Journal: Oncogene

Article Title: Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

doi: 10.1038/sj.onc.1209126

Figure Lengend Snippet: Figure 1 Different outcomes of the stress response in NIH3T3 cells. (a) FACS analysis: differently irradiated NIH3T3 cells were harvested at the indicated time points after irradiation and stained with propidium iodide to monitor DNA content. (b) Western blot: lysates derived from irradiated NIH3T3 cells at the indicated time points after irradiation were used to monitor the presence of cleaved caspase 3 and its substrate cdc6 as markers of apoptosis. Equal protein loading was confirmed by tubulin expression. (c) X-Gal staining: activity of senescence-associated b-galactosidase was monitored in untreated or 20 Gy gamma-irradiated NIH3T3 cells, harvested 12 days after irradiation. (d) Western blot: lysates from untreated () or irradiated ( þ ) NIH3T3 cells, harvested 8 h after irradiation, were probed for p53 and serine 18 phosphorylated p53. Equal loading is shown by a non-specific band of the anti p53- serine18-P antibody (control).

Article Snippet: Antibodies were obtained from Upstate: Bax (06- 499); Biomol: Cytochrome c (SA-226); Cell Signaling: Cleaved caspase 3 (9661) and Phospho-p53-Ser15 (9284); BD Pharmingen: p21 (556430); Oncogene: PIG3 (PC268) and Tubulin (CP06); Imgenex: Puma (IMG-459); Santa Cruz: 14-3-3s (sc7681).

Techniques: Irradiation, Staining, Western Blot, Derivative Assay, Expressing, Activity Assay, Control

Journal: Oncogene

Article Title: Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

doi: 10.1038/sj.onc.1209126

Figure Lengend Snippet: Figure 2 Influence of p53 and p21 in the response to 20 Gy gamma-irradiation. (a) Western blot: lysates derived from NIH3T3 (wtp53), 10-1 (p53/) and Balb/c3T3tx (mutp53) cells were analysed for p21 expression at the indicated time points after 20 Gy gamma-irradiation. C indicates a control lysate from NIH3T3 cells. Tubulin expression confirmed equal protein loading. (b, d) Western blot: the presence of cleaved caspase 3 was monitored in lysates derived from cells that were harvested 24 h after mock-treatment () or 20 Gy gamma-irradiation ( þ ). MEFs with different genotype (b) and cell lines 10-1 and Balb/c3T3tx (d) were analysed. (c, e) FACS analysis: DNA content was monitored in untreated or 20 Gy gamma-treated MEFs of the indicated genotype (c) and cell lines 10-1 and Balb/c3T3tx (e). Cells were harvested 24 h after irradiation.

Article Snippet: Antibodies were obtained from Upstate: Bax (06- 499); Biomol: Cytochrome c (SA-226); Cell Signaling: Cleaved caspase 3 (9661) and Phospho-p53-Ser15 (9284); BD Pharmingen: p21 (556430); Oncogene: PIG3 (PC268) and Tubulin (CP06); Imgenex: Puma (IMG-459); Santa Cruz: 14-3-3s (sc7681).

Techniques: Irradiation, Western Blot, Derivative Assay, Expressing, Control

Journal: Oncogene

Article Title: Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

doi: 10.1038/sj.onc.1209126

Figure Lengend Snippet: Figure 3 Expression level of p53 and p53 targets in irradiated NIH3T3 cells. (a-c) Western blot: expression level of anti- and pro- apoptotic p53 targets was analysed in lysates from NIH3T3 cells at the indicated time points after irradiation. Equal protein loading was confirmed by tubulin expression. (b) Puma was detected in lysates of non-irradiated () or irradiated ( þ ) NIH3T3 cells 8 h after irradiation. Equal protein loading is shown by a non-specific band of the anti-p53-ser18-P antibody (control). (c) Lysates of differently irradiated NIH3T3 cells were probed for total p53, serine 18 phosphorylated p53 and Mdm2 at the indicated time points after irradiation.

Article Snippet: Antibodies were obtained from Upstate: Bax (06- 499); Biomol: Cytochrome c (SA-226); Cell Signaling: Cleaved caspase 3 (9661) and Phospho-p53-Ser15 (9284); BD Pharmingen: p21 (556430); Oncogene: PIG3 (PC268) and Tubulin (CP06); Imgenex: Puma (IMG-459); Santa Cruz: 14-3-3s (sc7681).

Techniques: Expressing, Irradiation, Western Blot, Control

Journal: Oncogene

Article Title: Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

doi: 10.1038/sj.onc.1209126

Figure Lengend Snippet: Figure 4 High p53 amounts provide an apoptotic signal. (a) Western blot: 18 h after transfection with GFP or HA-wtp53, NIH3T3 cells were mock () or 20 Gy gamma ( þ ) irradiated and harvested 26 h after irradiation. Lysates were probed for cleaved caspase 3, p53 and p21. Equal protein loading was confirmed by analysis of tubulin expression. (b) Immunostaining: 18 h after transfection with HA-wtp53 or -Gal-GFP, NIH3T3 cells were 20 Gy gamma-irradiated and fixed 26 h after irradiation. Transfected cells were identified with an HA-specific antibody or by GFP-fluorescence. Apoptotic cells were detected by staining of cleaved caspase 3, nuclei were stained with DRAQ5.

Article Snippet: Antibodies were obtained from Upstate: Bax (06- 499); Biomol: Cytochrome c (SA-226); Cell Signaling: Cleaved caspase 3 (9661) and Phospho-p53-Ser15 (9284); BD Pharmingen: p21 (556430); Oncogene: PIG3 (PC268) and Tubulin (CP06); Imgenex: Puma (IMG-459); Santa Cruz: 14-3-3s (sc7681).

Techniques: Western Blot, Transfection, Irradiation, Expressing, Immunostaining, Staining

Journal: Oncogene

Article Title: Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

doi: 10.1038/sj.onc.1209126

Figure Lengend Snippet: Figure 5 Cytosolic p53 and Bax-activation in irradiated NIH3T3 cells. (a) Western blot: lysates from NIH3T3 cells were probed for cleaved caspase 3 at the indicated time points after 100 J/m2 UV-irradiation. Equal protein loading was confirmed by tubulin expression. (b) Western blot: NIH3T3 cells were irradiated as indicated and nuclear and cytosolic fractions were prepared 8 h after irradiation. Lysate (25 mg) were probed for p53. Purity of the fractions was confirmed by monitoring SAF-A and cytochrome c. (c–e) Confocal images: mitochondria and Bax were stained in untreated and irradiated NIH3T3 cells at different time points after irradiation. Representative cells are depicted. Bars are 5 mm. (c, d) Cells were fixed at different time points after 100 J/m2 UV- irradiation, either in an apoptotic (26 h) or pre-apoptotic state (8 and 12 h). (e) Gamma-irradiated cells (20 Gy) were fixed at the time of maximal Bax expression (26 h).

Article Snippet: Antibodies were obtained from Upstate: Bax (06- 499); Biomol: Cytochrome c (SA-226); Cell Signaling: Cleaved caspase 3 (9661) and Phospho-p53-Ser15 (9284); BD Pharmingen: p21 (556430); Oncogene: PIG3 (PC268) and Tubulin (CP06); Imgenex: Puma (IMG-459); Santa Cruz: 14-3-3s (sc7681).

Techniques: Activation Assay, Irradiation, Western Blot, Expressing, Staining

Journal: Oncogene

Article Title: Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

doi: 10.1038/sj.onc.1209126

Figure Lengend Snippet: Figure 7 3T3tx mutp53 triggers Bax-activation. (a) Western blot: The presence of cleaved caspase 3 was analysed in lysates from Balb/ c3T3tx cells at the indicated time points after 100 J/m2 UV-irradiation. (b) FACS analysis: DNA content was monitored in propidium iodide stained Balb/c3T3tx cells at the indicated time points post 100 J/m2 UV-irradiation. (c) Western blot: lysates from Balb/c3T3tx cells were probed for p53, serine 18 phosphorylated p53, and p53 targets Mdm2 (antibody 4B2) and Bax at the indicated time points after 100 J/m2 UV-irradiation. (d) Confocal images: mitochondria and Bax were stained in Balb/c3T3tx cells before or 6 h after 100 J/m2 UV-irradiation. Representative cells are depicted. Bars are 5 mm. (e) Confocal images: mitochondria, Bax and p53 were stained in Balb/ c3T3tx cells that were transiently transfected with p53-specific siRNA. A cell with downregulated mutp53 is depicted in higher magnification. Bars are 10 mm.

Article Snippet: Antibodies were obtained from Upstate: Bax (06- 499); Biomol: Cytochrome c (SA-226); Cell Signaling: Cleaved caspase 3 (9661) and Phospho-p53-Ser15 (9284); BD Pharmingen: p21 (556430); Oncogene: PIG3 (PC268) and Tubulin (CP06); Imgenex: Puma (IMG-459); Santa Cruz: 14-3-3s (sc7681).

Techniques: Activation Assay, Western Blot, Irradiation, Staining, Transfection

Journal: Clinical and Translational Medicine

Article Title: Integrated analysis of genes encoding ATP‐dependent chromatin remodellers identifies CHD7 as a potential target for colorectal cancer therapy

doi: 10.1002/ctm2.953

Figure Lengend Snippet: Depletion of chromodomain helicase DNA‐binding protein 7 (CHD7) activates the p53 signalling pathway. (A) The mRNAs from cells expressing control short hairpin RNAs (shRNA) or CHD7 shRNA‐2 were extracted and subjected to RNA sequencing (RNA‐seq). At the significance of q < 0.05 and with foldchange ≥1.5, the number of dysregulated genes are shown. (B) Volcano plot shows the differentially expressed genes between RKO cells expressing control shRNAs and CHD7 shRNA‐2. At the significance of q < 0.05 and with foldchange ≥1.5, significantly upregulated genes are shown as red dots, and downregulated as blue dots. (C) Heat maps show the expression of significantly dysregulated genes. (D) Gene ontology (GO) enrichment analyses of significantly dysregulated genes in CHD7 knockdown RKO cells. (E) Hallmark enrichment analyses of dysregulated genes in CHD7 knockdown RKO cells. (F) Total mRNA from RKO cells expressing indicated shRNAs was extracted and quantitative real‐time RT‐PCR assays were performed. Each bar represents the mean ± SD for n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001 versus control (Student's t test). (G) Quantitative real‐time RT‐PCR assays were performed using primers targeting p53. Each bar represents the mean ± SD for n = 3, *** P < 0.001 versus control (Student's t test). (H) Western blot analysis of the expression of p53, CDKN1A, TP53BP1, and β‐actin in RKO and HCT116 cells expressing control or CHD7 shRNAs. (I) RKO and HCT116 cells expressing control shRNA or CHD7 shRNA‐2 were treated with cycloheximide (CHX) (50 mg/ml) for 0, 30, 60, 90, 120, and 150 min, and then cell lysates were subjected to Western blotting

Article Snippet: Anti‐CDKN1A (A5952), anti‐β‐actin (AC038), anti‐TP53INP1 (A5952), anti‐p‐p53‐S46 (AP046), anti‐p‐p53‐S392 (AP0860), anti‐p‐p53‐S376 (AP0987), anti‐p‐p53‐S15 (AP0950), anti‐AMPKα (A12718), anti‐p‐AMPK (AP0116), and anti‐AK4 (19854) were purchased from ABclonal Technology Co. (Wuhan, Hubei, CN).

Techniques: Binding Assay, Expressing, Control, shRNA, RNA Sequencing, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Integrated analysis of genes encoding ATP‐dependent chromatin remodellers identifies CHD7 as a potential target for colorectal cancer therapy

doi: 10.1002/ctm2.953

Figure Lengend Snippet: Chromodomain helicase DNA‐binding protein 7 (CHD7) knockdown increases the stability and activity of p53 through AK4‐AMPK‐p53 axis. (A) CHD7 knockdown decreases AK4 protein levels. The cell lysates from RKO and HCT116 cells expressing control or CHD7 short hairpin RNAs (shRNAs) were subjected to Western blotting using indicated antibodies. (B) AK4 knockdown activated AMPK and increased the phosphorylation and stability of p53 protein. (C) CHD7 depletion increased the phosphorylation of AMPK and p53 protein. (D) Knockdown efficiency of shAK4 was detected by Western blotting. (E) Depletion of AK4 decreases the viability of colorectal cancer cells. Cell viability was measured using MTT assays. (F) RKO and HCT116 cells expressing control or AK4 shRNAs were maintained in culture media for 2 weeks and stained with crystal violet, and the number of colonies was counted. Scale bar, 5 mm. (G) Western blot analysis of the expression of CHD7, FLAG‐AK4, p53, and β‐actin in indicated cells. (H) The viability of indicated cells was measured using MTT assays. (I) Colony formation assays were performed in indicated cells. Scale bar, 5 mm. (J) Cell apoptosis was detected by flow cytometry. For parts ) ), data are mean ± SD for n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t test). (K) Western blot analysis of the expression of CHD7, FLAG‐CHD7, AK4, p53, and β‐actin in indicated cells. (L) The viability of indicated cells was measured using MTT assays. (M) Colony formation assays were performed in indicated cells. Scale bar, 5 mm. (N) Cell apoptosis was detected by flow cytometry. For parts – and –, data are mean ± SD for n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t test)

Article Snippet: Anti‐CDKN1A (A5952), anti‐β‐actin (AC038), anti‐TP53INP1 (A5952), anti‐p‐p53‐S46 (AP046), anti‐p‐p53‐S392 (AP0860), anti‐p‐p53‐S376 (AP0987), anti‐p‐p53‐S15 (AP0950), anti‐AMPKα (A12718), anti‐p‐AMPK (AP0116), and anti‐AK4 (19854) were purchased from ABclonal Technology Co. (Wuhan, Hubei, CN).

Techniques: Binding Assay, Knockdown, Activity Assay, Expressing, Control, Western Blot, Phospho-proteomics, Staining, Flow Cytometry

Journal:

Article Title: Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

doi: 10.1093/emboj/cdg579

Figure Lengend Snippet: Fig. 1. Nucleolar disruption is independent of p53. (A–G′) Single confocal sections of nuclei of NDFs (A–G) and 041 cells (A′–G′) stained for fibrillarin, 6 h after treatment with each indicated agent. Each image corresponds to a single nucleus. (A′′–G′′′) wide field images of NDFs (A′′–G′′) and 041 cells (A′′′–G′′′) stained for NPM after the same treatments. A pseudo-colour scale (indicated) was applied to each image to highlight all intensity ranges. (H) Dual plot of nuclear p53 expression level (DO-1 staining) and NPM translocation index for NDFs treated for 6 h with the indicated agents.

Article Snippet: Membranes were probed with either DO-1 antibody, anti-p53 Ser15-P rabbit polyclonal (Santa Cruz) or anti-p53 Ser392-P mouse monoclonal antibodies (AutogenBioclear, Calne, UK), followed by horseradish peroxidase (HRP)-labelled secondary antibody (Dako, Ely, UK).

Techniques: Staining, Expressing, Translocation Assay

Journal:

Article Title: Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

doi: 10.1093/emboj/cdg579

Figure Lengend Snippet: Fig. 2. Effects of micropore irradiation on p53 expression and NPM translocation in NDFs. (A) Distribution of the fraction of irradiated areas on NDF nuclei (n = 1699) observed by the ratio between the area exposed to UV irradiation through 3 µm Isopore filters (irradiated area detected by antibody labelling of photolesions) and the total nuclear projected area (Hoechst 33324). The insert shows an example field of micropore-irradiated nuclei, with CPDs labelled red and nuclei blue (Hoechst). (B) p53 expression levels in NDFs whole-nucleus irradiated at 10 J/m2 (red), micropore irradiated at 40 (green), 60 (yellow) and 80 J/m2 (blue), and non-irradiated (black). The fractions of nuclei receiving WED ≥10 J/m2 (see text) under each micropore irradiation condition are indicated in (A). (C) NPM translocation indext for NDFs irradiated in the same conditions as in (B). Cells in (A) were fixed immediately after irradiation, while cells in (B) and (C) were fixed 6 h after irradiation.

Article Snippet: Membranes were probed with either DO-1 antibody, anti-p53 Ser15-P rabbit polyclonal (Santa Cruz) or anti-p53 Ser392-P mouse monoclonal antibodies (AutogenBioclear, Calne, UK), followed by horseradish peroxidase (HRP)-labelled secondary antibody (Dako, Ely, UK).

Techniques: Irradiation, Expressing, Translocation Assay

Journal:

Article Title: Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

doi: 10.1093/emboj/cdg579

Figure Lengend Snippet: Fig. 3. Correlation of MRD for p53 expression and NPM translocation in NDFs and Cockayne syndrome complementation group A (CS-A) fibroblasts. NDFs (A–D) and CS-A cells (E–H) were irradiated at the indicated UV densities, fixed 6 h later and stained for p53 expression (A–D and E–H, with positions of nuclei indicated by Hoechst staining in A′–D′ and E′–H′) or NPM (A′′–D′′ and E′′–H′′). All images are wide-field. Pseudo-colour is used in NPM images.

Article Snippet: Membranes were probed with either DO-1 antibody, anti-p53 Ser15-P rabbit polyclonal (Santa Cruz) or anti-p53 Ser392-P mouse monoclonal antibodies (AutogenBioclear, Calne, UK), followed by horseradish peroxidase (HRP)-labelled secondary antibody (Dako, Ely, UK).

Techniques: Expressing, Translocation Assay, Irradiation, Staining

Journal:

Article Title: Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

doi: 10.1093/emboj/cdg579

Figure Lengend Snippet: Fig. 4. Effect of nucleolar disruption by microinjection of an anti-UBF antibody in NDFs. (A, C and E) Microinjection of anti-UBF (IgG1) with fibrillarin distribution (A) and p53 expression (C) assayed 6 h post-injection, and p21 expression (E) 18 h post-injection. (B, D and F) Microinjection of control purified mouse IgG1. (A′–F′) Reference Hoechst images. Arrows indicate microinjected cells. Lower magnification was used in p21 images in order to incorporate more cells into the field of view. (G–J) p53 phosphorylation in response to microinjection of anti-UBF (G and I) and non-specific IgG (H and J). p53 phosphorylation was detected for Ser15 (G and H) and Ser392 (I and J). (G′–J′) reference Hoechst images. (K–N) Phosphorylation at Ser15 and Ser392 in control and UV-irradiated NDFs (14 J/m2) as indicated. (O) Western blot analysis of p53 expression and phosphorylation (Ser15 and Ser392) in control and UV-irradiated NDFs (1–2, 4 and 14 J/m2). Cells were harvested 6 h post-irradiation.

Article Snippet: Membranes were probed with either DO-1 antibody, anti-p53 Ser15-P rabbit polyclonal (Santa Cruz) or anti-p53 Ser392-P mouse monoclonal antibodies (AutogenBioclear, Calne, UK), followed by horseradish peroxidase (HRP)-labelled secondary antibody (Dako, Ely, UK).

Techniques: Expressing, Injection, Purification, Irradiation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: C 2 -Ceramide-Induced Rb-Dominant Senescence-Like Phenotype Leads to Human Breast Cancer MCF-7 Escape from p 53-Dependent Cell Death

doi: 10.3390/ijms20174292

Figure Lengend Snippet: C 2 -ceramide-induced apoptosis-related and senescence-related signaling pathway. ( A ) The expression changes of SA- and pro-apoptotic proteins in C 2 -ceramide-treated breast cancer cells. The two breast cancer cell lines were treated with 20 μM of C 2 -ceramide for 6, 12, and 24 h respectively. β-actin as an internal control. All fold changes were normalized by the level of internal control. ( B ) The investigation of p 53 activator NSC59984 (1 µM) co-treated with C 2 -ceramide (5 and 20 µM respectively) 24 h for cell viability analysis. * p < 0.05.

Article Snippet: Briefly, 1 × 10 3 cells were seeded and pretreated with the indicated concentrations of p 53 activator NSC59984 (Cat No. B6045 APExBIO, Houston, TX, USA) prior to the treatment of indicated concentrations of C 2 -ceramide (Sigma-Aldrich) for 24 h. Afterward, the MTS solution was added to the cells and further incubated for 1 h at 37 °C according to the manual of the manufacturer.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: C 2 -Ceramide-Induced Rb-Dominant Senescence-Like Phenotype Leads to Human Breast Cancer MCF-7 Escape from p 53-Dependent Cell Death

doi: 10.3390/ijms20174292

Figure Lengend Snippet: A proposed mechanism whereby breast cancer cells escape C 2 -ceramide-induced apoptosis through modulating senesce-like phenotype. Exogenous C 2 -ceramide inhibits growth arrest and induces apoptosis in breast cancer MDA-MB-231 cells through down-regulating the expression of mutant p 53 while up-regulating pro-apoptosis pathways, including the expression of Bax and Bad and the proteolytic activation of caspase-3. In contrast, C 2 -ceramide treatments that favor the induction of senescence-like phenotype might occur through the activation of Rb rather than the activation of p 53-signaling of senescence. Rb-mediated senescence-like phenotype (SLP) might be reversible and confer more resistance of breast cancer MCF-7 cells to C 2 -ceramide. In contrast, the activation of wild type p 53 using a p 53 activator resensitizes MCF-7 cells to C 2 -ceramide, suggesting the critical role of wild type p 53 in C 2 -ceramide-induced death in breast cancer cells. Therefore, the proposed model suggests that some cancer cells escape apoptosis induction through modulating senescence-like phenotype, whereas the p 53 activation can overcome the chemoresistance and should be a promising strategy for treating cancer cells which favor stress-induced SLP. (↑, increase or upregulation; ↓, decrease or downregulation; T, attenuation or blockade).

Article Snippet: Briefly, 1 × 10 3 cells were seeded and pretreated with the indicated concentrations of p 53 activator NSC59984 (Cat No. B6045 APExBIO, Houston, TX, USA) prior to the treatment of indicated concentrations of C 2 -ceramide (Sigma-Aldrich) for 24 h. Afterward, the MTS solution was added to the cells and further incubated for 1 h at 37 °C according to the manual of the manufacturer.

Techniques: Expressing, Mutagenesis, Activation Assay