Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: cAMP inhibits the IR-induced p53 accumulation in an Epac-independent fashion. (A) Reh cells were treated with forskolin or 8CPT-cAMP for 30 minutes before exposure to 10 Gy of IR. Cells were harvested at the indicated times after IR, lysed, and subjected to immunoblot analysis with DO-1 and antiactin antibodies. p53 bands were subsequently quantified by densitometric analysis, and fold increase of p53 band intensity was calculated relative to the unirradiated control. One representative experiment of three is shown. (B) Human peripheral CD4+ T cells, U2OS and MCF-7 were treated with forskolin or 8-CPT-cAMP for 30 minutes before exposure to IR. After 4 hours, cells were harvested, lysed, and subjected to immunoblot analysis with DO-1 and antiactin antibodies. One representative experiment of three is shown. (C) Reh cells were treated with the indicated concentrations of 8-CPT-cAMP or 8-pCPT-2′-O-Me-cAMP for 30 minutes before exposure to IR. After 4 hours, the cells were harvested, lysed, and subjected to immunoblot analysis with DO-1 and antiactin antibodies.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: p53 mRNA steady-state levels are not affected by IR or cAMP. Reh cells were treated with forskolin 30minutes before 10 Gy IR. Cells were harvested at the indicated times after IR, RNA was isolated and subjected to Northern blot analysis with a p53 cDNA probe. RNA isolated formHCT116 p53+/+ and p53-/- cells was used as control for the specificity of the cDNA probe.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Isolation, Northern Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: cAMP inhibits the IR-induced stabilization of the p53 protein. Reh cells were treated with forskolin (60 µM) or 8-CPT-cAMP (200 µM) for 30 minutes before IR. Four hours after IR, cells were treated with cycloheximide (CHX; 25 µg/ml) and then harvested at the indicated times. Whole-cell lysates were prepared and analyzed by immunoblot analysis with DO-1 and antiactin antibodies. Upper panel shows one representative experiment of four. Lower panel: the immunoblots represented in the upper panel were scanned, and the intensity of the p53 protein bands was quantitated and plotted with the value obtained for cells not treated with CHX set as 1. Values were normalized with those of actin (n = 4).

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: cAMP inhibits p53 accumulation in a proteasome-dependent manner and counteracts IR-induced reduction of p53 ubiquitination. (A) Reh cells were preincubated with (upper panel) or without (lower panel) MG-132 for 2 hours before treatment with forskolin or 8-CPT-cAMP for 30 minutes. Cells were then exposed to IR, harvested at the indicated times post-IR, and subjected to immunoblot analysis with DO-1 and antiactin antibodies. Vertical lines have been inserted to indicate repositioned gel lanes. Lower panel is shown as comparative data on the effect of MG-132 on p53 levels. (B) Upper panel: Reh cells were pretreated with MG-132 for 2 hours before addition of forskolin. After 30 minutes, cells were exposed to IR, harvested after 4 hours, and then subjected to immunoblot analysis with DO-1 and antiactin antibodies. Lower panel: Reh cells were treated as described for the upper panel. Whole-cell extracts were prepared and immunoprecipitated (IP) with FL-393 antibody. The recovered proteins were resolved on SDS-PAGE and then subjected to immunoblot analysis with antibodies against ubiquitin. Subsequently, the blot was stripped and then reprobed with DO-1.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Western Blot, Immunoprecipitation, SDS Page

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: The inhibitory effect of cAMP on p53 accumulation requires functional HDM2 and involves inhibition of IR-induced dissociation of the p53-HDM2 complex. (A) Reh cells were treated with Nutlin-3a for 10 minutes before the addition of 8-CPT-cAMP, forskolin, or corresponding volumes of their solvents dH2O or DMSO, respectively. After 30 minutes, cells were exposed to IR and incubated for an additional 4 hours. Whole-cell lysates were then prepared and analyzed by immunoblot analysis with the DO-1 and antiactin antibodies. The immunoblot shows one representative experiment of three. The histogram depicts the average densitometric value of the p53 protein bands. Values from cAMP-treated samples have been normalized to their relevant solvent controls, whose values were set to 100% (n = 3). (B) Reh cells were transfected with control siRNA or siRNA against HDM2. After 24 hours, cells were cultured in the presence or absence of forskolin for 30 minutes before exposure to IR and incubated for an additional 4 hours. Whole-cell lysates were then prepared and analyzed by immunoblot analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2), DO-1, and antiactin antibodies. (C) Reh cells were treated with forskolin for 30 minutes before exposure to IR. Cells were then harvested at the indicated times after IR and subjected to immunoblot analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2) and antiactin antibodies. The immunoblot shows one representative experiment of seven. The immunoblots represented above were scanned, and the intensity of the p53 protein bands was quantitated and plotted with the value obtained for untreated cells set as 1 (n = 7). (D) Reh cells were treated as in C and analyzed by immunoblot analysis with the indicated antibodies. One representative experiment of three is shown. (E) Reh cells were pretreated with MG-132 for 2 hours before addition of forskolin. After 30 minutes of forskolin treatment, cells were exposed to IR and then harvested at the indicated times. Whole-cell extracts were prepared and immunoprecipitated with FL-393. The recovered proteins were resolved on SDS-PAGE and then subjected to immunoblot analysis with the anti-HDM2 (a mixture of SMP14, IF2, and 4B2) and DO-1 antibodies. The immunoblot shows one representative experiment of seven. The intensity of protein bands was quantified densitometrically, and the ratio of signal intensity for HDM2 relative to p53 was calculated and plotted with the value obtained for untreated cells set as 1 (n = 7; at all three time points, there is a significant difference between cells treated with IR alone and those treated with IR + forskolin, P < .05). Panel marked input represents Western blots for HDM2, p53, and actin in cell extracts before immunoprecipitation. Vertical lines have been inserted to indicate repositioned gel lanes.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Functional Assay, Inhibition, Incubation, Western Blot, Transfection, Cell Culture, Immunoprecipitation, SDS Page

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: Effects of cAMP on p53 phosphorylation. Upper panel: Reh cells were treated with MG-132 (20 µM) for 2 hours before addition of forskolin (60 µM). After 30 minutes, cells were exposed to 10 Gy of IR and harvested at the indicated times. For the examination of p53 phosphorylation at S15 and S20, whole-cell lysates were subjected to immunoblot analysis with phospho-specific antibodies against p53 phosphorylated at S15 or S20. Subsequently, the membranes were stripped and reprobed with DO-1 to detect total p53 protein. One representative experiment of five is shown. For detection of p53 phosphorylated at T18, whole-cell lysates were immunoprecipitated with DO-1, and the recovered proteins were immunoblotted with phospho-specific antibodies against p53 phosphorylated at T18. The blot was then stripped and reprobed with total p53 (FL-393) antibody. One representative experiment of four is shown. Lower panel: The immunoblots represented in the upper panel were scanned, and the intensity of protein bands was quantified densitometrically. The ratio of signal intensity for phosphorylated p53 at S15, S20, or T18 relative to total p53 was then calculated, and the obtained values were normalized to the value obtained for cells at time 0 (S15 and S20, n = 5; T18, n = 4).

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Western Blot, Immunoprecipitation

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: Inhibition of p53-induced cell death requires binding of HDM2 to p53. Reh cells were treated with forskolin for 30 minutes before addition of Nutlin-3a or exposure to 10 Gy IR. After 4 hours, a portion of cells were harvested and subjected to immunoblot analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2), DO-1, and antiactin antibodies. The immunoblot shows one representative experiment of four. The remaining cells were analyzed for cell death by PI staining at 20 hours after IR (n = 4, *P < .01, relative to cells treated with IR only).

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques: Inhibition, Binding Assay, Western Blot, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of cAMP Signaling Interferes with Stress-Induced p53 Accumulation in ALL-Derived Cells by Promoting the Interaction between p53 and HDM2 1 2

doi:

Figure Lengend Snippet: Model depicting how cAMP regulates the DNA damage-induced accumulation of p53.

Article Snippet: Antibodies were as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2 (IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX); phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling (Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin (PW8810) from R&D Systems (Minneapolis, MN) and Biomol (Plymouth Meeting, PA), respectively.

Techniques:

Journal: Archives of Biological Sciences

Article Title: Quercetin and lithium chloride modulate Wnt signaling in pluripotent embryonal carcinoma NT2/D1 cells

doi: 10.2298/abs1301201m

Figure Lengend Snippet: Fig. 2. LiCl decreases the proliferation rate of NT2/D1 cells in a p53-dependent manner. (A) MTT proliferation assay of NT2/D1 treated with lithium chloride-7, 10 and 20 mM, for 24 h. The results are shown as per- centages of the negative control, untreated NT2/D1 cells. Values are presented as the means ±S.E.M. of at least three independent experiments. Mean values of relative proliferation rates were compared using Student’s t test. Values of p<0, 05 are presented by *. (B) Western blot analysis of p53 protein expression in NT2/ D1 cells treated with 7, 10 and 20 mM lithium chloride for 24 h. The level of GAPDH was used as a control for equal amounts of input proteins.

Article Snippet: Western blots were performed using anti c-myc (9E10) (Santa Cruz Biotechnology), anti-p53 (DO1) (Gene Spin), anti α-tubulin (DM1A) (Calbi- ochem), and anti-GAPDH (AM20337PU-S) (Acris Antibodies, Inc).

Techniques: Proliferation Assay, Negative Control, Western Blot, Expressing, Control

Journal: Pharmaceuticals

Article Title: Anti-Colorectal Cancer Effects of a Novel Camptothecin Derivative PCC0208037 In Vitro and In Vivo

doi: 10.3390/ph16010053

Figure Lengend Snippet: Effects of PCC0208037 on molecules related to DNA damage in CRC cells. Western blotting analysis was conducted to detect the expression of the battery of proteins that are known to be related to DNA damage, such as γ-H2AX, ATR, p-ATR, ATM, p-ATM, Chk1, p-Chk1, Chk2, p-Chk2, and p-P53 in HCT116 and LS180 cells treated with PCC0208037 (0.01 and 0.03 μM) for 8 h.

Article Snippet: Antibodies against ATM (2873), p-ATM (13050), ATR (13934), p-ATR (2853), Chk1 (2360), p-Chk1 (2348), Chk2 (2662), p-Chk2 (2348), P53 (18032), p-P53 (9284), Bcl2 (15071), β-actin (3700), and GAPDH (5174) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Battery