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: Nature Communications

Article Title: Leveraging the multivalent p53 peptide-MdmX interaction to guide the improvement of small molecule inhibitors

doi: 10.1038/s41467-022-28721-x

Figure Lengend Snippet: a Alignment of the amino acid sequence of N-MdmX with that of N-Mdm2. Non-identical residues on N-MdmX are shown in red. b A cartoon model representing N-MdmX and N-Mdm2 structures, based on their crystal structures in complex with p53p. c Each ligand-binding pocket on N-MdmX or N-Mdm2 is composed of three subsites, i.e., the F19 p53p , W23 p53p , and L26 p53p subsites, with reference to the three key binding residues of p53p. d The amino acid sequence of the p53p peptide. Three key residues, i.e., F19 p53p , W23 p53p and L26 p53p , are in red. e The structure of p53p with three key residues highlighted. f Nutlin-3a can be docked tightly into the three subsites on N-Mdm2, mimicking p53p.

Article Snippet: The p53p ΔF19 gene was synthesized by GenScript (Wuxi, China) and sub-cloned to the pET28-MdmX plasmid.

Techniques: Sequencing, Ligand Binding Assay, Binding Assay

Journal: Nature Communications

Article Title: Leveraging the multivalent p53 peptide-MdmX interaction to guide the improvement of small molecule inhibitors

doi: 10.1038/s41467-022-28721-x

Figure Lengend Snippet: a The structures of the N-MdmX/nutlin-3a complexes superimposed on those of the N-MdmX/p53p complexes. Green stick: nutlin-3a ; purple stick: F19 p53p , W23 p53p , and L26 p53p . b Major side-chain conformational deviations occurred at the residues in the F19 p53p subsite and L26 p53p subsite of N-MdmX in complex with nutlin-3a and p53p. c – f Fast backbone dynamics ( ps–ns ) of N-MdmX in complex with nutlin-3a ( c , d ) or p53p ( e , f ). The order parameter ( S 2 ) and timescale of motion (τ e ) of N-MdmX in both complexes are plotted against the backbone amide residue. The secondary structure is shown on the top of the graph. R, S, and H denote the flexible region, β-sheet and α-helix, respectively. Regions with enhanced dynamics are shaded in gray. The data are represented as mean values ± SD and error bars for each parameter represent the propagated uncertainty determined from Monte Carlo simulations. Source data for ( c – f ) are provided as a Source Data file.

Article Snippet: The p53p ΔF19 gene was synthesized by GenScript (Wuxi, China) and sub-cloned to the pET28-MdmX plasmid.

Techniques: Residue

Journal: Nature Communications

Article Title: Leveraging the multivalent p53 peptide-MdmX interaction to guide the improvement of small molecule inhibitors

doi: 10.1038/s41467-022-28721-x

Figure Lengend Snippet: a A representative crystal structure of N-Mdm2 in complex with nutlin-3a (red line). b The structure N-MdmX/nutlin-3a complexes (in purple) are superimposed on those of the N-Mdm2/nutline-3a complexes (in yellow). c Comparison of major side-chain configurations between the F19 p53p subsites of N-MdmX and N-Mdm2. d A 2Fo-Fc map contoured at 1.5 σ of a section of the H72 side chain on N-Mdm2 revealing two configurations. e , f Comparison of major side chain configurations in the W23 p53p and L26 p53p subsites, respectively, between N-MdmX and N-Mdm2.

Article Snippet: The p53p ΔF19 gene was synthesized by GenScript (Wuxi, China) and sub-cloned to the pET28-MdmX plasmid.

Techniques: Comparison

Journal: Nature Communications

Article Title: Leveraging the multivalent p53 peptide-MdmX interaction to guide the improvement of small molecule inhibitors

doi: 10.1038/s41467-022-28721-x

Figure Lengend Snippet: a Affinity comparison of p53p F19 with p53p and p53p ΔF19 . n = 3 independent experiments and the data are represented as mean values ± SD. Source data are provided as a Source Data file. b The crystal structure of N-MdmX in complex with p53p F19 (purple) is superimposed on that of N-MdmX in complex with p53p (green). c A p53p ΔF19 -MdmX fusion protein was constructed for screening pharmacophores that could specifically target the F19 p53p subsite on N-MdmX. d , e Four benzodiazepine analogs ( d ) and their pharmacophores ( e ).

Article Snippet: The p53p ΔF19 gene was synthesized by GenScript (Wuxi, China) and sub-cloned to the pET28-MdmX plasmid.

Techniques: Comparison, Construct

Journal: Nature Communications

Article Title: Leveraging the multivalent p53 peptide-MdmX interaction to guide the improvement of small molecule inhibitors

doi: 10.1038/s41467-022-28721-x

Figure Lengend Snippet: a The perturbation of the 15 N- 1 H HSQC resonances of the W23 p53p side chain by four benzodiazepine compounds are compared. Protein and ligand ratios: black, 1:0; green, 1:0.5; blue, 1:1; yellow, 1:1.5 and red, 1:2. b , c The perturbation of the 15 N– 1 H HSQC resonances of the W23 p53p side chain by five pharmacophores are compared. The protein and ligand ratios: black, 1:0; green, 1:0.5; blue, 1:1; yellow, 1:1.5; red, 1:2, orange, 1:3 and purple, 1:5.

Article Snippet: The p53p ΔF19 gene was synthesized by GenScript (Wuxi, China) and sub-cloned to the pET28-MdmX plasmid.

Techniques:

Journal: Nature Communications

Article Title: Leveraging the multivalent p53 peptide-MdmX interaction to guide the improvement of small molecule inhibitors

doi: 10.1038/s41467-022-28721-x

Figure Lengend Snippet: a The structure and activity relationships of p53p and nutlin analogs are compared based on Fg1 and Fg5. n = 3 independent experiments and the data are represented as mean values ± SD. Source data are provided as a Source Data file. b Molecular structures of nutlin analogs H202 and H203. c , d Structure models of N-MdmX interacting with H202 and H203, evaluated by molecular docking.

Article Snippet: The p53p ΔF19 gene was synthesized by GenScript (Wuxi, China) and sub-cloned to the pET28-MdmX plasmid.

Techniques: Activity Assay