Journal: Oncotarget

Article Title: AGER rs2070600 polymorphism elevates neutrophil-lymphocyte ratio and mortality in metastatic lung adenocarcinoma

doi: 10.18632/oncotarget.21764

Figure Lengend Snippet: Associations of the AGER polymorphisms with the risk of lung adenocarcinoma

Article Snippet: The PCR mixture contained TaqMan SNP Genotyping Assay (C__15867521_20, C___3293837_1_, C___8848033_1_; Life Technologies Corp., Carlsbad, CA, USA), genomic DNA (2.0 g/mL), and TaqMan Fast Universal PCR Master Mix (Life Technologies Corp.).

Techniques: Mutagenesis

Journal: Oncotarget

Article Title: AGER rs2070600 polymorphism elevates neutrophil-lymphocyte ratio and mortality in metastatic lung adenocarcinoma

doi: 10.18632/oncotarget.21764

Figure Lengend Snippet: Box plots showing the range of neutrophil-lymphocyte ratios with/without the AGER rs2070600 minor allele (T) in the patients with metastatic lung adenocarcinoma (n = 96) (A), and exploratory subgroup analysis in patients with EGFR mutation-positive and EGFR mutation-negative lung adenocarcinoma, separately (B). Each box represents the 25 th to 75 th percentiles; solid lines within the boxes indicate the median values; whiskers are the 10 th and 90 th percentiles; and points represent outliers. The p -values were evaluated using Mann–Whitney U -tests.

Article Snippet: The PCR mixture contained TaqMan SNP Genotyping Assay (C__15867521_20, C___3293837_1_, C___8848033_1_; Life Technologies Corp., Carlsbad, CA, USA), genomic DNA (2.0 g/mL), and TaqMan Fast Universal PCR Master Mix (Life Technologies Corp.).

Techniques: Mutagenesis, MANN-WHITNEY

Journal: Oncotarget

Article Title: AGER rs2070600 polymorphism elevates neutrophil-lymphocyte ratio and mortality in metastatic lung adenocarcinoma

doi: 10.18632/oncotarget.21764

Figure Lengend Snippet: Kaplan-Meier analysis showing (A) the survival rate and (B) progression free survival (PFS) with platinum-based chemotherapy in patients with metastatic lung adenocarcinoma, on the presence or absence of the AGER rs2070600 minor allele (T) (n = 96). Solid line indicates patients with the C/C genotype. Dotted line indicates patients with the C/T or T/T genotype. Patients with the AGER rs2070600 minor allele (T) showed significantly poorer survival and shorter PFS compared to those without the minor allele.

Article Snippet: The PCR mixture contained TaqMan SNP Genotyping Assay (C__15867521_20, C___3293837_1_, C___8848033_1_; Life Technologies Corp., Carlsbad, CA, USA), genomic DNA (2.0 g/mL), and TaqMan Fast Universal PCR Master Mix (Life Technologies Corp.).

Techniques:

Journal: Biomolecules

Article Title: Coumarin Ameliorates Impaired Bone Turnover by Inhibiting the Formation of Advanced Glycation End Products in Diabetic Osteoblasts and Osteoclasts

doi: 10.3390/biom10071052

Figure Lengend Snippet: Inhibition of glucose-induced advanced glycation end products (AGE) secretion ( A ) and receptor for AGE (RAGE) expression ( B ), and elevation of AGE-induced osteoclast differentiation ( C ), and bone resorption ( D ) by coumarin in Raw 264.7 cells. Cells were cultured in α-MEM media containing 5.5 mM glucose, 33 mM glucose, or 100 μg/mL AGE with 50 ng/mL RANKL in the absence and presence of 1–20 μM coumarin for five days. Cell media and lysates were subject to western blot analysis with a primary antibody against AGE and RAGE. Representative blot data were obtained from three independent experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panel represent quantitative results obtained from a densitometer. Values not sharing a letter are different at p < 0.05. After cells were cultured with 100 μg/mL AGE–bovine serum albumin (BSA) for five days, cells were fixed and stained using a leukocyte acid phosphatase kit ( C ). TRAP-positive multinucleated osteoclasts were visualized under light microscopy. The osteoclast bone resorption was assayed by using a commercially available bone resorption assay kit ( D ). The resorbed areas on the plate were visualized under light microscopy. Magnification: 20-fold.

Article Snippet: Advanced glycation end product-bovine serum albumin (AGE-BSA) protein was obtained from Merck Millipore (Burlington, MA, USA).

Techniques: Inhibition, Expressing, Cell Culture, Western Blot, Control, Staining, Light Microscopy

Journal: Biomolecules

Article Title: Coumarin Ameliorates Impaired Bone Turnover by Inhibiting the Formation of Advanced Glycation End Products in Diabetic Osteoblasts and Osteoclasts

doi: 10.3390/biom10071052

Figure Lengend Snippet: Inhibition of advanced glycation end-product (AGE) secretion and receptor for AGE (RAGE) induction ( A ) in osteoblastic MC3T3-E1 cells by coumarin. MC3T3-E1 cells were cultured for nine days in the presence of 1–20 μM of coumarin. Cell media and lysates were subject to western blot analysis with a primary antibody against AGE and RAGE. Representative blot data were obtained from three independent experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panel represent quantitative results obtained from a densitometer. MC3T3-E1 cells were cultured in differentiation media containing 5.5 mM glucose or 100 μg/mL AGE–bovine serum albumin (BSA) in the presence of 1–20 μM coumarin on glass chamber plates for six days ( B ). Alkaline phosphatase (ALP) staining was proportional to the content of ALP enzyme activity from MC3T3-E1 cells. The intensity of ALP staining (mean ± SEM, n = 4) was measured using an optical Axiomager microscope system ( B ). The ALP staining was visualized under light microscopy. Magnification: 10-fold. Values not sharing a letter are different at p < 0.05.

Article Snippet: Advanced glycation end product-bovine serum albumin (AGE-BSA) protein was obtained from Merck Millipore (Burlington, MA, USA).

Techniques: Inhibition, Cell Culture, Western Blot, Control, Staining, Activity Assay, Microscopy, Light Microscopy

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Replication Stress Response Modifies Sarcomeric Cardiomyopathy Remodeling

doi: 10.1161/JAHA.121.021768

Figure Lengend Snippet: ( A ) γH2AX Western blot from postnatal day (P) 7 control (Ctl) and Mybpc3 −/− myocardial tissue. ( B ) Relative quantification γH2AX in Ctl (n=6) and Mybpc3 −/− (n=6) myocardial tissue at P7 normalized to β‐actin. ( C ) Representative immunofluorescence staining of γH2AX (red) in Ctl and Mybpc3 −/− left ventricular (LV) tissue at P7. Cardiomyocyte (CM) nuclei were identified with pericentriolar material 1 (PCM1) (green), and nuclei were labeled by 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). Bars=100 µm. ( D ) Comparison of γH2AX staining between CM and noncardiomyocyte (Non‐CM) nuclei in Ctl (n=4–6) and Mybpc3 −/− (n=4–6) LV tissue at P7. Minimum 100 nuclei/sample. ( E ) Relative quantification of γH2AX fluorescence in CM nuclei in Ctl (n=6–9) and Mybpc3−/− (n=7–8) LV tissue at P2, P7, P25, and P180. Minimum 50 nuclei/sample. ( F ) Representative γH2AX foci (red, arrowhead) immunofluorescence staining with CM‐specific PCM1 (green) and nuclei labeled by DAPI (blue) in Ctl and Mybpc3 −/− LV tissue at P7. Bar=5 µm. ( G ) Quantitation of average γH2AX foci per CM nuclei in Ctl (n=6) and Mybpc3 −/− (n=6) LV tissue. Minimum 50 nuclei/sample. ( H ) Representative immunofluorescence staining of 53BP1 (p53‐binding protein 1) (green) and γH2AX (red) and nuclei labeled with DAPI (blue) in Ctl and Mybpc3 −/− myocardial tissue at P7. Bar=5 µm. ( I ) Average γH2AX‐53BP1 colocalized foci per nuclei in Ctl (n=6) and Mybpc3 −/− (n=6) LV tissue. Minimum 50 nuclei/sample. ( J ) Telomere peptide nucleic acid (PNA) fluorescence in situ hybridization assay from Ctl or Mybpc3 −/− mice at P7 with γH2AX (green), telomere (red), and nuclei DAPI (blue) staining. Bar=5 µm. ( K ) Percentage of telomere‐γH2AX colocalization foci per nuclei in Ctl (n=4) and Mybpc3 −/− (n=4) LV tissue was quantified. Minimum 100 nuclei/sample. ( L ) Neutral comet assay of nuclei from Ctl or Mybpc3 −/− myocardial tissue at P7. ( M ) Relative quantitation of comet tail length of nuclei from Ctl (n=4) and Mybpc3 −/− (n=4) myocardial tissue. Minimum 100 nuclei/sample. All results are shown as mean±SEM.

Article Snippet: Sections were then incubated with blocking solution (1% BSA in PBS) for 1 hour at room temperature, followed by primary antibodies targeting p‐Ser139 Histone H2AX (γH2AX) (Millipore Sigma, 05‐636‐I), p53‐binding protein 1 (53BP1) (Novus Biologicals, NB100‐304), pericentriolar material 1 (PCM1) (Sigma, HPA023370), Ki67 (CST, 9129), p‐Ser428 ATR (CST, 2853), and sarcomeric α‐actinin (Abcam, ab9465), either alone or in combination, at 4°C overnight.

Techniques: Western Blot, Immunofluorescence, Staining, Labeling, Comparison, Fluorescence, Quantitation Assay, Binding Assay, In Situ Hybridization, Neutral Comet Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Replication Stress Response Modifies Sarcomeric Cardiomyopathy Remodeling

doi: 10.1161/JAHA.121.021768

Figure Lengend Snippet: ( A ) Western blot of phosphorylated ataxia telangiectasia and rad3 related (ATR) (p‐Ser428 ATR), total ATR, phosphorylated ataxia telangiectasia mutated –(ATM) (p‐Ser1981 ATM), and total ATM from postnatal day (P) 7 control (Ctl) and Mybpc3 −/− myocardial tissue. β‐Actin was the loading control. ( B ) Relative quantification of p‐Ser428 ATR and p‐Ser1981 ATM in Ctl (n=8) and Mybpc3 −/− (n=8) myocardial tissue normalized to total ATR and total ATM, respectively. ( C ) Western blot of p53 in Ctl and Mybpc3 −/− myocardial tissue at P7 ( top ). Relative quantification of p53 in P7 Ctl (n=7) and Mybpc3 −/− (n=7) myocardial tissue normalized to β‐actin ( bottom ). ( D ) Western blot of p53 in Ctl and Mybpc3 −/− myocardial tissue at P25 ( top ). Relative quantification of p53 in P25 Ctl (n=7) and Mybpc3 −/− (n=7) myocardial tissue normalized to β‐actin ( bottom ). ( E ) Measurement of p53 gene expression from Ctl and Mybpc3 −/− myocardial tissue at P7 (n=7) and P25 (n=6). The genes of interest were normalized to Rpl32 expression. Fold changes are shown relative to Ctl gene expression. ( F ) Representative terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling (TUNEL) assay images from Ctl and Mybpc3 −/− mouse left ventricular (LV) tissue section stained with TUNEL (green), α‐actinin (red), and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue) staining. DNase I–treated control LV tissue section was used as positive control for the assay (DNAse I Positive Ctl). Bar=50 µm. All results are shown as mean±SEM.

Article Snippet: Sections were then incubated with blocking solution (1% BSA in PBS) for 1 hour at room temperature, followed by primary antibodies targeting p‐Ser139 Histone H2AX (γH2AX) (Millipore Sigma, 05‐636‐I), p53‐binding protein 1 (53BP1) (Novus Biologicals, NB100‐304), pericentriolar material 1 (PCM1) (Sigma, HPA023370), Ki67 (CST, 9129), p‐Ser428 ATR (CST, 2853), and sarcomeric α‐actinin (Abcam, ab9465), either alone or in combination, at 4°C overnight.

Techniques: Western Blot, Expressing, End Labeling, TUNEL Assay, Staining, Positive Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Replication Stress Response Modifies Sarcomeric Cardiomyopathy Remodeling

doi: 10.1161/JAHA.121.021768

Figure Lengend Snippet: ( A ) Schematic illustration of ATR kinase inhibition from postnatal day (P) 4 to P7 with the drug AZD6738. ( B ) M‐mode echocardiography at P7 from control (Ctl) and Mybpc3 −/− mice administered vehicle (Veh.) or 25 mg/kg per day AZD6738. Echocardiography assessment of interventricular septal thickness at end diastole (IVSd) ( C ), left ventricular posterior wall thickness at end diastole (LVPWd) ( D ), left ventricular internal diameter at end diastole (LVIDd) ( E ), and fractional shortening (FS) ( F ) in Ctl (n=5–7) and Mybpc3 −/− (n=9–12) mice administered Veh. or AZD6738. Heart weight (HW) ( G ) and HW/body weight (BW) ratio ( H ) for Ctl (n=5–7) and Mybpc3 −/− (n=9–12) mice administered Veh. or AZD6738. ( I ) Representative immunohistochemical staining with wheat‐germ agglutinin (green) and 4′,6‐diamidino‐2‐phenylindole (blue) of left ventricular (LV) tissue from Ctl and Mybpc3 −/− mice administered Veh. or AZD6738. Bar=10 µm. ( J ) LV cross‐sectional area of Ctl (n=5) and Mybpc3 −/− (n=5) mice administered Veh. or AZD6738. Minimum 50 cells/sample measured. ( K ) Western blot of p53 in myocardial tissue lysate from Mybpc3 −/− administered Veh. or AZD6738. ( L ) Relative quantification of p53 protein expression from Mybpc3 −/− administered Veh. (n=4) or AZD6738 (n=5) normalized to β‐actin. All results are shown as mean±SEM.

Article Snippet: Sections were then incubated with blocking solution (1% BSA in PBS) for 1 hour at room temperature, followed by primary antibodies targeting p‐Ser139 Histone H2AX (γH2AX) (Millipore Sigma, 05‐636‐I), p53‐binding protein 1 (53BP1) (Novus Biologicals, NB100‐304), pericentriolar material 1 (PCM1) (Sigma, HPA023370), Ki67 (CST, 9129), p‐Ser428 ATR (CST, 2853), and sarcomeric α‐actinin (Abcam, ab9465), either alone or in combination, at 4°C overnight.

Techniques: Inhibition, Immunohistochemical staining, Staining, Western Blot, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Replication Stress Response Modifies Sarcomeric Cardiomyopathy Remodeling

doi: 10.1161/JAHA.121.021768

Figure Lengend Snippet: ( A ) Schematic of Mybpc3 −/− /p53 fl/fl Myh6Cre +/− double‐null murine model generated by crossing a p53 fl/fl Myh6Cre +/− mouse with an Mybpc3 −/− mouse. ( B ) Western blot of p53 from control (Ctl) (n=3), Mybpc3 −/− (n=3), and Mybpc3 −/− /p53 fl/fl Myh6Cre +/− (n=3) myocardial tissue at postnatal day (P) 25. Western blot of β‐actin used as loading control. ( C ) Measurement of p53 target gene expression (mouse double minute 2 [Mdm2], cyclin‐dependent kinase inhibitor 1 [Cdnk1a], growth differentiation factor 15 [Gdf15], and growth arrest and DNA damage inducible α [Gadd45a]) at P25 in Ctl (n=6), Mybpc3 −/− (n=6), and Mybpc3 −/− /p53 fl/fl Myh6Cre +/− (n=6) left ventricular (LV) tissue RNA. The genes of interest were normalized to Rpl32 expression. Fold changes are shown relative to Ctl gene expression. Echocardiography assessment of interventricular septal thickness at end diastole (IVSd) ( D ), LV posterior wall thickness at end diastole (LVPWd) ( E ), LV internal diameter at end diastole (LVIDd) ( F ), and fractional shortening (FS) ( G ) in Ctl (n=6), p53 fl/fl Myh6Cre +/− (n=4), Mybpc3 −/− (n=13), and Mybpc3 −/− /p53 fl/fl Myh6Cre +/− (n=13) mice at P25. Heart weight (HW) ( H ) and HW/tibia length (TL) ratio ( I ) from Ctl (n=7), p53 fl/fl Myh6Cre +/− (n=6), Mybpc3 −/− (n=7), and Mybpc3 −/− /p53 fl/fl Myh6Cre +/− (n=6) mice at P25. ( J ) Representative immunohistochemical staining using wheat‐germ agglutinin (green) and 4′,6‐diamidino‐2‐phenylindole (blue) of LV tissue from Ctl, Mybpc3 −/− , and Mybpc3 −/− /p53 fl/fl Myh6Cre +/− mice. Bar=10 µm. ( K ) LV cardiomyocyte cross‐sectional area from Ctl (n=5), Mybpc3 −/− (n=5), and Mybpc3 −/− /p53 fl/fl Myh6Cre +/− (n=5) mice. Minimum 50 cells/sample measured. All results are shown as mean±SEM.

Article Snippet: Sections were then incubated with blocking solution (1% BSA in PBS) for 1 hour at room temperature, followed by primary antibodies targeting p‐Ser139 Histone H2AX (γH2AX) (Millipore Sigma, 05‐636‐I), p53‐binding protein 1 (53BP1) (Novus Biologicals, NB100‐304), pericentriolar material 1 (PCM1) (Sigma, HPA023370), Ki67 (CST, 9129), p‐Ser428 ATR (CST, 2853), and sarcomeric α‐actinin (Abcam, ab9465), either alone or in combination, at 4°C overnight.

Techniques: Generated, Western Blot, Targeted Gene Expression, Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: Opioids Impair Intestinal Epithelial Repair in HIV-Infected Humanized Mice

doi: 10.3389/fimmu.2019.02999

Figure Lengend Snippet: Morphine treatment induced mucosal cell apoptosis and tissue inflammation in the intestine of HIV-infected BLT mice. (A) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay was used to detect apoptotic cells in the intestinal sections. Apoptotic cells were stained red. Scale bar: 50 μm; white arrow: apoptotic cells. (B) Quantification of the TUNEL assay. The number of apoptotic cells in each villus was quantified. Quantification from three different villi for each animal is depicted ( n = 18). ANOVA followed by Bonferroni correction, F (3,68) = 52.32, ANOVA P < 0.0001. P -values for group comparison are shown in the figure if smaller than 0.05. (C) ELISA was used to determine the expression levels of IL-6 in the intestine. Each dot represents one animal ( n = 6). ANOVA followed by Bonferroni correction, F (3, 20) = 19.13, ANOVA P < 0.0001. P -values for group comparison are shown in the figure if smaller than 0.05.

Article Snippet: Patient-derived HIV isolates (ADA-M) were obtained from National Institutes of Health AIDS repository and expanded on human monocytes (donor-derived; Red Cross of America) for 14 days, with media and monocyte replenishment every 48–72 h. At the end, media were harvested and centrifuged at 500 g, and HIV concentration was measured using HIV p24 antigen ELISA kit (ZeptoMetrix Corporation, Buffalo, NY).

Techniques: Infection, End Labeling, TUNEL Assay, Staining, Comparison, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Oncogene

Article Title: Cyclin D1 in low-dose radiation-induced adaptive resistance

doi: 10.1038/onc.2008.265

Figure Lengend Snippet: Low-dose ionizing radiation (LDIR)-induced adaptive radioresistance was eliminated by inhibition of cyclin D1. (a) Induction of cyclin D1. HK18 cells were irradiated with 0.5-cGy X-ray for 10, 20, 30 or 40 times (total accumulated doses were 5, 10, 15 or 20-cGy; upper panel). Sham-IR cells (0-cGy) were collected at the same fractions as irradiated cells. Western blot was performed with cellular proteins prepared at 24 h after the final radiation exposure. Lower panel shows western blot and densitometry of cyclin D1 expression in HK18 cells after exposure to a single dose of 0.5-cGy X-ray (C, sham-IR control; β-actin as loading control used for normalization of cyclin D1 levels). (b) Induction of cyclin D1 by 10-cGy and 5-Gy γ-ray. HK18 cells were irradiated with a single dose of 10-cGy X-ray or 5-Gy γ-ray and western blot was performed at indicated times after radiation or sham-IR cells (0-cGy; C, sham-IR control; β-actin as loading control). HK18 cells were left untreated (c) or treated with 10 nM of cyclin D1 short interfering RNA (siRNA) or scramble (d) for 60 h before irradiation with 10-cGy X-ray, and 6 h later a group of dishes were further exposed to 5-Gy γ-ray. Clonogenic survival was measured at 24 h after radiation. Colonies consisting of more than 50 cells were scored as surviving colonies and normalized with the clone numbers observed on nonirradiated cells (n = 3). The inset of (d) shows a western blot of cyclin D1 and β-actin in cells treated with cyclin D1 siRNA or scramble siRNA.

Article Snippet: The antibody preparations against 14-3-3ζ (sc-1019), actin (sc-8432), cyclin D1 (sc-20044), cytochrome c (sc-13560), Bax (sc-493) and Bcl-2 (sc-509) were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA).

Techniques: Inhibition, Irradiation, Western Blot, Expressing, Control, Small Interfering RNA

Journal: Oncogene

Article Title: Cyclin D1 in low-dose radiation-induced adaptive resistance

doi: 10.1038/onc.2008.265

Figure Lengend Snippet: Differential distribution of cyclin D1 induced by 10-cGy X-ray and 5-Gy γ-ray. (a) Cytoplasmic (C) and nuclear (N) proteins were prepared from HK18 cells at 24h after last exposure to 10 fractions of 0.5-cGy X-ray and protein levels were measured with western blotting with α-tubulin and histone H3 as cytoplasmic and nuclear markers (W, sham-LDIR (low-dose ionizing radiation) control). (b) Western blotting of cyclin D1 with cytoplasmic (C) and nuclear (N) proteins prepared at indicated time points from HK18 cells after radiation with a low- or high-dose of radiation (α-tubulin and histone H3 as markers for cytoplasmic and nuclear fractions; W, sham-LDIR control). HK18 cells were treated with sham, 10-cGy X-ray or 5-Gy γ-ray, and subjected to immunostaining of cyclin D1 and 4,6-diamidino-2-phenylindole (DAPI) staining of DNA at 8h after radiation (c). Skin tissue sections prepared from mice whole-body-irradiated with sham, 10-cGy X-ray or 5-Gy γ-ray were subjected to immunostaining (d). Right panels indicate the percentage of cells with nuclear staining of cyclin D1 (100 cells per field were counted and the average of five fields was used to generate the graph).

Article Snippet: The antibody preparations against 14-3-3ζ (sc-1019), actin (sc-8432), cyclin D1 (sc-20044), cytochrome c (sc-13560), Bax (sc-493) and Bcl-2 (sc-509) were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA).

Techniques: Western Blot, Control, Immunostaining, Staining, Irradiation

Journal: Oncogene

Article Title: Cyclin D1 in low-dose radiation-induced adaptive resistance

doi: 10.1038/onc.2008.265

Figure Lengend Snippet: Cyclin D1 and 14-3-3ζ formed a complex that was dissociated by low-dose ionizing radiation (LDIR). (a) Detection of cyclin D1/14-3-3ζ complex. Whole-cell lysates of resting and irradiated HK18 cells were immunoprecipitated with anti-cyclin D1 antibody followed by immunoblotting (IB) with 14-3-3ζ or cyclin D1 antibody. Immunoprecipitation (IP) and IB without cell extracts were included as negative control (NC; right panel, cyclin D1/14-3-3ζ interaction estimated by densitometry). (b) Cyclin D1/14-3-3ζ complex dissociation. The upper panel (IP) shows cyclin D1/14-3-3ζ interaction and the lower panel (IB) shows the increased free cyclin D1 in the supernatant (β-actin served as loading control) 8h after LDIR treatment. (c) Dissociation of cyclin D1/14-3-3ζ in cytoplasm. Upper panel, cyclin D1/14-3-3ζ interaction was detected in cytoplasmic (C) and nuclear (N) extracts by IP. Extracts of sham-IR cells were preincubated with cyclin D1 antibody included as NC (W, sham-LDIR). Lower panel, western blotting of cyclin D1 (α-tubulin and histone H3 as cytoplasmic and nuclear markers) and the densitometry estimated cyclin D1/14-3-3ζ complex shown in the upper panel. (d) Inhibition of 14-3-3ζ by short interfering RNA (siRNA). Upper panel, western blot of 14-3-3ζ in HK18 cells after transfection with 14-3-3ζ (50 or 100 nM) or scramble siRNA (control; 100 nM) for 60 h. Lower panel, HK18 cells were treated with 100 nM 14-3-3ζ or scramble siRNA before 5-Gy radiation. Cytoplasmic and nuclear cyclin D1 were detected by western blotting. (e) Upper panel, HK18 cells transfected with 14-3-3ζ siRNA for 60 h were subjected to immunohistochemistry (IHC) of 14-3-3ζ 8 h after 5-Gy radiation (nucleus visualized by 4,6-diamidino-2-phenylindole (DAPI)). Lower panel, HK18 cells treated with 14-3-3ζ siRNA, scramble or transfection agent (Lipo), and cyclin D1 was detected by IHC 8 h after 5-Gy radiation.

Article Snippet: The antibody preparations against 14-3-3ζ (sc-1019), actin (sc-8432), cyclin D1 (sc-20044), cytochrome c (sc-13560), Bax (sc-493) and Bcl-2 (sc-509) were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA).

Techniques: Irradiation, Immunoprecipitation, Western Blot, Negative Control, Control, Inhibition, Small Interfering RNA, Transfection, Immunohistochemistry

Journal: Oncogene

Article Title: Cyclin D1 in low-dose radiation-induced adaptive resistance

doi: 10.1038/onc.2008.265

Figure Lengend Snippet: Ser-58 of 14-3-3ζ was required for cyclin D1 interaction. (A) HK18 cells were co-transfected with fusion vectors pFlag-14-3-3ζ-YN173 and pHA-cyclin D1-YC156 or pFlag-14-3-3ζ S58D-YN173 and pHA-cyclin D1-YC156 as indicated. Cells were transfected for 6h and fluorescence images were visualized 24 h after irradiation with 10-cGy. Panels c and d indicate higher magnification of images shown in the red box of a and b, respectively (arrows indicate the formation of 14-3-3ζ/cyclin D1 complex). Right panel shows the estimated difference of cell numbers with fluorescence in panel a versus b. Five fields (100 cells per field) were counted and the average was used to generate the graph. (B) Total cell lysates of (A) prepared 24h after irradiation with 10-cGy and cyclin D1/14-3-3ζ interaction was detected by IP/IB with indicated antibodies (NC = total cell lysates of HK18 cells of panel A were preincubated with cyclin D1 antibody).

Article Snippet: The antibody preparations against 14-3-3ζ (sc-1019), actin (sc-8432), cyclin D1 (sc-20044), cytochrome c (sc-13560), Bax (sc-493) and Bcl-2 (sc-509) were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA).

Techniques: Transfection, Fluorescence, Irradiation

Journal: Oncogene

Article Title: Cyclin D1 in low-dose radiation-induced adaptive resistance

doi: 10.1038/onc.2008.265

Figure Lengend Snippet: Cyclin D1/Bax interaction linked with mitochondrial membrane potential (Δψm) and apoptosis. (a) Western blot of Bcl-2 and Bax in HK18 cells treated with sham-IR, 5-Gy, 10-cGy or 10-cGy + 5-Gy IR. Lower panel, the Bax:Bcl-2 ratio determined by relative levels of densitometry. (b) IP of whole-cell extracts prepared at indicated times after radiation (W, sham IR). Lower panel, IP and IB were performed with antibodies of cyclin D1 and Bax in HK18 cells treated with 10 nM cyclin D1 or scramble short interfering RNA (siRNA) for 60 h (NC, extracts preincubated with the antibody used for IP; W, sham-IR control). (c) Western blotting of mitochondrial and cytosolic cytochrome c of HK18 cells treated with as in a (Cox IV and CuZnSOD as markers for mitochondrial and cytosolic fractions; C, cytoplasmic fraction; M, mitochondrial fraction). Right panel shows the relative amount of cytosolic cytochrome c estimated by densitometry. (d and e) Cyclin D1 siRNA inhibited low-dose ionizing radiation (LDIR)-induced Δψm and apoptosis. HK18 cells were treated with 10 nM of cyclin D1 siRNA or scramble for 60 h before irradiation as in (a). Cells were then treated with JC-1 probe, harvested, and the changes in Δψm was measured, using a fluorescent spectrophotometer (d) or stained by terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) method (e) 24h after radiation (mean±s.e., n = 3).

Article Snippet: The antibody preparations against 14-3-3ζ (sc-1019), actin (sc-8432), cyclin D1 (sc-20044), cytochrome c (sc-13560), Bax (sc-493) and Bcl-2 (sc-509) were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA).

Techniques: Membrane, Western Blot, Small Interfering RNA, Control, Irradiation, Spectrophotometry, Staining, End Labeling, TUNEL Assay