Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: Dkk3 overexpression suppressed the aggressive phenotypes of colorectal cancer cells. After transfection of pcDNA3.1 -Dkk3 , Dkk3 expression became strong in HCT-15 and HCT-116 cells using anti-Dkk3 and anti-His antibodies by Western blot (A) , real-time RT-PCR (B) , and immunofluorescence ( C , green: positive for Dkk3, blue: DAPI for nuclei). The cell culture supernatant of Dkk3 transfectants showed a higher Dkk3 concentration in comparison with those of the control and mock (D) . Colorectal cancer patients showed a higher serum Dkk3 level than healthy individuals regardless of their age, evidenced by ELISA assay (E) . The transfectants showed a lower proliferation (F) and G 2 arrest (G) in comparison with the control and mock. There was an apoptosis-induced effect of Dkk3 overexpression in the transfectants of HCT-15 and HCT-116, evidenced by annexin V assay (H) . JC-1 staining displayed that the mitochondrial membrane potential was decreased in the abovementioned cells with Dkk3 overexpression by fluorescence (I) and flow cytometry (J) . Dkk3-overexpressing cells had a weaker ability to migrate (K) and invade (L) . We also treated the Dkk3 transfectants with anti-Dkk3 antibody (0, 30, and 60 ng/ml) and found that this antibody blocked the effects of Dkk3 overexpression on these phenotypes ( F–L , p < 0.05). mock, cells transfected with pcDNA3.1 vector; * P < 0.05, compared with the control and treatment with anti-Dkk3 antibody.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Over Expression, Transfection, Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence, Cell Culture, Concentration Assay, Comparison, Control, Enzyme-linked Immunosorbent Assay, Annexin V Assay, Staining, Membrane, Fluorescence, Flow Cytometry, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: Recombinant Dkk3 inhibited the aggressive phenotypes of colorectal cancer cells. Treatment with recombinant Dkk3 protein significantly suppressed the proliferation of HCT-15 and HCT-116, compared with the control cells (A) . Recombinant Dkk3 protein could induce G 2 arrest (B) and cell apoptosis (C) of both kinds of cells in a dose-dependent manner. JC-1 staining also demonstrated that recombinant Dkk3 exposure significantly reduced mitochondrial membrane potential of the abovementioned cells treated with 150 ng/mL recombinant protein by fluorescence (D) and flow cytometry (E) . After being exposed to recombinant Dkk3, both colorectal carcinoma cells exhibited a low ability to migrate (F) and invade (G) , compared with the control cells. * P < 0.05, compared with the control.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Recombinant, Control, Staining, Membrane, Fluorescence, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The phenotype-related proteins were screened by Western blot. Dkk3 overexpression and treatment decreased the expression of Bcl-2, cdc25B, cdc25c, N-cadherin, Slug, and Twist and increased the expression of Bax and E-cadherin in HCT-15 and HCT-116 cells. The anti-Dkk3 antibody suppressed the effects of Dkk3 overexpression on the expression of the phenotype-related proteins.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Western Blot, Over Expression, Expressing

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The effects of Dkk3 on tumor growth and carcinogenesis of colorectal cancer. PCR primers were designed (A) and subjected to PCR of tail and stomach DNA (B) with the same color for the corresponding primers and products. Expression of Dkk3, N-cadherin, and E-cadherin was confirmed by real-time PCR (C) and Western blot (D) . Immunohistochemistry was employed to observe the maker proteins’ expression in the intestinal adenoma of target villin-cre/Dkk3 knockout mice induced by sodium desoxycholate (E) . * p < 0.05, compared with the transfectant or knockout mice. WT, wild-type mice; VD, villin-cre +; Dkk3 -/-.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Knock-Out, Transfection

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The clinicopathological significance of Dkk3 mRNA expression in colorectal cancer. Real-time RT-PCR was employed to detect Dkk3 mRNA expression in colorectal cancer samples (A) . Its expression was compared with pathological behaviors of colorectal cancer (B) . N, normal; C, cancer, TA, tubular adenocarcinoma; MA, mucinous adenocarcinoma. * p < 0.05.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The bioinformatics analysis of Dkk3 mRNA expression in colorectal cancer. Oncomine (A) and TCGA (B) data sets were employed to analyze Dkk3 expression in colorectal cancer, and its expression was compared with pathological parameters of cancers. Dkk3 mRNA expression was higher in cancer-associated fibroblasts (CAF) than CD133-positive (CD133+) and -negative (CD133-) colorectal cancer cells (C) . Kaplan-Meier curves were used to analyze the prognostic significance of Dkk3 mRNA expression according to KM plotters (D) . HR, hazard ratio; N, lymph node metastasis. * p < 0.05; **** p < 0.001.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The clinicopathological significance of Dkk3 protein expression in colorectal cancer. Western blot was employed to detect Dkk3 protein expression in colorectal cancer samples (A) . Its expression was compared with pathological behaviors of colorectal cancer (B) . N, normal; C, cancer, TA, tubular adenocarcinoma; MA, mucinous adenocarcinoma. * p < 0.05.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Expressing, Western Blot

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: Dkk3 -enriched signal pathway in colorectal cancer according KEGG analysis.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Migration, Coagulation, Infection

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Dickkopf-3 (Dkk3) is protective against ischemic neuronal death. (A) Nissl staining in 10 μm coronal mouse brain sections collected every 320 μm along the rostro-caudal extension of the ischemic lesion in wild-type and Dkk3 knockout mice 1 day after middle cerebral artery occlusion (MCAO). (B) Infarct size in wild-type and Dkk3 knockout mice 1 day, 3 days or 7 days following MCAO. Values are means + SEM. * p < 0.05 vs. wild-type mice (Student’s t -test; day 1: n = 7–9, p = 0.0073, t (14) = 3.135; day 3: n = 4–5, p = 0.0143, t (7) = 3.24; day 7: n = 4–7, p = 0.0387, t (9) = 2.42). (C) Immunohistochemical analysis of Dkk3 in the cerebral cortex of wild-type mice at 6 h and 12 h following MCAO. Low and high magnification images are shown. (D) Double fluorescence immunostaining of Dkk3 and GFAP in the ischemic cortex of wild-type mice 12 h following MCAO. Arrowheads show Dkk3 expression in astrocytes. Arrows show Dkk3 expression in GFAP-negative cells.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Staining, Knock-Out, Immunohistochemical staining, Fluorescence, Immunostaining, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Protective activity of Dkk3 in cultured astrocytes exposed to oxidative damage or glucose deprivation. (A) Flow cytometry analysis (FACS) of live cells in cultured cortical astrocytes from wild-type and Dkk3 knockout mice (C57BL/6J strain) following 1 h of incubation with H 2 O 2 (250 μM). Values are means + SEM ( n = 3–4 per group). p < 0.05 vs. the respective control cultures not exposed to H 2 O 2 (*), or vs. wild-type cultures exposed to H 2 O 2 ( # two-way ANOVA + Fisher’s least significant difference (LSD); genotype, p < 0.001, F (1,11) = 15.79; treatment, p < 0.001, F (1,11) = 28.24; genotype × treatment, F (1,11) = 23.43). (B) Same as in (A) but after exposure to 4 h of glucose deprivation. Values are means + SEM. ( n = 6–9 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,25) = 26.73; treatment, p < 0.001, F (1,25) = 19.33; genotype × treatment, F (1,25) = 18.26). Dkk3 protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (C) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p = 0.003, F (1,12) = 8.18; treatment, p = 0.004, F (1,12) = 6.53; genotype × treatment, F (1,12) = 5.82). VEGF protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (D) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,12) = 38.54; treatment, p = 0.003, F (1,12) = 6.60; genotype × treatment, F (1,12) = 6.90). Phospho-AKT protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (E) . Values are means + SEM ( n = 6 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,20) = 19.40; treatment, p = 0.016, F (1,20) = 3.09; genotype × treatment, F (1,20) = 3.93). Bcl2 protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (F) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,12) = 25.46; treatment, p = 0.038, F (1,12) = 1.74; genotype × treatment, F (1,12) = 3.93). VEGF protein levels in the whole brain of wild-type and Dkk3 knockout mice are shown in (G) . Values are means + SEM ( n = 3–4 per group). * p < 0.05 vs. wild-type mice (Student’s t-test ; p = 0.0005, t 5 = 7.98). Dkk3 and VEGF protein levels in the whole brain of CD1 and C57BL/6J mice are shown in (H) . Values are means ± SEM ( n = 3 per group). * p < 0.05 vs. CD1 mice (Student’s t-test ; Dkk3, p = 0.0099, t (4) = 4.62; VEGF, p = 0.0384, t (4) = 3.04). (I) FACS analysis of live cells in cultures of cortical astrocytes from CD1 mice incubated for 12 h with hrDkk3 (10 ng/ml) prior to a 1 h-exposure to H 2 O 2 (100 μM). Values are means + SEM ( n = 3 per group). p < 0.05 vs. the respective control cultures not exposed to H 2 O 2 (* p = 0.0001), or vs. the respective cultures not treated with hrDkk3 ( # p = 0.0013; one-way ANOVA + Fisher’s LSD; F (3,8) = 26.2). VEGF protein levels in lysates of cultured astrocytes from CD1 mice treated with hrDkk3 (10 ng/ml) for 12 h are shown in (J) . Values are means ± SEM ( n = 3 per group). * p < 0.05 vs. controls (Student’s t-test ; p = 0.0119, t (4) = 4.38). Ctrl = untreated controls.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Activity Assay, Cell Culture, Flow Cytometry, Knock-Out, Incubation, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Dkk3 is protective against excitotoxic neuronal death in mixed cortical cultures. Neuronal death in mixed cortical cultures prepared from different mouse genotypes (all C57BL/6J strain) and challenged with 100 μM N -methyl-D-aspartate (NMDA) is shown in (A) . Values are means + SEM ( n = 4–8 per group). p < 0.05 vs. the respective cultures not treated with NMDA (*), or vs. cultures treated with NMDA from wild-type mice in both neurons astrocytes and neurons ( # two-way ANOVA + Fisher’s LSD; genotype, p = 0.002 for astrocytes knockout/neurons knockout; p = 0.026 for astrocytes knockout/neurons wild-type, F (3,40) = 2.06; treatment, p < 0.001 for astrocytes wild-type/neurons wild-type, astrocytes knockout/neurons wild-type and astrocytes knockout/neurons knockout; p = 0.003 for astrocytes wild-type/neurons knockout, F (1,40) = 77.52; genotype × treatment, F (3,40) = 2.07). Neuronal death in cultures from CD1 mice challenged with 45 μM NMDA followed or not by 20-h exposure to hrDkk3 (10 ng/ml) is shown in (B) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. cultures not treated with NMDA (* p < 0.0001) or vs. cultures challenged with NMDA not exposed to hrDkk3 ( p = 0.0009; one-way ANOVA + Fisher’s LSD; F (3,12) = 83.69). Data obtained in cultures from CD1 mice challenged with 80 μM NMDA followed or not by 20-h exposure to hrDkk3 (10 ng/ml) w/wo ZM3238881 (10 μM) is shown in (C) . Values are means + SEM ( n = 5–8 per group). p < 0.05 vs. cultures not treated with NMDA (* p = 0.0005 for NMDA, p < 0.0001 for NMDA+ hrDkk3 + ZM323881, p = 0.0012 for NMDA+ ZM323881) or vs. cultures challenged with NMDA and not exposed to hrDkk3 ( # p = 0.0045; one-way ANOVA + Fisher’s LSD; F (5,32) = 8.39). Neuroprotection by hrVEGF against NMDA toxicity (here, 150 μM) in mixed cortical cultures from CD1 mice is shown in (D) . Cells were exposed to hrVEGF for 20 h following the NMDA pulse. Values are means + SEM ( n = 4–11 per group). p < 0.05 vs. cultures not treated with NMDA (* p < 0.0001) or vs. cultures challenged with NMDA not exposed to hrVEGF ( # p < 0.0001; one-way ANOVA + Fisher’s LSD; F (5,31) = 22.26).

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Knock-Out

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Schematic representation of the protective feedback Dkk3-mediated in astrocytes and/or neurons exposed to cellular stress (glucose deprivation, oxidative stress or ischemia). Dkk3 leads to VEGF upregulation with ensuing VEGFR2 activation, AKT phosphorylation and upregulation of the antiapoptotic factor Bcl2.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Activation Assay, Phospho-proteomics

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Dickkopf 3 attenuates xanthine dehydrogenase expression to prevent oxidative stress-induced apoptosis.

doi: 10.1111/gtc.12484

Figure Lengend Snippet: Figure 1 DKK3 expression in human cell lines. (A) DKK3 mRNA expression was normalized that of 18S ribosomal RNA in real-time PCR analysis. (B) DKK3 protein levels were assessed by immunoblotting 10 or 40 lg protein from conditioned medium or cell lysates, respectively, after culturing for 24 h. b-actin was used as a loading control. Recombinant human DKK3 protein produced by a mouse myeloma cell line (rhDKK3) and sterilized water were used as positive and negative (N) controls, respec- tively. Hepatocellular carcinoma (HCC) cells lines: KIM-1, Li7HM, Li7NM, KYN-1, KYN-2, KYN-3, Li21, PLC/PRF/5, HuH7, tPH5T; Hepatoblastoma (HBL) cell lines: Li24, HepG2, HuH6; immortalized human non-neoplastic hepatocyte cell line: tPH5CH; fetal-type rhabdomyosarcoma cell line: RD.

Article Snippet: Immunoblotting was carried out according to standard procedures with primary antibodies for DKK3 (goat polyclonal, R&D Systems, Minneapolis, MN, USA) and b-actin (mouse monoclonal, Sigma-Aldrich), and horseradish peroxidase-conjugated anti-goat or anti-mouse IgG secondary antibodies (Life Technologies).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Recombinant, Produced

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Dickkopf 3 attenuates xanthine dehydrogenase expression to prevent oxidative stress-induced apoptosis.

doi: 10.1111/gtc.12484

Figure Lengend Snippet: Figure 2 Induction of apoptosis in Li21 and tPH5CH DKK3-knockdown cells. (A) DKK3 mRNA levels were analyzed by real- time PCR 48 h after siRNA transfection. (B) DKK3 protein knockdown confirmed by Western blot analysis 48 h after siRNA transfection. b-actin was used as a loading control. (C) Phase-contrast micrographs of cells 48 h after siRNA transfection. (D) Attached cells were counted using a hemocytometer. (E) Cell proliferation was assessed 48 h after siRNA transfection by WST-1 assay. (F) Caspase 3/7 activity was measured 48 h after siRNA transfection. (G) PS exposure was detected with the Cell-APOPer- centage Apoptosis Assay 48 h after siRNA transfection in Li21 cells. PCtr, positive control; siCtr, siRNA control; NCtr, non- treated control. (H) Mitochondrial membrane depolarization was monitored 48 h after DKK3-knockdown by JC-1 staining in Li21 cells. Scale bars, 50 lm; *P < 0.05.

Article Snippet: Immunoblotting was carried out according to standard procedures with primary antibodies for DKK3 (goat polyclonal, R&D Systems, Minneapolis, MN, USA) and b-actin (mouse monoclonal, Sigma-Aldrich), and horseradish peroxidase-conjugated anti-goat or anti-mouse IgG secondary antibodies (Life Technologies).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Control, WST-1 Assay, Activity Assay, Apoptosis Assay, Positive Control, Membrane, Staining

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Dickkopf 3 attenuates xanthine dehydrogenase expression to prevent oxidative stress-induced apoptosis.

doi: 10.1111/gtc.12484

Figure Lengend Snippet: Figure 3 ROS accumulation in DKK3-knockdown cells. (A) ROS accumulation was measured by CellROX green staining at 14 and 24 h after siRNA transfection following treatment with N-acetyl-cysteine (NAC, 4.0 mM) or menadione (100 lM). (B) Representative fluorescence microscopy images of Li21 cells 24 h after siRNA transfection. ROS (green, white arrowheads); nuclei (blue). (C) siRNA-transfected cells were treated with increasing concentrations of NAC (0, 0.4 and 4.0 mM) and counted 48 (left) or 72 h (right) later. (D) Cell morphology and adherence were assessed by phase-contrast microscopy 48 h after siRNA transfection. Scale bar, 50 lm; *P < 0.05, **P < 0.01.

Article Snippet: Immunoblotting was carried out according to standard procedures with primary antibodies for DKK3 (goat polyclonal, R&D Systems, Minneapolis, MN, USA) and b-actin (mouse monoclonal, Sigma-Aldrich), and horseradish peroxidase-conjugated anti-goat or anti-mouse IgG secondary antibodies (Life Technologies).

Techniques: Knockdown, Staining, Transfection, Microscopy

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Dickkopf 3 attenuates xanthine dehydrogenase expression to prevent oxidative stress-induced apoptosis.

doi: 10.1111/gtc.12484

Figure Lengend Snippet: Figure 4 DKK3 over-expression increases resistance to H2O2-induced oxidative stress in Li21 and tPH5CH cells. (A, B) Forced DKK3 over-expression was confirmed in Li21 and tPH5CH cells at the (A) mRNA and (B) protein levels. D, DKK3-transfected cells; M, mock-transfected cells; N, negative control. (C) DKK3- and mock-transfected cells were cultured for 24 h before treat- ment with increasing concentrations of H2O2 (0–1000 lM). Cells were counted 18 h later. (D) Phase-contrast microscopy of siDKK3- and mock-transfected Li21 and tPH5CH cells 18 h after treatment with 500 and 400 lM H2O2, respectively. Scale bar, 50 lm; *P < 0.05 vs. mock-transfected controls.

Article Snippet: Immunoblotting was carried out according to standard procedures with primary antibodies for DKK3 (goat polyclonal, R&D Systems, Minneapolis, MN, USA) and b-actin (mouse monoclonal, Sigma-Aldrich), and horseradish peroxidase-conjugated anti-goat or anti-mouse IgG secondary antibodies (Life Technologies).

Techniques: Over Expression, Transfection, Negative Control, Cell Culture, Microscopy