Journal: RSC advances

Article Title: Unveiling anticancer, antimicrobial, and antioxidant activities of novel synthesized bimetallic boron oxide-zinc oxide nanoparticles.

doi: 10.1039/d3ra03413e

Figure Lengend Snippet: Fig. 5 Effect of bimetallic B2O3–ZnO NPs on cell viability of Vero, and Wi38 normal cell line (A) and cell inhibition of Caco 2 cell line (B and C).

Article Snippet: Bimetallic B2O3–ZnONPs cytotoxicity was assessed using a slightly modied version of the MTT technique.33,34 The American Type Culture Collection (ATCC) provided normal Vero, Wi38 normal cell lines and the malignant Caco 2 (Caucasian colon adenocarcinoma) cell line.

Techniques: Inhibition

Journal: Molecular and Cellular Biology

Article Title: In vivo evidence of interaction between interferon-stimulated gene factors and the interferon-stimulated response element.

doi: 10.1128/mcb.9.8.3533

Figure Lengend Snippet: FIG. 2. Comparison of ISG15 constitutive and IFN-a-induced DNase-hypersensitive sites in WI38VA and Daudi cells at different times after IFN-a treatment. (A) WI38VA cells were treated with 500 U of IFN-a per ml for the time shown. Nuclei were isolated and treated with the indicated concentrations of DNase, and then DNA was extracted, digested with TaqI, electrophoresed, blotted, and probed with a radiolabeled ISG15 EcoRI-Taql fragment (+510 to +850). Fragment sizes (base pairs) were calculated with the equa- tion obtained from linear regression analysis of the marker mobili- ties (R2 > 0.99), and the centers of sites were then estimated on the basis of known location of the probe within the gene. Resolution in the size range of the promoter was very good, and the centers of the DNase-hypersensitive sites were reproducibly mapped to within +10 base pairs. The uppermost site is the full-length restriction fragment detected by the probe. The markers (M) used were 1-kilobase and 123-base-pair ladders (Bethesda Research Laborato- ries) labelled with 32P and mixed with 20 ,ug of sheared salmon sperm DNA prior to electrophoresis. (B) Daudi cells were treated with 250 U of IFN-a per ml for the time shown. The analysis was performed as described for panel A. (C) The results illustrated by panels A and B are depicted schematically. Restriction sites and DNase-hyper- sensitive sites I through IV are shown above the horizontal line. The induced hypersensitive site is marked by the heavy vertical line. The positions given for the hypersensitive sites represent the centers of the analogous pair of sites in the two cell lines. A site observed in only one cell line is shown with that cell line designated underneath. The ISRE (-) and the first exon (U) are shown. The subcloned fragment used as a probe (restriction sites and =) is indicated.

Article Snippet: WI38VA (ATCC CCL 75.1) or Daudi

Techniques: Comparison, Isolation, Marker, Electrophoresis

Journal:

Article Title: Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

doi:

Figure Lengend Snippet: Human cyclin K is associated with a potent CTD kinase activity. (A) Western blot analysis of human cell lysates probed with antibodies (α) to cyclin K. Sixty micrograms of protein from each of the following whole-cell extracts was probed with affinity-purified α-Kpep antibodies: U-2 OS, osteogenic sarcoma; WI-38, human diploid fibroblast; HepG2, hepatocellular carcinoma; and RPE (gift of A. Davis). (B) Immunoprecipitation of endogenous cyclin K protein from the [35S]methionine-labeled RPE cell line. Two microliters (2 μg) of affinity-purified α-Kpep antibodies or 5 μl of NRS was used to immunoprecipitate protein from lysates (500 μg). One microgram of cyclin K peptide (80-fold molar excess) was used to inhibit 2 μg of α-Kpep antibody. Asterisks correspond to bands associated with the cyclin K immunoprecipitation. (C) GST-CTD in vitro kinase activity associated with cyclin K. RPE whole-cell extracts (100 μg) were immunoprecipitated with either affinity-purified α-Kpep (lanes 1 to 6) or preimmune (P.I.) sera (lanes 7 and 8) that had been preincubated with (lanes 5 and 6) or without (lanes 1, 2, 3, 4, 7, and 8) peptide competitor, and the resultant complexes were used for in vitro kinase assays with (lanes 1, 2, 3, 5, and 7) or without (lanes 4, 6, and 8) the addition of a bacterially produced GST-CTD fusion protein. (D) Association of cyclin K with RNAP II in RPE. Immunoprecipitations were performed with 100 μg of RPE cell extract by using 20 μl each of protein A-Sepharose beads, protein A-Sepharose beads coupled with preimmune (P.I.) sera, and protein A-Sepharose beads covalently cross-linked with crude α-Kpep antibodies, with or without preincubation with cyclin K peptide competitor. Western blots were then performed with anti-RNAP II (ARNA3) antibodies (upper panel) and α-KFL antibodies (lower panel) as probes.

Article Snippet: Immunoprecipitations were performed from RPE and WI-38 cell lysates by using 2 μg each of NRS, affinity-purified α-K pep antibodies, affinity-purified α-K pep antibodies preincubated with peptide competitor, and anti-Cdk7 (Santa Cruz Biotechnology, Inc.) antibodies as described in Materials and Methods.

Techniques: Activity Assay, Western Blot, Affinity Purification, Immunoprecipitation, Labeling, In Vitro, Produced

Journal:

Article Title: Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

doi:

Figure Lengend Snippet: Cyclin K is a CAK in vitro. (A) Cyclin K is associated with a CAK activity towards cyclin A-Cdk2. Immunoprecipitations were performed from RPE and WI-38 cell lysates by using 2 μg each of NRS, affinity-purified α-Kpep antibodies, affinity-purified α-Kpep antibodies preincubated with peptide competitor, and anti-Cdk7 (Santa Cruz Biotechnology, Inc.) antibodies as described in Materials and Methods. IPs were incubated with 0.1 μg of bacterially expressed and purified cyclin A, HA-Cdk2, and ATP. After activation, the complexes were assayed for H1 kinase activity with [γ-32P]ATP as previously described (9), and reaction products were electrophoresed by SDS-PAGE (10% polyacrylamide) and visualized by autoradiography. (B) The cyclin K-associated CAK activity requires T161 of Cdk2. CAK assays were performed as described for panel A with IPs from RPE cell extracts, except that instead of HA-Cdk2, the T161A mutant of HA-Cdk2 was used.

Article Snippet: Immunoprecipitations were performed from RPE and WI-38 cell lysates by using 2 μg each of NRS, affinity-purified α-K pep antibodies, affinity-purified α-K pep antibodies preincubated with peptide competitor, and anti-Cdk7 (Santa Cruz Biotechnology, Inc.) antibodies as described in Materials and Methods.

Techniques: In Vitro, Activity Assay, Affinity Purification, Incubation, Purification, Activation Assay, SDS Page, Autoradiography, Mutagenesis

Journal: Cancer Science

Article Title: Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

doi: 10.1111/j.1349-7006.2012.02203.x

Figure Lengend Snippet: Effect of transforming growth factor‐β1 (TGF‐β1) on expression of angiomodulin (AGM), fibronectin (FN) and α‐smooth muscle actin (α‐SMA) in two kinds of cultured human fibroblasts. Human natal dermal fibroblasts (HDFs) (a) and WI38 cells (b) were incubated with the indicated concentrations (ng/mL) of TGF‐β1 in serum‐free medium for 2 days. From each culture, the conditioned medium and cell lysates were prepared, as described in Materials and Methods. AGM and fibronectin were analyzed with the conditioned media, while α‐SMA and β‐actin as an internal loading control were done with the cell lysates. The results were reproduced in at least three separate experiments.

Article Snippet: Human fetal lung fibroblast cell line WI38 and human bladder carcinoma cell line T24 (EJ‐1 strain) were provided from Japanese Collection of Research and Bioresources (JCRB, Osaka, Japan).

Techniques: Expressing, Cell Culture, Incubation, Control

Journal: Cancer Science

Article Title: Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

doi: 10.1111/j.1349-7006.2012.02203.x

Figure Lengend Snippet: Effects of angiomodulin (AGM) and transforming growth factor‐β1 (TGF‐β1) on growth of human fibroblasts. (a,b) Human natal dermal fibroblasts (HDFs) were incubated with the indicated concentrations of TGF‐β1 for 5 days (a) or AGM for 4 days (β) in DMEM/F12+5% FCS medium on 24‐well plates. Each point represents the mean ± SD of the numbers of cells in triplicate wells. (c) Time course of HDF growth in presence (●) or absence (○) of 10 μg/mL AGM. (d) Effect of varied concentrations of AGM on the growth of HDFs was examined in the presence (●) or absence (○) of 10 μM Smad inhibitor SB431542 (Smad inh.) for 6 days on a 96‐well plate. The cell growth was measured by the crystal violet staining. Each point represents the mean ± SD in triplicate wells. (e,f) Effects of varied concentrations of TGF‐β1 (e) or ΑGΜ (f) on the growth of WI38 cells were examined for 5 days as described in (a) and (b). Other experimental conditions are described in Materials and Methods.

Article Snippet: Human fetal lung fibroblast cell line WI38 and human bladder carcinoma cell line T24 (EJ‐1 strain) were provided from Japanese Collection of Research and Bioresources (JCRB, Osaka, Japan).

Techniques: Incubation, Staining