Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Western blotting of endogenous PACAP in CHO-K1 and neuro2a cells. As shown CHO-K1 had no detectable endogenous PACAP, while neuro2a cells produced endogenous PACAP. (B) The expression of PAC1-YFP and M-PAC1-YFP detected by immunofluorescence. Shown were immunofluorescence results of PAC1-CHO and M-PAC1-CHO cells cultured in DMEM with 0.5% CS-FBS at 37°C overnight, which indicated that both PAC1 and M-PAC1 trafficked normally to the plasma membrane and 0.5% CS-FBS induced no significant receptors endocytosis. (C) Fluorescence densities assays. Shown were the YFP fluorescence densities in the whole cell lysate detected using the Victor3 1420 multi-label counter with excitation (460±30 nm) and emission (535±30 nm), indicating that the expression levels of PAC1-YFP in CHO cells were equal to those of M-PAC1-YFP. (D) Western blotting assays using reductive SDS-PAGE. Western blotting with a goat polyclonal IgG against the C-terminus of PAC1 in the reductive condition showed that there were similar bands with the molecular weight about 160 kD in PAC1-CHO and M-PAC1-CHO, but not in CHO. (E) The cell viabilities of PAC1-CHO and M-PAC1-CHO cells promoted by PACAP. The data were plotted as the fold changes of the treatment without PACAP (0 nM). After the cells were submitted the addition of PACAP (1–100 nM) in the absence of CS-FBS for 24 h, MTT assays showed that PACAP exerted more significant proliferative effects on M-PAC1-CHO than on PAC1-CHO (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO), indicating that the activation level of PAC1 by PACAP was lower than that of M-PAC1. (F) The intracellular cAMP levels induced by PACAP (1–100 nM) in PAC1-CHO and M-PAC1-CHO cells. After the data were plotted as the fold changes of the treatment with 0 nM PACAP, it was shown that the intracellular cAMP levels in M-PAC1-CHO cells induced by PACAP were significantly higher than the intracellular cAMP levels in PAC1-CHO cells induced by PACAP (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Western Blot, Produced, Expressing, Immunofluorescence, Cell Culture, Clinical Proteomics, Membrane, Fluorescence, SDS Page, Molecular Weight, Activation Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) The remaining cell viabilities of PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells 48 h after serum withdrawal. When the data were plotted as the percentage of the initial cell viability without serum withdrawal, it was shown that PAC1-CHO had remaining cell viability (57.34±5.91%) that was significantly higher than that of M-PAC1-CHO (36.96±6.85%) or pcDNA-CHO (37.89±7.11%) (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). (B) The intracellular caspase3 activities after serum withdrawal. The reactions of pcDNA-CHO were considered not result from PAC1 because pcDNA-CHO did not express PAC1 or PACAP; therefore, all the data were plotted as fold changes in pcDNA-CHO. As shown, PAC1-CHO had significantly lower caspase3 activity than M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO), whereas there was no significant difference between M-PAC1-CHO and pcDNA-CHO. (C) The intracellular Bcl-2 levels after serum withdrawal. After the data were plotted as the fold changes of pcDNA-CHO, it was shown that PAC1-CHO had significantly higher Bcl-2 level about 2 folds of that in M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments. (D) The detection of β-catenin, cyclin D1 and c-myc levels in PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells by western blotting. The western blotting results and the statistical analysis showed that the levels of β-catenin, cyclin D1 and c-myc (tow targets of β-catenin) in PAC1-CHO cells were significantly higher than those in M-PAC1-CHO or pcDNA-CHO cells (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). These findings indicated that overexpression of wild type PAC1 endowed CHO with anti-apoptotic activities against serum withdrawal, suggesting that PAC1 had ligand independent basal activity, while M-PAC1 did not. And Wnt/β-catenin signals were involved in the anti-apoptotic activity of PAC1-CHO. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Activity Assay, Western Blot, Over Expression

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Knockdown of endogenous PACAP and PAC1 with shRNA in Neuro2a. Western blotting assays showed that shRNA against PACAP significantly diminished the expression of endogenous PACAP in neuro2a/PACAP - , and further transfection with shRNA plasmids against PAC1 (+) to neuro2a/PACAP - cells decreased the PAC1 levels significantly, while control plasmids (-) did not interfere with expression of PAC1. The knockdown of PACAP and PAC1 in neuro2a produced a chance for the detection of the correlation of PAC1 down-regulation with its ligand independent basal activity. (B) The remaining cell viabilities of nero2a/PACAP - transfected with PAC1 shRNA plasmids (+) or control plasmid (-). After the data were plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that down-regulation of PAC1 with PAC1 shRNA plasmids (+) decreased the remaining cell viabilities to almost a half of the remaining cell viabilities transfected with control plasmids (-) 48 h after serum withdrawal (*, P<0.01, shRNA + vs. shRNA-). (C) Western blotting of β-catenin, cyclin D1 and c-myc in the nero2a/PACAP - cells transfected with PAC1 shRNA plasmids (+) or control plasmids (-). After the relative protein levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that PAC1 shRNA plasmids (+) significantly decreased the levels of β-catenin, cyclin D1 and c-myc compared with control plasmids (+)(*, P<0.01, shRNA+ vs. shRNA-). These data suggested that down-regulation of PAC1 in the natural cells such neuro2a with high expression of PAC1 inhibited the anti-apoptotic activities in the ligand free condition. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Knockdown, shRNA, Western Blot, Expressing, Transfection, Control, Produced, Activity Assay, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

doi: 10.1073/pnas.1801772115

Figure Lengend Snippet: Identification and validation of N6FFA as a modulator of N17 phosphorylation. Cells were methanol fixed and stained with a primary conjugate antibody recognizing N17 with phospho-groups at S13 and S16 (N17-S13pS16p). Five images per well were taken with a 40× air objective. Images were analyzed by PhenoRipper open source software. A unitless PCA plot and a summary of hits are presented in A and B, respectively. Compound 134, indicated by a red arrow, represents vehicle control; compound 50, encircled in green, represents N6FFA. (C) Chemical structure of N6FFA. (D and E) STHdhQ7/Q7 (D) and STHdhQ111/Q111 (E) cells were treated with the indicated concentrations of N6FFA for 24 h, and total cell lysates were separated by SDS/PAGE followed by immunoblotting with N17-S13pS16p antibody. Pixel intensity results from three independent replicates were quantified. Asterisks indicate concentrations of N6FFA that significantly increased N17 phosphorylation compared with control as determined by an unpaired two-tailed t test (*P < 0.05). Bars represent mean values ± SEM. (F) Mouse cortical neurons were transfected with N586-HttQ22 or N586-HttQ82 and were treated with various concentrations of N6FFA. A nuclear condensation assay was performed to determine percent of cell death. Asterisks indicate concentrations of N6FFA that significantly decreased cell death as determined by an unpaired two-tailed t test from three independent replicates (*P < 0.05; **P < 0.001).

Article Snippet: Additional antibodies used in this study were anti–N17-S13pS16p (New England Peptides), anti-CK2 alpha (Abcam), and anti-N6FFA/kinetin riboside (Agrisera) for immunofluorescence and anti-thiophosphate ester (Abcam), anti-GAPDH (Abcam), rabbit anti-huntingtin (PW0595; Enzo Life Sciences), and mouse anti-polyglutamine (mAB1574, Chand mouse anti-huntingtin) for Western blots.

Techniques: Staining, Software, SDS Page, Western Blot, Two Tailed Test, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

doi: 10.1073/pnas.1801772115

Figure Lengend Snippet: CK2 uses KTP as a phospho-donor to phosphorylate huntingtin N17 when primed by phosphorylation. (A and B) Representative blot (A) and quantification (B) of an in vitro N17 phosphorylation assay using CK2 with either ATP or KTP as phospho-donor. **P < 0.001, Mann–Whitney test; n = 3 independent replicates. (C) Representative blot (Upper) and quantification (Lower) of N17-S13pS16p levels in STHdhQ111/Q111 cells upon treatment with N6FFA with or without the addition of the CK2 inhibitor DMAT (4.5 µM). *P < 0.05, Mann–Whitney test (n = 4 independent replicates). (D) Representative blot (Upper) and quantification (Lower) of N17-S13pS16p levels in STHdhQ111/Q111 cells upon treatment with the nonhydrolysable N6FFA derivative 9DK (analysis by Mann–Whitney test) (Left) or treatment with N6FFA (analysis by unpaired t test) (Right). n = 3 independent replicates for both experiments; *P < 0.05. For all data, error bars show mean ± SEM.

Article Snippet: Additional antibodies used in this study were anti–N17-S13pS16p (New England Peptides), anti-CK2 alpha (Abcam), and anti-N6FFA/kinetin riboside (Agrisera) for immunofluorescence and anti-thiophosphate ester (Abcam), anti-GAPDH (Abcam), rabbit anti-huntingtin (PW0595; Enzo Life Sciences), and mouse anti-polyglutamine (mAB1574, Chand mouse anti-huntingtin) for Western blots.

Techniques: In Vitro, Phosphorylation Assay, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

doi: 10.1073/pnas.1801772115

Figure Lengend Snippet: Components of the N6FFA–APRT–KTP–CK2–huntingtin pathway colocalize at sites of DNA damage. (A, Upper) Human fibroblasts were stained with anti-CK2 (green) and anti–N17-S13pS16p (magenta) and were imaged using SR-SIM. (Lower) Magnified views of the boxed areas. (Scale bar: 1 μm.) (B) Human fibroblasts were irradiated with a 405-nm laser in the indicated region to induce DNA damage. After a 20-min incubation period, immunofluorescence was performed against CK2 (green) and N17-S13pS16p (magenta), and the x and y coordinates were revisited to image the irradiated cells by Z-stacked wide-field microscopy and deconvolution. We observed a 100% correlation between CK2 and N17-S13pS16p localization to irradiated regions in two experiments (n = 20–30 cells per experiment). (C) Human fibroblasts were irradiated as in B, stained with anti-N6FFA riboside (green), and imaged by Z-stacked wide-field microscopy and deconvolution. (D) Human fibroblasts were treated with vehicle control (mock) or 10 μM KU55933 for 25 min and then were irradiated as in B, stained with anti-APRT (green) and anti–N17-S13pS16p (magenta), and imaged by Z-stacked wide-field microscopy and deconvolution. (Scale bars: 10 μm in B–D.)

Article Snippet: Additional antibodies used in this study were anti–N17-S13pS16p (New England Peptides), anti-CK2 alpha (Abcam), and anti-N6FFA/kinetin riboside (Agrisera) for immunofluorescence and anti-thiophosphate ester (Abcam), anti-GAPDH (Abcam), rabbit anti-huntingtin (PW0595; Enzo Life Sciences), and mouse anti-polyglutamine (mAB1574, Chand mouse anti-huntingtin) for Western blots.

Techniques: Staining, Irradiation, Incubation, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) BiFC assays. Shown were YFP fluorescence intensity re-produced by the transfection of the receptor constructs as indicated. The cells without transfection were used as negative control and the cells transfected with PAC-YFP as positive control. Exogenous NAC (10 nM) decreased the YFP fluorescence intensity produced by PAC-Y/N+PAC-Y/C significantly (*, P<0.01 PAC-Y/N+PAC-Y/C+NAC vs. PAC-Y/N+PAC-Y/C), while the transfection of M+PAC-Y/N+M+PAC-Y/C produced no YFP fluorescence signals. Data were presented as means ± S.E. of three independent experiments. (B) Saturation BRET. Shown were the BRET saturation curves plotted as a ratio of YFP fluorescence to Rlu luminescence that were observed for tagged receptor constructs studied with a fixed amount of donor and increasing amounts of acceptor. PAC-Rluc/PAC-YFP receptor constructs yielded exponential curves that reached asymptotes indicating significant homo-dimerization of PAC1, while M-PAC-Rluc/M-PAC-YFP yielded curves not different from a straight line, indicating that D-PAC1 lost the ability to form dimers. The addition with NAC (10 nM) at 2 h before the BRET signal assay lowered the curves significantly (*, P<0.01 PAC-Rluc/PAC-YFP+NAC vs. PAC-Rluc/PAC-YFP). The data were represented as the means ± S.E. of three independent experiments. (C) Static BRET. BRET ratios for CHO cells expressing receptor constructs as indicated. For static BRET, a total of 1.0 µg of DNA per well divided equally among the noted constructs in each condition was utilized. The shaded area represents the nonspecific BRET signal generated between PAC-Rlu and soluble YFP protein, with BRET signals above this area considered to be significant. As shown the BRET ratio in PAC-Rluc/PAC-YFP CHO cells incubated with NAC (10 nM) was significantly lower than that in cells without treatment with NAC (*, P<0.01 PAC-Rluc/PAC-YFP+NAC vs. PAC-Rluc/PAC-YFP). The data were presented as the means ± S.E. of three independent experiments. (D) Western blotting analysis with a goat polyclonal IgG against the C-terminus of PAC1 using non-reductive SDS-PAGE. When PAC-YFP expressing cells incubated with exogenous NAC (10 nM), as shown, the band with the molecular weight (about 160 kD) consistent with the molecular weight of the PAC1 dimer was weakened by the presence of NAC (10 nM). All these results showed that NAC was an inhibitor of the dimerization of PAC1, which offered us a tool to analysis the relation of the dimerization of PAC1 with its basal activity.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Fluorescence, Produced, Transfection, Construct, Negative Control, Positive Control, Expressing, Generated, Incubation, Western Blot, SDS Page, Molecular Weight, Activity Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Western blotting of endogenous PACAP in CHO-K1 and neuro2a cells. As shown CHO-K1 had no detectable endogenous PACAP, while neuro2a cells produced endogenous PACAP. (B) The expression of PAC1-YFP and M-PAC1-YFP detected by immunofluorescence. Shown were immunofluorescence results of PAC1-CHO and M-PAC1-CHO cells cultured in DMEM with 0.5% CS-FBS at 37°C overnight, which indicated that both PAC1 and M-PAC1 trafficked normally to the plasma membrane and 0.5% CS-FBS induced no significant receptors endocytosis. (C) Fluorescence densities assays. Shown were the YFP fluorescence densities in the whole cell lysate detected using the Victor3 1420 multi-label counter with excitation (460±30 nm) and emission (535±30 nm), indicating that the expression levels of PAC1-YFP in CHO cells were equal to those of M-PAC1-YFP. (D) Western blotting assays using reductive SDS-PAGE. Western blotting with a goat polyclonal IgG against the C-terminus of PAC1 in the reductive condition showed that there were similar bands with the molecular weight about 160 kD in PAC1-CHO and M-PAC1-CHO, but not in CHO. (E) The cell viabilities of PAC1-CHO and M-PAC1-CHO cells promoted by PACAP. The data were plotted as the fold changes of the treatment without PACAP (0 nM). After the cells were submitted the addition of PACAP (1–100 nM) in the absence of CS-FBS for 24 h, MTT assays showed that PACAP exerted more significant proliferative effects on M-PAC1-CHO than on PAC1-CHO (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO), indicating that the activation level of PAC1 by PACAP was lower than that of M-PAC1. (F) The intracellular cAMP levels induced by PACAP (1–100 nM) in PAC1-CHO and M-PAC1-CHO cells. After the data were plotted as the fold changes of the treatment with 0 nM PACAP, it was shown that the intracellular cAMP levels in M-PAC1-CHO cells induced by PACAP were significantly higher than the intracellular cAMP levels in PAC1-CHO cells induced by PACAP (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Western Blot, Produced, Expressing, Immunofluorescence, Cell Culture, Clinical Proteomics, Membrane, Fluorescence, SDS Page, Molecular Weight, Activation Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: The fluorescence microscopic observation (A) and the fluorescence density assays (B) of the PAC1-YFP expression induced by Dox (0–100 ng/mL) in Tet-on inducible system. The fluorescence microscopic images showed that the numbers of cells with YFP fluorescence increased following increases in the concentration of Dox (1–100 ng/mL), whereas there was no fluorescence observed without induction by Dox (0 ng/mL). Bar, 20 µm. The YFP fluorescence densities, assayed using the Victor3 1420 multi-label counter, increased with the concentration of Dox (1–100 ng/mL), indicating that the expression levels of PAC1 were controlled by Dox in a concentration-dependent manner. (C) Western blotting of the inducible expression of PAC1-YFP. The western blotting with goat polyclonal IgG against the C-terminus of PAC1 using reductive SDS-PAGE showed the bands corresponding to PAC1-YFP deepened with the increase of Dox (1–100 ng/mL), while no band corresponding to PAC1-YFP was found in the treatment without Dox (0 ng/mL). The remaining cell viabilities (D), the caspase3 activity (E) and the Bcl-2 levels (F) after serum withdrawal in the double-stable Tet-on advanced inducible cells treated with Dox (1–100 ng/mL) were plotted as the fold changes in the data from the cells treated without Dox (0 ng/mL). It was shown that the higher concentrations of Dox induced higher expression levels of PAC1-YFP, which in turn led to the higher anti-apoptotic activity of the cells, including higher remaining cell viability, lower caspase3 activity and higher Bcl-2 level. (G) Top-flash assays. In double-stable Tet-on advanced inducible cells, after the transfection with Top-flash + pRluc or Fop-flash + pRluc, cells were submitted to serum-withdraw induced apoptosis with Dox (1–100 ng/mL) or without Dox for another 24 h. And then cells were lysed and luciferase activities were measured. Relative luciferase activities were expressed as the ratio of TOP-flash/FOP-flash luciferase activity and the data were plotted as fold changes in the data from the cells treated without Dox (0 ng/mL). It was shown that the relative luciferase activities increased following the increase of Dox (1–100 ng/mL), indicating that the higher expression levels of PAC1-YFP induced by higher concentration of Dox resulted into stronger Wnt/β-catenin signals. (H) Western blotting of β-catenin, cyclin D1 and c-myc corresponding to Wnt/β-catenin pathway. After the protein expression levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes of the cells treated without Dox, it was shown that in the cells expressing a range of PAC1-YFP induced by Dox (0–100 ng/mL), β-catenin, cyclin D1 and c-myc levels increased following the increases of the PAC1 levels. All these data suggested the significant positive correlation of the PAC1 levels with the anti-apoptotic activities involved with Wnt/β-catenin signals. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Fluorescence, Expressing, Concentration Assay, Western Blot, SDS Page, Activity Assay, Transfection, Luciferase, Control

Journal: The Journal of Biological Chemistry

Article Title: Molecular Cloning and Characterization of First Organic Matrix Protein from Sclerites of Red Coral, Corallium rubrum *

doi: 10.1074/jbc.M112.352005

Figure Lengend Snippet: Silver staining of electrophoresis gels and Western blots on organic matrix extracted from sclerites of C. rubrum . a , one-dimensional SDS-PAGE (10 μg protein/lane; BisTris 12% polyacrylamide gel, silver stain molecular mass marker (M6539; Sigma) as protein marker). b , one-dimensional SDS-PAGE (10 μg protein/lane, Tris-Tricine 16.5% polyacrylamide gel, kaleidoscope polypeptide standards (161-0325; Bio-Rad) as protein marker); c , two-dimensional electrophoresis gel (Tris-Tricine 16.5% polyacrylamide gel, kaleidoscope polypeptide standards (161-0325) as protein marker) of the major protein band (18*) excised from one dimensional SDS-PAGE. d , Western blot (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC TM standard (161-0376; Bio-Rad) as protein marker) with antibodies against phosphorylated serine. e , Western blot with antibodies against the phosphorylated threonine (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC TM standard (161-0376) as protein marker). f , Western blot with antibodies against phosphorylated tyrosine (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC TM standard (161-0376) as protein marker). g , Western blot with anti-scleritin antibodies (Tris-Tricine 16.5% polyacrylamide gel, Precision Plus Protein WesternC TM standard (161-0376) as protein marker). MW , molecular mass; M , protein marker; and triangles show apparent molecular mass of proteins (in kDa).

Article Snippet: The protein markers used were: Silver Stain Molecular Weight Marker (M6539; Sigma) for the BisTris silver-stained electrophoresis gel, Kaleidoscope Polypeptide Standards (Bio-Rad 161-0325) for the Tris-Tricine silver-stained electrophoresis gels, and Precision Plus Protein WesternC TM standard (161-0376 Bio-Rad) both for the BisTris and Tris-Tricine gels of Western blots.

Techniques: Silver Staining, Electrophoresis, Western Blot, SDS Page, Marker

Journal: PLoS ONE

Article Title: A Prokaryotic S1P Lyase Degrades Extracellular S1P In Vitro and In Vivo : Implication for Treating Hyperproliferative Disorders

doi: 10.1371/journal.pone.0022436

Figure Lengend Snippet: (A) Quiescent MCF-7 cells were treated for 10 min with either vehicle (DMEM, -) or S1P (1 µM) in the absence or presence of WT StSPL (StSPL; 10 µg/ml) or the K311A mutant (10 µg/ml). Cell lysates were prepared and separated by SDS-PAGE, transferred to nitrocellulose and subjected to Western blotting using antibodies against phospho-p42/p44 (dilution of 1∶1000, upper panel) and total p42/p44-MAPK (dilution each 1∶6000, lower panel). (B) Quiescent MCF-7 cells were treated for 24 h with either vehicle (Co) or S1P (1 µM), which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL; 10 µg/ml) or the K311A mutant (10 µg/ml), in the presence of [ 3 H]thymidine. Incorporated radioactivity was measured as described in the section. Results are expressed as cpm/well of incorporated [ 3 H]thymidine and are means ±S.D. (n = 4). (C) Quiescent MCF-7 cells were treated for 24 h with DMEM (Co) or S1P (1 µM), which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL) or the K311A mutant. Thereafter, migrated cells were analysed as described in the section. Results are expressed as migrated cells per counted field and are means ±S.D. (n = 3). (D) Quiescent MCF-7 cells were treated for 24 h with DMEM (Co) or S1P (1 µM) which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL; 10 µg/ml), or the K311A mutant (10 µg/ml). Thereafter, supernatants were taken for a VEGF ELISA. Results are expressed as pg/ml of VEGF and are means ±S.D. (n = 4). *p<0.05, ***p<0.001 considered statistically significant when compared to the vehicle treated control values; # p<0.05, ### p<0.001 when compared to the S1P-treated values.

Article Snippet: Secretion of vascular endothelial growth factor (VEGF) into cell culture medium was quantified by ELISA (R&D Systems Europe Ltd., Abingdon, U.K.) as recommended by the manufacturer.

Techniques: Mutagenesis, SDS Page, Western Blot, Radioactivity, Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: A Prokaryotic S1P Lyase Degrades Extracellular S1P In Vitro and In Vivo : Implication for Treating Hyperproliferative Disorders

doi: 10.1371/journal.pone.0022436

Figure Lengend Snippet: (A) Quiescent HCT 116 cells were treated for 10 min with either vehicle (DMEM, -) or S1P (1 µM) in the absence or presence of WT StSPL (StSPL; 10 µg/ml) or the K311A mutant (10 µg/ml). Cell lysates were prepared and separated by SDS-PAGE, transferred to nitrocellulose and subjected to Western blotting using antibodies against phospho-p42/p44 (dilution of 1∶1000, upper panel) and total p42/p44-MAPK (dilution each 1∶6000, lower panel). (B) Quiescent HCT 116 cells were treated for 28 h with either vehicle (Co) or S1P (1 µM), which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL; 10 µg/ml) or the K311A mutant (10 µg/ml), in the presence of [ 3 H]thymidine. Incorporated radioactivity was measured as described in the section. Results are expressed as cpm/well of incorporated [ 3 H]thymidine and are means ±S.D. (n = 4). (C) Quiescent HCT 116 cells were treated for 14 h with DMEM (Co) or S1P (1 µM), which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL) or the K311A mutant. Thereafter, migrated cells were analysed as described in the section. Results are expressed as migrated cells per counted field and are means ±S.D. (n = 3). (D) Quiescent HCT-116 cells were treated for 14 h with DMEM (Co) or S1P (1 µM) which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL; 10 µg/ml), or the K311A mutant (10 µg/ml). Thereafter, supernatants were taken for a VEGF ELISA. Results are expressed as pg/ml of VEGF and are means ±S.D. (n = 4). *p<0.05, ***p<0.001 considered statistically significant when compared to the vehicle treated control values; # p<0.05, ### p<0.001 when compared to the S1P-treated values.

Article Snippet: Secretion of vascular endothelial growth factor (VEGF) into cell culture medium was quantified by ELISA (R&D Systems Europe Ltd., Abingdon, U.K.) as recommended by the manufacturer.

Techniques: Mutagenesis, SDS Page, Western Blot, Radioactivity, Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene . (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, A3A shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA APOBEC target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.

Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

Techniques: Mutagenesis, Expressing, In Vitro, CRISPR, shRNA, Activity Assay, Plasmid Preparation, Incubation, Quantitative RT-PCR, Western Blot

Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: The average mutation profiles of (A) 14 non-APOBEC-mutated and (B) 14 APOBEC-mutated BRCA cell lines. Specific cell lines in each category are defined in . (C) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression was measured by qRT-PCR in non-APOBEC-mutated (N) and APOBEC-mutated (M) BRCA cell lines. Similar results were obtained comparing APOBEC expression to TBP . Each circle represents the mean of 3 replicate measurements for an individual cell line. Horizontal bars indicate the median expression for each APOBEC3 family member among the non-APOBEC-mutated or APOBEC-mutated cell lines. Data points corresponding to cell lines without detectable expression of individual APOBECs are not shown on the graph but are included in the calculation of the median. Statistical significance for differences in the expression of a given APOBEC family member between non-APOBEC-mutated and APOBEC-mutated lines was assessed by Mann-Whitney Summed Rank test. ** indicates p = 0.0067. Correlations between A3A expression (blue dots) or A3B expression (red dots) measured by qRT-PCR and the minimum estimate of APOBEC-induced mutations for each of the 28 BRCA cell lines were determined by a Pearson correlation test using mutation lists obtained from (D) the Cancer Cell Line Encyclopedia and (E) the Catalogue of Somatic Mutations in Cancer (COSMIC).

Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: (A) The mutation profile of BT474 cells. (B) The number of APOBEC-induced (black bars) and non-APOBEC-induced (grey bars) mutations in BT474 cells. (C) mRNA expression levels of individual APOBEC3 family members relative to HPRT1 expression in BT474 as measured by qRT-PCR. Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean of these measurements. n.d. indicates “not detected.” (D) In vitro cytidine deaminase assay (conducted similarly to ) of whole-cell extracts generated BT474 cells or BT474 cells transduced with lentiviral vectors to express scramble control, A3A-targeting, or A3B targeting shRNAs. Deaminase reactions were supplemented with either 2 units UGI (NEB #M0281S) or an equal volume of 50% glycerol. Specificity of each shRNA was confirmed by qRT-PCR, and equal protein amounts used in deaminase assays were verified by α-GAPDH western.

Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, In Vitro, Generated, Transduction, shRNA, Western Blot

Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: (A) In vitro cytidine deaminase assays conducted similarly to those in , but using extracts for BT474, CAMA-1 and MDA-MB-453 cells transduced to express scramble (Scram) shRNA controls or A3A- and A3B-targeted shRNAs. Prior to incubating the extracts with the hairpin DNA substrate, extracts where either mock-treated or treated with 2.5 μM RNaseA at 37°C for 30 min. (B) Coomassie stained SDS-PAGE showing the purity of A3A (23 kDa) and A3B (42 kDa) over-expressed in and purified from HEK293T cells. (C) The activity of purified A3A and A3B was compared in vitro by incubating 0.25 μM hairpin DNA substrate with decreasing concentrations of A3A or A3B for 30 min at 37°C in the presence of uracil DNA glycosylase. Reactions were heated at 95°C for 10 min in formamide loading buffer prior to resolving on a denaturing polyacrylamide gel. (D) Increasing amounts of total RNA isolated from BT474 cells were added to in vitro cytidine deaminase assays containing 100 nM of either A3A or A3B and 0.25 μM of hairpin DNA substrate to assess the relative impact of RNA on A3A and A3B activity. Additionally, reactions containing 100 ng/μL of RNA were either mock treated or treated with RNaseA to ensure inhibition of cytidine deaminase activity was due to interactions with RNA. (E) To assess RNA inhibition of A3A and A3B at similar enzymatic activities, 0.8 nM A3A and 100 nM A3B where incubated with 0.25 μM of hairpin DNA substrate and increasing RNA concentrations. Samples in (D) and (E) were processed as in (C).

Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

Techniques: In Vitro, shRNA, Staining, SDS Page, Purification, Activity Assay, Isolation, Inhibition, Incubation

Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: (A) The pairwise alignment of the A3A (blue) and A3B (red) transcripts shows a central region of high sequence identity with two unique regions in each transcript. Green, yellow, and red indicate 100%, ≥30%, and <30% identity respectively. Arrows indicate the position of qRT-PCR primers. (B) Correlations between qRT-PCR measurements of A3A or A3B mRNA abundance and corresponding RSEM normalized RNA-seq measurements or RNA-seq measurements limited to reads mapping within the defined unique regions of A3A and A3B transcripts (unambiguous RNA-seq) for BRCA cell lines were assessed by Pearson correlation test. A3A and A3B expression measured by RSEM normalized RNA-seq or unambiguous RNA-seq was compared to the minimum estimate of APOBEC-induced mutations in (C) 973 TCGA sequenced primary BRCA tumors or (D) a subset of 229 APOBEC-mutated TCGA sequenced primary BRCA tumors by Pearson correlation test.

Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

Techniques: Sequencing, Quantitative RT-PCR, RNA Sequencing Assay, Expressing

Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: RSEM normalized RNA-seq values for A3A (blue) and A3B (red) were obtained for 410 bladder (BLCA), 197 cervical (CESC), and 549 head and neck (HNSC) cancers assessed by the Cancer Genome Atlas. Expression of each APOBEC was normalized to HPRT1 and compared to the minimum estimate of APOBEC-induced mutations in the corresponding samples. A3A expression strongly correlates with mutagenesis in each tumor type by Pearson correlation analysis even when only evaluating APOBEC mutated tumors.

Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

Techniques: RNA Sequencing Assay, Expressing, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 1. Regardless of p53 status, G0/G1 synchronized HCT116 cells traverse into and accumulate in S phase in response to de novo purine synthesis inhibition by DDATHF. Human colon car- cinoma HCT116 cells with p53 wild-type (p53/) or null (p53/) status were synchronized in G0/G1 and analyzed by two parameter flow cytometry prior to release from synchrony (A) into media containing 65 M BrdU and either PBS (B), 10 M DDATHF (C), or 400 M PALA (D). After 48 h in drug and BrdU, cells were fixed and stained with pro- pidium iodide and anti-BrdU FITC, and cell cycle progression was monitored via flow cytometry, gating out any sub-G1 debris.

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Inhibition, Flow Cytometry, Staining

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 2. Despite the increase in p53 expression induced by pu- rine nucleotide depletion, the immediate downstream tran- scriptional target p21 fails to accumulate in human carcinoma cells. A, Western blot analysis of p53 and p21 expression in HCT116 p53/ and p53/ cells exposed to 10 M DDATHF for the indicated time. Cell lysate protein (20 g) was subjected to SDS-PAGE and immunoblotting with antibodies against p53 or p21. The first lane of each panel was loaded with protein from HCT116 p53/ cells exposed to 10 M etoposide (VP16) for 24 h. Western blots were stripped and reprobed with an actin antibody. B, HCT116 p53/ cells were treated with either 10 M VP16 for 24 h, or exposed to 12 or 15 Gy irradiation and harvested after 3.5 h, and Western blotting was performed. C, Western blot analysis of p53 protein levels in HCT116 p53/ cells upon treatment with 10 M DDATHF in the presence or absence of the purine salvage metabolite inosine (50 M). The first two lanes were loaded with lysates from p53/ cells treated with 10 M VP16, or untreated. D, cell lysates of human breast carcinoma cells (MCF7) and human lung carcinoma cells (A549) were prepared after treatment with 10 M DDATHF for the indicated periods, and Western blot analysis was conducted. Actin levels were equal in D.

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Expressing, Western Blot, SDS Page, Irradiation

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 3. The transcriptional activat- ing and repressing functions of p53 are impaired after inhibition of de novo purine synthesis by DDATHF. A, Northern blot analysis of p21 and mdm2 induction following treatment of HCT116 p53/ cells with 10 M DDATHF. Total RNA was isolated after the indicated times of DDATHF exposure and 2 g poly(A) RNA were subjected to Northern blotting. The sample in the first lane was 2 g of mRNA from HCT116 p53/ cells treated with 10 M VP16 for 24 h. The blot was stripped and reprobed for gapdh. B, RPA of p53-regulated genes following exposure of p53/ cells to DDATHF. Twenty micrograms of total RNA were hybridized to a probe synthe- sized from a stress-inducible multiprobe template. Shown in the first lane are the migration positions of the unhybridized probe and, in the last lane, yeast tRNA hybridized with probe as a test of speci- ficity of hybridization. For details, see “Experimental Procedures.” C, Western blot analysis of p53 downstream tran- scriptional targets Mdm2, Gadd45, and Bax was performed on lysates of HCT116 p53/ and p53/ cells exposed to 10 M DDATHF for the indicated times. Stripping and reprobing of the blots with actin was used to assess protein loading.

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Inhibition, Northern Blot, Isolation, Migration, Hybridization, Western Blot, Stripping Membranes

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 4. DDATHF-induced stabilization of p53. A, Western blot displaying p53 protein levels following inhibition of protein synthesis. HCT116 p53/ cells were treated with 10 M DDATHF for 8 h, or were exposed to 15 Gy ionizing radiation and incubated for 2.5 h, at which time 20 g/ml cycloheximide was added. Lysates were prepared at the indicated times and Western blotting was performed with the p53 antibody Ab-6. B, in vivo ubiquitination assay showing laddering of ubiquitinated p53 conjugates. HCT116 p53/ or p53/ cells were either untreated or exposed to 10 M DDATHF for 6 or 12 h, or to 10 M VP16 for 24 h. p53 was immunoprecipitated from whole cell lysates and ubiquitination was assessed by Western blotting with a p53 antibody, Ab-6. The heavy (H) and light (L) chains of the immunoprecipitating antibody are indicated.

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Western Blot, Inhibition, Incubation, In Vivo, Ubiquitin Proteomics, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 5. Synchronous entrance of HCT116 cells into S phase correlates with an elevation in p53 protein and an increase in p53 nuclear staining. p53/ cells were released from a G0/G1 synchrony into media containing either PBS or DDATHF, and cells were collected and analyzed via Western blotting, flow cytometry, or immunofluorescence. A, cell lysates were harvested at the indicated times after release from synchrony, and p53 or p21 protein expression was deter- mined by immunoblotting. In the first lane are lysates from asynchronous p53/ cells treated with VP16, which demonstrate a positive response to p53 and p21. B, cells were collected and stained with propidium iodide, and DNA content was assessed by flow cytometry. C, cells were fixed and stained with p53 antibody (Ab-6) and imaged at 200 mag- nification. D, HCT116 p53/ cells were treated with VP16 for 24 h, fixed, and stained with the p53 antibody Ab-6. E, untreated HCT116 p53/ cells were fixed and stained with p53 antibody, as shown in the left panel, and imaged at 300 magnification. The right panel is a phase contrast image.

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Staining, Western Blot, Flow Cytometry, Immunofluorescence, Expressing

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 6. GART inhibitors, as a class, induce p53 accumulation without concomitant p21 induction, but this phenomenon is not common to all antifolates. A, Western blot analysis of p53 and p21 expression following exposure of HCT116 p53/ cells to the second generation GART inhibitors LY309887 (10 M) and AG2034 (10 M), and the third generation GART inhibitor, AG2037 (10 M). B, Western blot analysis of p53 and p21 protein levels after treatment of HCT116 p53/ cells with the thymidylate synthase inhibitor, D1694 (10 M), or the dihydrofolate reductase inhibitor, methotrexate (10 M).

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 7. Analysis of the post-translational modifications of the p53 that accumulates following GART inhibition indicates lack of serine phosphorylation and lack of lysine acetylation. A, HCT116 p53/ cells were exposed to either 10 M DDATHF, 10 M VP16, 10 M D1694, or 10 M methotrexate for the indicated times. SDS-PAGE and immunoblotting were performed on 25 g of protein, phosphorylation of p53 was determined by probing with an antibody specific to phosphorylated p53 at serine residue 15, and the membranes were stripped and reprobed for actin. B, cells were treated with 10 M DDATHF (12 h), 10 M VP16 (24 h), or irradiated at 15 Gy and harvested after 3.5 h. A preliminary Western blot was performed to quantitate total p53 levels, and equivalent amounts of p53-containing lysate were subsequently loaded and subjected to immunoblotting with antibodies specific for p53 phosphorylated at serines 6, 15, or 20. C, immunoprecipitation with an antibody to p53 was performed on HCT116 p53/ cell lysates prior to immunoblotting. p53 protein lev- els were determined, and equal amounts of p53-containing immunopre- cipitate were subjected to Western blotting. Acetylation of C-terminal residues of p53 was examined utilizing an acetylation-specific antibody to lysines 373/382. Blots were stripped and reprobed with an antibody to p53 to confirm p53 loading.

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Inhibition, Phospho-proteomics, SDS Page, Western Blot, Residue, Irradiation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 8. The p53 accumulating in the nucleus after GART inhibition is capable of binding an oligonucleotide containing the p53 binding element of the p21 promoter. A, EMSA examining the in vitro binding of p53 to its 5 consensus element on the p21 promoter. The first three lanes show binding of recombinant p53, while the remaining lanes show binding of p53 extracted from nuclei of DDATHF or VP16-treated HCT116 p53/ cells exposed to drug as in Fig. 2. The bottom of the film, which had bands indicating free probe and nonspecific binding, is not shown. B, EMSA displaying the specificity of binding to the p21 oligonucleotide. The top gel shows data from VP16-treated nuclei, and the bottom gel, from DDATHF-treated nuclei. Either 20- or 40-fold molar excess of cold wild-type or mutant p21 oligonucleotide were added simultaneously with 32P labeled wild-type p21 probe. Specific p53 binding is supershifted by the addition of the p53 antibody Ab-6. For details, see “Experimental Procedures.”

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Inhibition, Binding Assay, In Vitro, Recombinant, Mutagenesis, Labeling

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 9. DDATHF-stabilized binds the p21 promoter in vivo, but histone acetylation at the p21 promoter is compromised. A, sche- matic of the p21 promoter, indicating the 5 and 3 p53 consensus binding motifs. The pentameric p53 binding elements (43) in the p21 promoter are underlined. The arrows depict relative positions of the PCR primers flanking the two p53 binding sites. B and C, in vivo ChIP analysis of the p21 promoter in HCT116 p53/ and p53/ cells. HCT116 p53/ cells were treated with 10 M DDATHF for 12 h, 10 M VP16 for 24 h, or p53/ and p53/ cells were left untreated. Protein-DNA complexes were cross-linked, and the chromatin was sheared and subjected to im- munoprecipitation with antibodies to p53 (B), RNA polymerase II, CBP/ p300, or acetyl-lysine histone H3 (C) as described under “Experimental Procedures.” DNA was subjected to semi-quantitative and real-time PCR using primers flanking the 5 (black bars) or 3 (gray bars) p53 binding elements on the p21 promoter. For semi-quantitative analysis, the ratio of immunoprecipitated DNA to input DNA is indicated beneath each gel. For real-time analysis, fold induction or binding of the indicated proteins to the p21 promoter is shown, along with standard deviation. For details, see “Experimental Procedures.”

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: In Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition

doi: 10.1074/jbc.m304844200

Figure Lengend Snippet: FIG. 10. Defects in the p53 response following GART inhibition by DDATHF. DDATHF inhibits the enzyme GART, resulting in inhi- bition of de novo purine synthesis and a reduction in ATP and GTP. These decreases in ribonucleotide pools may be sensed by p53, causing stabilization and accumulation of a p53 protein that is hypophosphory- lated at key N-terminal serine residues, such as serines 6, 15, and 20, and hypoacetylated at C-terminal lysines 373/382. Despite its lack of post-translational modifications, this p53 species is capable of nuclear retention and p21 promoter binding, but there is a lack of chromatin acetylation at the p21 promoter, notably of histone H3, and a failure to stimulate transcription.

Article Snippet: For the phosphorylation-specific antibodies, 100 g or greater lysate (digested with 40 g/ml DNase I for 10 min at room temperature) were loaded, and polyclonal antibodies against p53 peptides with phosphorylated residues were serine 6 (AnaSpec) at 1:500, serine 15 (Cell Signaling) at 1:1000, and serine 20 (Santa Cruz Biotechnology) at 1:200.

Techniques: Inhibition, Binding Assay