Journal: Technology in cancer research & treatment

Article Title: miRNA-130a-3p/CPEB4 Axis Modulates Glioblastoma Growth and Progression.

doi: 10.1177/15330338231218218

Figure Lengend Snippet: Figure 1. The expression of CPEB4 and miR-130a-3p in glioblastoma. (A) Real-time PCR analysis of CPEB4 expression in T98G and U87MG cell lines. β-Actin was used as the reference gene. (B) Real-time PCR analysis of miR-130a-3p expression in T98G and U87MG cell lines. U6 was used as the reference gene. (C) and (D) The expression distribution of CPEB4 and miR-130a-3p in gliomas tissues with different histological grades based on the transcriptome data from TCGA database. **P < .01, ***P < .001, and ****P < .0001. Abbreviations: CPEB4, cytoplasmic polyadenylation element binding protein 4; PCR, polymerase chain reaction; TCGA, The Cancer Genome Atlas.

Article Snippet: The western blotting test was performed, as explained earlier.24 The membranes were treated with horseradish peroxidaseconjugated IgG antibody (BA1070, Boster) after being incubated with antibodies against CPEB4 (25342-1-AP, 1:500, Proteintech), E-cadherin (BM3903, 1:1000, Boster), N-cadherin (BM3921, 1:1000, Boster), Vimentin (BM4029, 1:500, Boster), or β-actin (81115-1-RR, 1:2000, Proteintech) at 4 °C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Polymerase Chain Reaction

Journal: Technology in cancer research & treatment

Article Title: miRNA-130a-3p/CPEB4 Axis Modulates Glioblastoma Growth and Progression.

doi: 10.1177/15330338231218218

Figure Lengend Snippet: Figure 2. CPEB4 is a target of miR-130a-3p in glioblastoma. (A) Effect of miR-130a-3p expression on protein levels of CPEB4 and EMT-related biomarkers. Glioblastoma cells were transfected with miR-130a-3p mimic, inhibitor, or negative control, then western blotting analyses were conducted and the expression of β-actin was used as internal reference. (B) Predicted miR-130a-3p target sequence in the 3’-UTR of CPEB4 (CPEB4-WT) and CPEB4-MUT containing 7 altered nucleotides. (C) Luciferase reporter assay was applied to detect the binding between miR-130a-3p and 3’-UTR of CPEB4 in T98G cells. Co-transfection of mimic and CPEB4-WT decreased the firefly/renilla luciferase activity, while co-transfection of mimic and CPEB4-MUT did not change the firefly/renilla luciferase activity. Co-transfection of NC mimic with CPEB4-WT or CPEB4-MUT did not change the firefly/renilla luciferase activity. ***P < .001. Abbreviations: CPEB4, cytoplasmic polyadenylation element binding protein 4; EMT, epithelial-mesenchymal transition; NC, negative controls; UTR, untranslated region.

Article Snippet: The western blotting test was performed, as explained earlier.24 The membranes were treated with horseradish peroxidaseconjugated IgG antibody (BA1070, Boster) after being incubated with antibodies against CPEB4 (25342-1-AP, 1:500, Proteintech), E-cadherin (BM3903, 1:1000, Boster), N-cadherin (BM3921, 1:1000, Boster), Vimentin (BM4029, 1:500, Boster), or β-actin (81115-1-RR, 1:2000, Proteintech) at 4 °C overnight.

Techniques: Expressing, Transfection, Negative Control, Western Blot, Sequencing, Luciferase, Reporter Assay, Binding Assay, Cotransfection, Activity Assay

Journal: Technology in cancer research & treatment

Article Title: miRNA-130a-3p/CPEB4 Axis Modulates Glioblastoma Growth and Progression.

doi: 10.1177/15330338231218218

Figure Lengend Snippet: Figure 5. CPEB4 overexpression resists the antiproliferation effect of miR-130a-3p. The miR-130a-3p mimic-transfected glioblastoma cells were introduced into CPEB4-pcDNA3.1 or empty vector pcDNA3.1, then MTT assay was performed.; *P < .05, **P < .01 miR-130a-3p mimic + CPEB4-pcDNA3.1 group compared with miR-130a-3p mimic + pcDNA3.1 group; #P < .05, ##P < .01 miR-130a-3p mimic group compared with untreated group. Abbreviation: CPEB4, cytoplasmic polyadenylation element binding protein 4; MTT, methylthiazolyl tetrazolium.

Article Snippet: The western blotting test was performed, as explained earlier.24 The membranes were treated with horseradish peroxidaseconjugated IgG antibody (BA1070, Boster) after being incubated with antibodies against CPEB4 (25342-1-AP, 1:500, Proteintech), E-cadherin (BM3903, 1:1000, Boster), N-cadherin (BM3921, 1:1000, Boster), Vimentin (BM4029, 1:500, Boster), or β-actin (81115-1-RR, 1:2000, Proteintech) at 4 °C overnight.

Techniques: Over Expression, Transfection, Plasmid Preparation, MTT Assay, Binding Assay

Journal: Technology in cancer research & treatment

Article Title: miRNA-130a-3p/CPEB4 Axis Modulates Glioblastoma Growth and Progression.

doi: 10.1177/15330338231218218

Figure Lengend Snippet: Figure 6. CPEB4 overexpression resists the antimigration effect of miR-130a-3p. The miR-130a-3p mimic-transfected glioblastoma cells were introduced into CPEB4-pcDNA3.1 or empty vector pcDNA3.1, then transwell assays were conducted in culture plates. The cells were stained with crystal violet and the number of migrating cells was counted in 5 random fields under a microscope (magnification, × 200). **P < .01, ***P < .001. Abbreviation: CPEB4, cytoplasmic polyadenylation element binding protein 4.

Article Snippet: The western blotting test was performed, as explained earlier.24 The membranes were treated with horseradish peroxidaseconjugated IgG antibody (BA1070, Boster) after being incubated with antibodies against CPEB4 (25342-1-AP, 1:500, Proteintech), E-cadherin (BM3903, 1:1000, Boster), N-cadherin (BM3921, 1:1000, Boster), Vimentin (BM4029, 1:500, Boster), or β-actin (81115-1-RR, 1:2000, Proteintech) at 4 °C overnight.

Techniques: Over Expression, Transfection, Plasmid Preparation, Staining, Microscopy, Binding Assay

Journal: Technology in cancer research & treatment

Article Title: miRNA-130a-3p/CPEB4 Axis Modulates Glioblastoma Growth and Progression.

doi: 10.1177/15330338231218218

Figure Lengend Snippet: Figure 10. CPEB4 was upregulated in gliomas cell lines based on CCLE database. The cell lines mRNA expression matrix of gliomas was obtained from the CCLE dataset. The abscissa represented the expression of CPEB4 and the ordinate represented different cell lines, different colors and the size of dots represented expression levels. Abbreviations: CCLE, Cancer Cell Line Encyclopedia; CPEB4, cytoplasmic polyadenylation element binding protein 4; mRNA, messenger RNA.

Article Snippet: The western blotting test was performed, as explained earlier.24 The membranes were treated with horseradish peroxidaseconjugated IgG antibody (BA1070, Boster) after being incubated with antibodies against CPEB4 (25342-1-AP, 1:500, Proteintech), E-cadherin (BM3903, 1:1000, Boster), N-cadherin (BM3921, 1:1000, Boster), Vimentin (BM4029, 1:500, Boster), or β-actin (81115-1-RR, 1:2000, Proteintech) at 4 °C overnight.

Techniques: Expressing, Binding Assay

Journal: Technology in cancer research & treatment

Article Title: miRNA-130a-3p/CPEB4 Axis Modulates Glioblastoma Growth and Progression.

doi: 10.1177/15330338231218218

Figure Lengend Snippet: Figure 11. The single-cell expression analysis of CPEB4 with TISCH database. The correlation between CPEB4 expression and tumor microenvironment was explored by using the TISCH database. (A) The heatmap showing the value of CPEB4 expression in different cells from different datasets. (B) Violin plot displaying the distribution of CPEB4 expression in different cells in GSE102130 dataset. Abbreviations: CPEB4, cytoplasmic polyadenylation element binding protein 4; TISCH, Tumor Immune Single-cell Hub.

Article Snippet: The western blotting test was performed, as explained earlier.24 The membranes were treated with horseradish peroxidaseconjugated IgG antibody (BA1070, Boster) after being incubated with antibodies against CPEB4 (25342-1-AP, 1:500, Proteintech), E-cadherin (BM3903, 1:1000, Boster), N-cadherin (BM3921, 1:1000, Boster), Vimentin (BM4029, 1:500, Boster), or β-actin (81115-1-RR, 1:2000, Proteintech) at 4 °C overnight.

Techniques: Expressing, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

doi: 10.1128/mcb.01035-07

Figure Lengend Snippet: FIG. 1. Nap1-interacting proteins. PrA- or TAP-tagged proteins as indicated were isolated from whole-cell lysates using IgG-Sepharose and eluted with MgCl2. The coprecipitation of Nap1 with these pro- teins was analyzed by Western blotting with an anti-Nap1 antibody (Nap1). For Cka2-TAP, the blot was probed with rabbit IgG antibody (IgG) to detect Cka2-TAP followed by Nap1 antibody. The white arrowhead indicates Nap1 band.

Article Snippet: Coprecipitating proteins were eluted with a MgCl2 gradient, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and visualized by Western blotting using a Nap1 antibody purchased from Santa Cruz Biotechnology Inc.

Techniques: Isolation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

doi: 10.1128/mcb.01035-07

Figure Lengend Snippet: FIG. 2. Nap1 interacts directly with the bud neck-associated pro- tein, Nba1. (A) MBP-Nba1 (200 nM), MBP (200 nM), GST (1 M), GST-Nap1 (1 M), and GST-Nap1 fragments (indicated by amino acid numbers above the lanes; 1 M) were immobilized and incubated with 250 nM GST-Nap1 (top blot) or 500 nM MBP-Nba1 (bottom blot). Bound proteins were visualized by Western blotting with anti-Nap1 (-Nap1) or anti-MBP (-MBP) (for Nba1) antibodies. (B) Nba1- GFP2 fusion protein was expressed in wild-type (WT) or nap1 cells and visualized by fluorescence microscopy. The coincident differ- ential interference contrast (DIC) image is also shown. (C) Nba1 fragments (indicated by amino acid number) were expressed as GFP2 fusion proteins in wild-type yeast and visualized as described above for panel B.

Article Snippet: Coprecipitating proteins were eluted with a MgCl2 gradient, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and visualized by Western blotting using a Nap1 antibody purchased from Santa Cruz Biotechnology Inc.

Techniques: Incubation, Western Blot, Microscopy

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

doi: 10.1128/mcb.01035-07

Figure Lengend Snippet: FIG. 3. Genetic interactions of NAP1. (A) Strains of the indicated genotype were equalized, spotted at 10-fold serial dilutions, and grown on YPD plates with and without benomyl. (B) Strains of the indicated genotypes were examined by differential interference contrast (DIC) microscopy, and the percentage of budded cells exhibiting elongated buds was quantified. The error bars indicate the standard error of the means for three independent experiments. Statistically significant dif- ferences between nap1 and the double deletion strains are indicated as follows: *, P 0.05; **, P 0.01. DIC images of selected strains as indicated are shown above the bar graph. WT. wild type.

Article Snippet: Coprecipitating proteins were eluted with a MgCl2 gradient, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and visualized by Western blotting using a Nap1 antibody purchased from Santa Cruz Biotechnology Inc.

Techniques: Microscopy

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

doi: 10.1128/mcb.01035-07

Figure Lengend Snippet: FIG. 4. Nap1 is phosphorylated. (A) Schematic of Nap1. Arrows illustrate the relative positions of the 11 identified phosphosites, with the amino acids of the CK2 sites indicated numerically. Black bars beneath the schematic show the regions contained within the solved structure (48). (B) Tertiary structure of the Nap1 dimer (left). The subunits are shown in purple and yellow, and phosphorylated residues on both subunits are green. The labeled subdomains A and B refer to the purple subunit. Close-up view (right) of the clamp region indicates phosphorylated residues in this domain, and the likely position of S177 is shown. An alternate projection of the dimer, illustrating S82, is shown for clarity (bottom view) (48). (C) Recombinant GST-Nap1 and the indicated Nap1 mutants were incubated with purified CK2 and [-32P]ATP for the time indicated. The proteins were separated by SDS-PAGE, Nap1 was visualized by Coomassie blue staining (CBB), and phosphorylation was measured by 32P incorporation detected us- ing a PhosphorImager. (D) In vitro kinase assay of GST-Nap1 or GST-Nap1(S159S177AS397A) (labeled Nap1 3S-A) was carried out for 30 min as described in Materials and Methods, and proteins in- cluded in the reaction mixture are as indicated. (E) In vitro kinase assay of GST-Nap1 or GST-Nap1 3S-A was carried out as described in Materials and Methods for the times indicated.

Article Snippet: Coprecipitating proteins were eluted with a MgCl2 gradient, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and visualized by Western blotting using a Nap1 antibody purchased from Santa Cruz Biotechnology Inc.

Techniques: Labeling, Recombinant, Incubation, SDS Page, Staining, Phospho-proteomics, In Vitro, Kinase Assay

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

doi: 10.1128/mcb.01035-07

Figure Lengend Snippet: FIG. 5. Reversible phosphorylation of Nap1 by CK2 may regulate normal progression through S phase. (A) Nap1, Nap1(S159A S177A S397A) (labeled Nap1 3S-A), or Nap1(S159D S177D S397D) (labeled Nap1 3S-D) were expressed in the Clb2-dependent strain and examined by differential interference contrast (DIC) microscopy. (B) The same strains were fixed, and the DNA was stained with Sytox green. Cells were analyzed for DNA content using the Amnis ImageStream instrument. Random images of cells within the peak corresponding to 4N DNA content (vector control sample only) are displayed on the right. BF, bright-field image, SYTOX, Sytox green-stained DNA. (C) Strains as in panel A were stained with Hoechst and observed by fluorescence and DIC microscopy and scored morphologically for their cell cycle distribution. The numbers of cells with no bud (G1), cells with a small bud and one nucleus (S phase), and cells with a large bud and two nuclei (G2/M) are indicated. Comparison of strains showed statistically significant differences (*, P 0.05; **, P 0.01). (D) The numbers in the boxes indicate the percentage of cells in each phase of the cell cycle derived from panel C.

Article Snippet: Coprecipitating proteins were eluted with a MgCl2 gradient, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and visualized by Western blotting using a Nap1 antibody purchased from Santa Cruz Biotechnology Inc.

Techniques: Phospho-proteomics, Labeling, Microscopy, Staining, Plasmid Preparation, Control, Comparison, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

doi: 10.1128/mcb.01035-07

Figure Lengend Snippet: FIG. 6. Phosphorylation of Nap1 by CK2 does not prevent histone binding by Nap1. GST, GST-Nap1, GST-Nap1(S159A S177A S397A) (labeled Nap1 3S-A), or GST-Nap1(S159D S177D S397D) (labeled Nap1 3S-D) (all at 1 M) was immobilized and incubated with purified core histones (1 M). Bound proteins were visualized by Coomassie blue staining. The black asterisk indicates a band corresponding to bovine serum albumin. Inputs of each protein (25 mol) are shown.

Article Snippet: Coprecipitating proteins were eluted with a MgCl2 gradient, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and visualized by Western blotting using a Nap1 antibody purchased from Santa Cruz Biotechnology Inc.

Techniques: Phospho-proteomics, Binding Assay, Labeling, Incubation, Staining

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

doi: 10.1128/mcb.01035-07

Figure Lengend Snippet: FIG. 7. Phosphorylation of Nap1 by CK2 regulates nuclear import. (A) Nap1, Nap1(S159A S177A S397A) (labeled Nap1 3S-A), or Nap1(S159D S177D S397D) (labeled Nap1 3S-D) were expressed as GFP2 fusion in nap1 cells and visualized by fluorescence microscopy. The coincident differential interference contrast (DIC) and Hoechst-stained images are shown. (B) Export-deficient Nap1-L99S, Nap1 3S-A-L99S, and Nap1 3S-D-L99S GFP2 fusion proteins were expressed and visualized as described above for panel A. (C) MBP-Kap114 (500 nM) was immobilized and incubated with recombinant Nap1 or Nap1 3S-D (both 250 nM). Bound proteins were visualized by Western blotting and quantitated using the Odyssey infrared imaging system as described in Materials and Methods. Relative binding of Nap1 to Kap114 is expressed in arbitrary units (AU). (D) The indicated GFP fusions were expressed in kap114 cells and visualized as described above for panel A.

Article Snippet: Coprecipitating proteins were eluted with a MgCl2 gradient, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and visualized by Western blotting using a Nap1 antibody purchased from Santa Cruz Biotechnology Inc.

Techniques: Phospho-proteomics, Labeling, Microscopy, Staining, Incubation, Recombinant, Western Blot, Imaging, Binding Assay