Journal: PLoS ONE

Article Title: Cardiac glycoside-mediated turnover of Na, K-ATPases as a rational approach to reducing cell surface levels of the cellular prion protein

doi: 10.1371/journal.pone.0270915

Figure Lengend Snippet: Summary of information that guided the shortlisting of cathepsins for determining their possible involvement in the CG-dependent reduction of PrP C levels.

Article Snippet: In determining which of the cathepsins are plausible candidates, we noted that a recent manuscript reporting on the levels of expression of individual cathepsins in the human brain [ ] mostly corroborated Human Protein Atlas (HPA) cathepsin protein expression data, and therefore decided to shortlist all cathepsins for testing that the HPA reported to be expressed in the brain at moderate to high levels, ignoring others which were reported to exhibit no or very low expression in this tissue ( ).

Techniques: In Vitro

Journal: Molecular Medicine Reports

Article Title: Downregulation of FOXK2 is associated with poor prognosis in patients with gastric cancer

doi: 10.3892/mmr.2018.9466

Figure Lengend Snippet: Association between FOXK2 expression and gastric cancer clinicopathological features.

Article Snippet: FOXK2 expression data in gastric cancer were collated from the Human Protein Atlas ( ).

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Downregulation of FOXK2 is associated with poor prognosis in patients with gastric cancer

doi: 10.3892/mmr.2018.9466

Figure Lengend Snippet: Univariate and multivariate analyses of prognostic parameters in patients with gastric cancer in terms of overall survival.

Article Snippet: FOXK2 expression data in gastric cancer were collated from the Human Protein Atlas ( ).

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Downregulation of FOXK2 is associated with poor prognosis in patients with gastric cancer

doi: 10.3892/mmr.2018.9466

Figure Lengend Snippet: Expression of FOXK2 in gastric cancer tissues. (A) Kaplan-Meier analysis of OS and PFS based on FOXK2 expression in patients with gastric cancer (log-rank test, P<0.01). (B) Kaplan-Meier analysis for survival was performed with data obtained from The Human Protein Atlas (log-rank test, P=0.0224). (C) Immunohistochemical staining of FOXK2 in various gastric cancer grades (magnification, ×200). The results were analyzed with one-way analysis of variance followed by Student-Newman-Keuls method; *P<0.05. (D) Compared with para-tumor tissue samples, relative FOXK2 mRNA expression was significantly lower in tumor tissue samples. FOXK2, forkhead box K2; OS, overall survival; PFS, progression free survival.

Article Snippet: FOXK2 expression data in gastric cancer were collated from the Human Protein Atlas ( ).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Molecular Medicine Reports

Article Title: Downregulation of FOXK2 is associated with poor prognosis in patients with gastric cancer

doi: 10.3892/mmr.2018.9466

Figure Lengend Snippet: FOXK2 expression affects c-caspase-3 expression and BGC-823 cell apoptosis. (A) si-FOXK2 transfection effectively reduced FOXK2 mRNA expression in BGC-823 cells, as well as (B) FOXK2 and c-caspase-3 protein expression. (C) FOXK2 overexpression plasmid transfection successfully increased FOXK2 mRNA. (D) FOXK2 and c-caspase-3 protein expression also increased. (E) si-FOXK2 decreased BGC-823 cell apoptosis, whereas (F) FOXK2 overexpression increased BGC-823 cell apoptosis. *P<0.05, **P<0.01 vs. the corresponding control group. c-, cleaved; FITC, fluorescein isothiocyanate; FOXK2, forkhead box K2; NC, negative control; PI, propidium iodide; si-, small interfering RNA.

Article Snippet: FOXK2 expression data in gastric cancer were collated from the Human Protein Atlas ( ).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Control, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Downregulation of FOXK2 is associated with poor prognosis in patients with gastric cancer

doi: 10.3892/mmr.2018.9466

Figure Lengend Snippet: FOXK2 regulates gastric cancer cell invasion. Western blot analysis demonstrated alterations in N-cadherin and E-cadherin expression following (A) si-FOXK2 or (B) FOXK2 plasmid transfection. Transwell assays were performed to assess the invasion of cells transfected with (C) si-FOXK2 or (D) FOXK2 plasmid. Magnification, ×200. *P<0.05, **P<0.01 vs. the corresponding control group. FOXK2, forkhead box K2; NC, negative control; si-, small interfering RNA.

Article Snippet: FOXK2 expression data in gastric cancer were collated from the Human Protein Atlas ( ).

Techniques: Western Blot, Expressing, Plasmid Preparation, Transfection, Control, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Downregulation of FOXK2 is associated with poor prognosis in patients with gastric cancer

doi: 10.3892/mmr.2018.9466

Figure Lengend Snippet: FOXK2 regulates gastric cancer cell proliferation. (A) Growth curve of cultured BGC-823 cells and the colony formation assay demonstrated that si-FOXK2 transfection induced cell proliferation, whereas (B) FOXK2 overexpression inhibited cell proliferation. (C) Wound-healing assays demonstrated that si-FOXK2 transfection increased cell migration, whereas (D) FOXK2 plasmid transfection inhibited cell migration. Magnification, ×100. *P<0.05 vs. the corresponding control group. FOXK2, forkhead box K2; NC, negative control; si-, small interfering RNA.

Article Snippet: FOXK2 expression data in gastric cancer were collated from the Human Protein Atlas ( ).

Techniques: Cell Culture, Colony Assay, Transfection, Over Expression, Migration, Plasmid Preparation, Control, Negative Control, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a Immunofluorescence analysis of centriole and centrosome amplification in U2OS cells stably expressing HA-Cep131. Cells were stained with anti-Cent (red) and anti-γTub (green) antibodies to visualize centrioles and centrosomes, respectively. Insets are approximately fivefold magnified at the centrosomal region. Scale bar, 10 μm. b Quantification of the proportion of cells with >2 centrosomes or >4 centrioles induced by stably expressing HA-Cep131. c , d Multinucleated cells were induced by HA-Cep131 overexpression in U2OS cells. DNA is visualized in blue. Scale bar, 10 μm. e , f U2OS cells were treated to 2 mM hydroxyurea (HU) for 48 h to induce centrosome amplification and were transfected with siCon or siCep131 duplexes. The graph shows quantification of cells with centrosome amplification ( f ). Insets are approximately fivefold magnified at the centrosomal region. Scale bar, 10 μm. g Quantification of the proportion of cells with centriole number after treatment of siCon, siCep131, and siPlk4. h Endogenous Cep131 was depleted by siRNA, and cells were transfected with EGFP vector or EGFP-Cep131, containing resistant sequences to siCep131. The graph shows quantification of four-centriole cells. Error bars in b , d , f , g represent means ± SEM from three independent experiments ( N > 300 for each experiment). * P < 0.05 and ** P < 0.01, unpaired Student’s t test

Article Snippet: Cep131 protein expression data in several organs were obtained from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Immunofluorescence, Amplification, Stable Transfection, Expressing, Staining, Over Expression, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: Immunofluorescence analysis of direct localization of Cep131 at the centriole. U2OS cells were co-stained for Cep131 (red) with centriolar marker proteins, such as anti-Cent a and Plk4 b . Scale bar, 0.5μm. c Triple staining of U2OS cells with antibodies against anti-Cent (blue), Plk4 (green), and Cep131 (red). Schematic illustration of the localization of Cep131 at the outer wall of centriole (right panel). Scale bar, 0.5 μm. d Asymmetrical localization of Cep131 at the mother centriole. Cep131 co-stained with Cent (far-red) and mother centriole-enriched protein, hCenexin (green). Schematic illustration to recognize each centriole, mother (M) and daughter (D) centriole. Scale bar, 0.5 μm. e Quantification of fluorescence intensity of Cep131 at each centriole (mother and daughter). Around 50 centrioles from three independent experiments were measured for each condition. *** P < 0.001, unpaired Student’s t test. f , g U2OS cells were treated with shGL2 (control) or shPCNT duplexes and were fixed by two different methods, common fixation (No extraction) and signal extraction (Extraction), to verify the localization of Cep131 at centriolar satellites and the centriole, respectively. Around 50 cells from three independent experiments were measured for each condition. *** P < 0.001, unpaired Student’s t test

Article Snippet: Cep131 protein expression data in several organs were obtained from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Control, Extraction

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a HEK293T cells co-expressing Myc-Plk4 and GFP-Cep131 were immunoprecipitated using Myc-magnetic beads, and the protein levels were observed by immunoblotting. b Endogenous Plk4 extracted from U2OS cells was immunoprecipitated by anti-Plk4, and total and pulled down proteins were observed by immunoblotting. c Bacterially generated GST-Plk4-WT or KD were incubated with His-Cep131 (1-911 aa) and pulled down using GSH-agarose. d , e HEK293T cells co-expressing Myc-Plk4 and HA-Cep131 deletions, including #mut2, #mut2+3, and #mut3, as illustrated in e , were immunoprecipitated, and protein levels were observed. f Alignment of amino acids from 513–542 in human Cep131 corresponded to several other species. Plk4-binding motif (PBM) is highlighted in red. Asterisk, fully conserved residue; colon, conserved residue; period, semi-conserved residue. A Coomassie (CBB) staining use as a loading control in b – d

Article Snippet: Cep131 protein expression data in several organs were obtained from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Expressing, Immunoprecipitation, Magnetic Beads, Western Blot, Generated, Incubation, Binding Assay, Residue, Staining, Control

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a Alignment of amino acids in human Cep131 and several other species. The phosphorylation sites at residues S21 by Plk4 are highlighted in red. Asterisk, fully conserved residue; colon, conserved residue; period, semi-conserved residue. b Peptide competition assay to verify the specificity of anti-Cep131-pS21 antibody. Lysates from HEK293T cells expressing Myc-Plk4 with HA-Cep131-WT or S21A were incubated with non- or phosphopeptides targeting Cep131-S21. After incubation, phosphorylation status was analyzed by immunoblotting using Cep131-pS21 antibody. c HEK293T cells co-expressing HA-Cep131-WT with Myc-Plk4-WT or KD were immunoprecipitated using HA-magnetic beads, followed by incubation with λ-phosphatase at 30 °C for 30 min. Phosphorylation status was then observed by immunoblotting using anti-Cep131-pS21 antibody. d Immunofluorescence analysis of co-localization of phosphorylated Cep131 at the centriole. U2OS cells were treated with shGL2 (control) or shCep131 and were co-stained for Cep131-pS21 (red) and Cent (green). e Quantification of Cep131-pS21-positive cells. Error bars represent means ± SEM from three independent experiments ( N > 300 for each experiment). ** P < 0.01, unpaired Student’s t test. CBB staining use as a loading control in b , c

Article Snippet: Cep131 protein expression data in several organs were obtained from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Phospho-proteomics, Residue, Competitive Binding Assay, Expressing, Incubation, Western Blot, Immunoprecipitation, Magnetic Beads, Immunofluorescence, Control, Staining

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: Immunoblotting analysis of immunoprecipitated samples from HEK293T cells co-expressing GFP-STIL with several constructs, such as Myc-Plk4 or GFP-Cep131 a , Myc-Plk4 with GFP-Cep131 (WT or 2A) b , and Myc-Plk4-ND (nondegradable; constitutive active form) with GFP-Cep131 (WT or 2A) c . After immunoprecipitation using GFP-magnetic beads, protein levels were detected using the indicated antibodies. HEK293T cells co-expressing HA-Cep131-WT and GFP-STIL-ΔC (truncated C terminus of STIL, 1-831 aa) ( d ) or GFP-STIL-3A (alanine mutants on residue S1015, S1062, and S1070) ( e ) were immunoprecipitated using HA-magnetic beads, and the proteins levels were observed by immunoblotting. f Endogenous STIL extracted from U2OS cells was immunoprecipitated, and total and pulled down proteins were observed by immunoblotting. Quantification of band intensity was measured by ImageJ (NIH, Bethesda, MD, USA) and indicated as ratios in b – f . g Immunoblotting analysis shows protein levels of Cep131. U2OS cells with stable expression of untagged Cep131-WT and 2A containing resistant sequences to siRNA were transfected with siCon or siCep131 to eliminate endogenous Cep131. h Immunofluorescence analysis Plk4 variations (top panel) and STIL (bottom panel) levels at the centriole in U2OS cells transfected as in g . Insets are approximately fivefold magnified at the centrosomal region. Scale bar, 10 μm. i The graph shows quantification of the fluorescence intensity of Plk4 (left) and STIL (right) at the centriole. More than 50 centrioles were measured for each condition. * P < 0.05 and ** P < 0.01, unpaired Student’s t test. CBB staining use as a loading control in a – g

Article Snippet: Cep131 protein expression data in several organs were obtained from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Western Blot, Immunoprecipitation, Expressing, Construct, Magnetic Beads, Residue, Transfection, Immunofluorescence, Fluorescence, Staining, Control

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a , b HEK293T cells co-expressing Myc-Plk4 and HA-Ub and/or GFP-Cep131 were treated with MG132 for 6 h and immunoprecipitated. Ubiquitylation properties were analyzed by immunoblotting. c U2OS cells with doxycycline-inducible expression of Myc-Plk4 were treated with 10 μM cycloheximide (CHX) for 8 h. Every 2 h, cells were harvested and analyzed by immunoblotting. d Quantification of Plk4 band intensity measured with ImageJ. Error bars represent means ± SD from three independent experiments. ** P < 0.01, unpaired Student’s t test. NS, not significant. e HEK293T cells stably expressing shGL2 (control) or shSTIL were transfected with HA-Cep131-WT or 2A and immunoprecipitated. Ubiquitylation properties were analyzed by immunoblotting. f Immunofluorescence analysis of excessive accumulations of Plk4 (top panel) and STIL (bottom panel) at the centriole in U2OS cells transfected with Cep131-WT or 2A. Insets are approximately fivefold magnified at the centrosomal region. Scale bar, 10 μm. g Quantification of fluorescence intensity of Cent (left), Plk4 (middle), and STIL (right) at the centriole. Over 50 centrioles were measured for each condition. ** P < 0.01, unpaired Student’s t test. CBB staining use as a loading control in a , b , c , e

Article Snippet: Cep131 protein expression data in several organs were obtained from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Expressing, Immunoprecipitation, Western Blot, Stable Transfection, Control, Transfection, Immunofluorescence, Fluorescence, Staining

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a HCT116 cells stably expressing vector or untagged Cep131-WT or 2A were infected with shGL2 (control) or shCep131 to eliminate endogenous Cep131 and transplanted into nude mice. After 5 weeks, tumors had grown to maximum sizes and were removed. N/A, not available. Quantification of transplanted tumor volumes ( b ), measured weekly, and weight ( c ). Error bars represent means ± SD ( N = 6 for each group). * P < 0.05, unpaired Student’s t test. d Tumors were stained with hematoxylin (violet) and eosin (pink) to indicate the nucleus and cytoplasm, respectively. Scale bar, 100 μm. e Immunohistochemistry analysis of centrosome amplification (γTub) and Cep131 expression. Scale bar, 10μm. Quantification of Cep131 overexpression ( f ) and centrosome amplification ( g ) in tumor tissues. Error bars represent means ± SD from four tissue sections ( N = 2 for each group). * P < 0.05, unpaired Student’s t test

Article Snippet: Cep131 protein expression data in several organs were obtained from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Infection, Control, Staining, Immunohistochemistry, Amplification, Over Expression

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a At the beginning of centrosome duplication, Plk4 phosphorylates not only STIL but also Cep131 at residues S21 and T205. Cep131 phosphorylation facilitates STIL recruitment to elongate new procentrioles. b Cep131 overexpression increases excessive recruitment of STIL at the centriole, which leads to uncontrolled stabilization and activation of Plk4. This coordinated cycle promotes centrosome amplification and therefore cancer progression

Article Snippet: Cep131 protein expression data in several organs were obtained from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Phospho-proteomics, Over Expression, Activation Assay, Amplification