, HFF, and T98G cells (DSMZ, Braunschweig, Germany) as well as HCT116 wild-type, p53 -/- and p21 -/- cells [ ] were grown in DMEM (Lonza) supplemented with 10% FCS and penicillin/streptomycin and maintained at 37 °C and 10% CO 2 .

Techniques: Negative Control, Affinity Purification, Mutagenesis, Purification, Western Blot, Binding Assay, Protein Binding