Journal: Cell Death and Differentiation

Article Title: Functional interplay between p53 and Δ133p53 in adaptive stress response

doi: 10.1038/s41418-019-0445-z

Figure Lengend Snippet: Moderate stimulation induced a novel p53 isoform ME-Δ123p53 enhances cell resistance to acute stresses. a Mouse Trp53 gene structure was shown in the diagram. Trp53 gene has 11 exons and 10 introns, and each exon relative position is labeled. The start codon ATG of full-length p53 is located in exon 2. An alternative exon 5′ in intron 4 contains 5′UTR and the coding sequence for the first two amino acids for a novel p53 isoform—ME-Δ123p53. ME-Δ123p53 lacks all transcription activity domain (TAD) and part of DNA-binding domain (DBD). Except the first two amino acids, the following parts are the same as full-length p53 from the 123th amino acid as shown. b MEF cells were pretreated with different doses of arsenic for 12 h, then p53 and ME-Δ123p53 protein abundance was determined by immunoblot, and c ME-Δ123p53 mRNA level was measured by qRT-PCR. d MEF cells were pretreated with 0.1, 10, or 50 μM arsenic for 12 h, and then received 10 Gy X-ray high-dose irradiation (HDR). After 48 h of irradiation (hpt), relative cell viability was measured by trypan blue staining and counting. e MEF cells were pretreated with 0.1 μM arsenic or 0.5 μM VK3 for 12 h, and then subjected with 5 nM actinomycin D (ActD), 50 μM 5-fluorouracil (5-FU), or 0.5 μM Camptothecin (Campt) treatment. After 24 h of treatment (hpt), cell viability was measured. f MEFs were transfected with expression plasmids containing ME-Δ123p53 or empty vector, then subjected with HDR at 12 h after transfection. 48hpt, (top) p53 and ME-Δ123p53 protein level and (bottom) relative cell viability was measured. g MEF cells were transfected with ME-Δ123p53 plasmids or empty vector, and then treated with 5 nM ActD, 50 μM 5-FU, or 0.5 μM Campt at 12 h post transfection, (top) p53 and ME-Δ123p53 protein induction was measured by immunoblot. (Bottom) Cell viability was measured at 24hpt. h MEFs were transfected with siRNA targeting ME-Δ123p53 (ME-Δ123p53i) or nonsense siRNA (siNS), and then treated with 0.1 μM low dose arsenic (LDA), 50 μM H2O2, or 0.5 μM vitamin K3 (VK3) at 12 h post transfection. (Top) p53 and ME-Δ123p53 protein level was measured. After 12 h, cells were treated with HDR, and (bottom) relative cell viability was measured. Error bars denote standard deviation (*P < 0.05, **P < 0.01). For comparing with the corresponding control sample, “#” was used instead of “*” for indicating P value range

Article Snippet: For western blot, anti-mouse-p53 (F-8, Santa cruz, Santa Cruz, CA, sc-374087, 1:1000, for p53 and ME-Δ123p53), anti-human-p53 antibody DO-1 (Santa Cruz, sc-126, 1:2000), anti-human-p53 (for isoforms) antibody DO-11 (GeneTex, Irvine, CA, GTX5258, 1:1000), anti-mouse/human-p53 antibody Ab-1 (Millipore, Burlington, MA, OP03, 1:500), anti-hif1α (Novusbio, Centennial, CO, NB100-479, 1:1000), anti-sod1 (Abcam, Cambridge, MA, ab16831, 1:1000), anti-bcl2 (Santa cruz, sc-7382, 1:1000), anti-cleaved caspase3 (Cell Signaling, Danvers, MA, #9664, 1:1000), anti-parp (Cell Signaling, #9532, 1:1000), and anti-β-actin antibody (Sigma, AC-15, 1:2000) was used.

Techniques: Labeling, Sequencing, Activity Assay, Binding Assay, Quantitative Proteomics, Western Blot, Quantitative RT-PCR, Irradiation, Staining, Transfection, Expressing, Plasmid Preparation, Standard Deviation, Control

Journal: Cell Death and Differentiation

Article Title: Functional interplay between p53 and Δ133p53 in adaptive stress response

doi: 10.1038/s41418-019-0445-z

Figure Lengend Snippet: Low level ROS-induced ME-Δ123p53 adjusts p53 signaling. a MEF cells were pretreated with 10 mM NAC for 4 h, and then treated with 0.1 Gy X-ray (LDR), LDA, 50 μM H2O2, or 0.5 μM VK3. (Left) Protein level of p53, ME-Δ123p53, and HIF1α were measured. 12 h later, culture medium was changed and cells were subjected with HDR. (Right) 48hpt, relative cell viability was measured. b MEFs were transfected with plasmids expressing SOD1 or empty vector, and pretreated with LDA, 50 μM H2O2 or 0.5 μM VK3 at 12 h post transfection. Then cells were subjected with HDR treatment. (Left) Protein level of SOD1 was checked before HDR. (Right) Cell viability was checked at 48 hpt. c MEFs were transfected with different ratio of p53 plasmids and ME-Δ123p53 plasmids as shown (completed with empty vector). (Left) Protein level of SOD1 was checked. (Right) After 48h, relative cell viability was measured. d Human H1299 cells were transfected with different ratio of human p53 plasmids and Δ133p53 plasmids as shown (completed with empty vector). The tranfection efficiency was checked by immunoblot. e Then cells were treated with HDR. Relative cell viability was measured at 48hpt. f Cells in d were treated with 5 nM ActD for 12 h. Relative cell viability was measured at 24hpt. Data are presented as mean ± standard deviation, *P < 0.05, **P < 0.01, Anova test. For comparing with the corresponding control sample, “#” was used instead of “*” for indicating P value range

Article Snippet: For western blot, anti-mouse-p53 (F-8, Santa cruz, Santa Cruz, CA, sc-374087, 1:1000, for p53 and ME-Δ123p53), anti-human-p53 antibody DO-1 (Santa Cruz, sc-126, 1:2000), anti-human-p53 (for isoforms) antibody DO-11 (GeneTex, Irvine, CA, GTX5258, 1:1000), anti-mouse/human-p53 antibody Ab-1 (Millipore, Burlington, MA, OP03, 1:500), anti-hif1α (Novusbio, Centennial, CO, NB100-479, 1:1000), anti-sod1 (Abcam, Cambridge, MA, ab16831, 1:1000), anti-bcl2 (Santa cruz, sc-7382, 1:1000), anti-cleaved caspase3 (Cell Signaling, Danvers, MA, #9664, 1:1000), anti-parp (Cell Signaling, #9532, 1:1000), and anti-β-actin antibody (Sigma, AC-15, 1:2000) was used.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Standard Deviation, Control

Journal: Cell Death and Differentiation

Article Title: Functional interplay between p53 and Δ133p53 in adaptive stress response

doi: 10.1038/s41418-019-0445-z

Figure Lengend Snippet: ME-Δ123p53 induction is dependent on HIF pathway. a MEFs were transfected with HIF1α siRNA (HIF1αi) or siNS, then pretreated with LDA. (Left) Protein level of p53, ME-Δ123p53 and HIF1α were measured. After 12 h, (right) MEFs were treated with HDR, and cell viability was measured at 48hpt. b MEFs were transfected with HIF1α siRNA (HIF1αi) or siNS, then pretreated with 0.5 μM VK3 for 12 h. (Left) Protein level of p53, ME-Δ123p53, and HIF1α were measured. After 12 h, (right) MEFs were treated with HDR, and relative cell viability of MEFs was measured at 48hpt. c The 3000 bp sequence before ME-Δ123p53 start codon was cloned into pGL3 luciferase reporter backbone for ME-Δ123p53 promoter analysis. Then different parts of ME-Δ123p53 promoters were cloned to drive luciferase as shown. MEFs were transfected with luciferase reporters, and then treated with LDA. d Luciferase activity was checked at 12hpt, and normalized with co-transfected Renilla signal. e Two promoting response elements (RE) of p53 were predicted on −1000 to −500 and −2500 to −2000 regions of ME-Δ123p53 promoter. The green and red arrows indicate the orientations of the quarter sites. The numbers in brackets indicate the distance (bps) between two half parts of binding motif. R = A or G, W = A or T, Y = C or T. The sequences of REs were shown in the diagram. Promoter with mutated p53-binding sites was cloned as luciferase reporter, and checked by luciferase assay. f MEFs were transfected with indicated luciferase reporters, combining with HIF1α siRNA or nonsense siRNA, and treated with LDA. Luciferase signal was checked at 12hpt. g MEFs were transfected with HIF1αi or siNS, and treated with LDA. ChIP assay was performed with anti-p53-antibody (1C12) or anti-IgG antibody at 12hpt. Specific primer pairs were designed to amplify the corresponding REs. DNA was normalized with a pair of negative control primers for β-actin exon. The results are presented as the relative occupancies of different REs. Data are presented as mean ± standard deviation, *P < 0.05, **P < 0.01, Anova test. For comparing with the corresponding control sample, “#” was used instead of “*” for indicating P value range

Article Snippet: For western blot, anti-mouse-p53 (F-8, Santa cruz, Santa Cruz, CA, sc-374087, 1:1000, for p53 and ME-Δ123p53), anti-human-p53 antibody DO-1 (Santa Cruz, sc-126, 1:2000), anti-human-p53 (for isoforms) antibody DO-11 (GeneTex, Irvine, CA, GTX5258, 1:1000), anti-mouse/human-p53 antibody Ab-1 (Millipore, Burlington, MA, OP03, 1:500), anti-hif1α (Novusbio, Centennial, CO, NB100-479, 1:1000), anti-sod1 (Abcam, Cambridge, MA, ab16831, 1:1000), anti-bcl2 (Santa cruz, sc-7382, 1:1000), anti-cleaved caspase3 (Cell Signaling, Danvers, MA, #9664, 1:1000), anti-parp (Cell Signaling, #9532, 1:1000), and anti-β-actin antibody (Sigma, AC-15, 1:2000) was used.

Techniques: Transfection, Sequencing, Clone Assay, Luciferase, Activity Assay, Binding Assay, Negative Control, Standard Deviation, Control

Journal: Cell Death and Differentiation

Article Title: Functional interplay between p53 and Δ133p53 in adaptive stress response

doi: 10.1038/s41418-019-0445-z

Figure Lengend Snippet: ME-Δ123p53 suppresses apoptosis via switching p53 pathway to upregulate BCL2. a MEFs were pretreated with LDA, 50 μM H2O2, or 0.5 μM VK3 for 12 h, and then treated with HDR. Caspase inhibitor 20 μM Z-VAD-FMK was added in culture medium 30 min before irradiation. 48hpt, relative cell viability was measured as shown. b MEFs were transfected with ME-Δ123p53i or siNS, then pretreated with LDA for 12 h. At 12 h post LDA treatment, combining with Z-VAD-FMK or not, cells were subjected with HDR. (Left) Total apoptosis cells ratio was determined by Annexin V/ 7-AAD dual staining—FACS assay. (Right) Relative cell viability was measured at 48hpt. c Cleaved caspase3 and PARP were measured by immunoblots. d MEFs were transfected with wild-type (WT) ME-Δ123p53, ME-Δ123p53 (R172H), or ME-Δ123p53 (R270H) mutation. (Left) Transfection efficiency was determined at 12 h post transfection. (Right) Then cells were treated with HDR, and cell viability was determined at 48hpt. e MEFs transfected with ME-Δ123p53i, BCL2 expressing plasmids or their combination (completed with siNS and empty vector at 12 hpt as shown. f (Left) MEFs transfected with ME-Δ123p53i or siNS were treated with LDA at 12 h post transfection. After 12 h, cells were treated with HDR. Bcl2 mRNA level was measured by qRT-PCR at 12 hpt. (Right) MEFs transfected with ME-Δ123p53 or empty vector were treated with HDR at 12 h post transfection. After 12 h, Bcl2 mRNA level was measured by qRT-PCR. g MEFs transfected with ME-Δ123p53 plasmid, BCL2 siRNA (Bcl2i) or their combination (completed with siNS and empty vector) were treated with HDR at 12 h post transfection. (Left) Relative cell viability was measured at 48hpt. (Right) Protein level of p53 and ME-Δ123p53 was measured before HDR treatment and protein level of BCL2 was measured at 12hpt as shown. Data are presented as mean ± standard deviation, *P < 0.05, **P < 0.01, Anova test. For comparing with the corresponding control sample, “#” was used instead of “*” for indicating P value range

Article Snippet: For western blot, anti-mouse-p53 (F-8, Santa cruz, Santa Cruz, CA, sc-374087, 1:1000, for p53 and ME-Δ123p53), anti-human-p53 antibody DO-1 (Santa Cruz, sc-126, 1:2000), anti-human-p53 (for isoforms) antibody DO-11 (GeneTex, Irvine, CA, GTX5258, 1:1000), anti-mouse/human-p53 antibody Ab-1 (Millipore, Burlington, MA, OP03, 1:500), anti-hif1α (Novusbio, Centennial, CO, NB100-479, 1:1000), anti-sod1 (Abcam, Cambridge, MA, ab16831, 1:1000), anti-bcl2 (Santa cruz, sc-7382, 1:1000), anti-cleaved caspase3 (Cell Signaling, Danvers, MA, #9664, 1:1000), anti-parp (Cell Signaling, #9532, 1:1000), and anti-β-actin antibody (Sigma, AC-15, 1:2000) was used.

Techniques: Irradiation, Transfection, Staining, Western Blot, Mutagenesis, Expressing, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation, Control

Journal: Cell Death and Differentiation

Article Title: Functional interplay between p53 and Δ133p53 in adaptive stress response

doi: 10.1038/s41418-019-0445-z

Figure Lengend Snippet: The ME-Δ123p53/p53 complex directly binds to different sites of the Bcl2 promoter than p53 alone, enabling the fine regulation of Bcl2 transcription. a Diagram shows the 3000 bps of mouse Bcl2 promoter and in silico predicted p53 family members binding sites on it. The sequences and positions of p53 family REs on Bcl2 promoter are listed below. R = A or G, W = A or T, Y = C or T, here the red font indicates bases not perfectly match the motif pattern. b The 3000 bps of mouse Bcl2 promoter was cloned in luciferase backbone as the WT promoter reporter control. The REs on Bcl2 promoter were mutated for investigating their function. The WT and mutated REs sequences were listed as the diagram shown. c Reporter plasmids with WT or mutant Bcl2 promoter were transfected in MEFs pre-transfected with p53, ME-Δ123p53 or their combination. d Reporter plasmids were transfected in MEFs pretreated with LDA, pre-transfected with ME-Δ123p53i or the combination treatment. The luciferase activity was measured. Reporter luciferase signals were normalize with co-transfected Rellina signals. e ChIP of p53/ME-Δ123p53 specific REs (response elements) in Bcl2 promoters in MEF cells at 12 h post transfected with HA tagged p53, myc tagged ME-Δ123p53 or their combination. Anti-HA-tag or Anti-myc-tag antibody was used to co-immunoprecipitate the protein-DNA complex, while IgG was used as a non-specific binding control. Specific primer pairs were designed to amplify the corresponding REs. DNA was normalized with a pair of negative control primers for β-actin exon. The results are presented as the relative occupancies of different REs. f CoIP with Anti-HA-tag or Anti-myc-tag antibody in MEF cells transfected with HA-p53, myc-ME-Δ123p53 or their combination as indicated. Data are presented as mean ± standard deviation, *P < 0.05, **P < 0.01, Anova test. For comparing with the corresponding control sample, “#” was used instead of “*” for indicating P value range

Article Snippet: For western blot, anti-mouse-p53 (F-8, Santa cruz, Santa Cruz, CA, sc-374087, 1:1000, for p53 and ME-Δ123p53), anti-human-p53 antibody DO-1 (Santa Cruz, sc-126, 1:2000), anti-human-p53 (for isoforms) antibody DO-11 (GeneTex, Irvine, CA, GTX5258, 1:1000), anti-mouse/human-p53 antibody Ab-1 (Millipore, Burlington, MA, OP03, 1:500), anti-hif1α (Novusbio, Centennial, CO, NB100-479, 1:1000), anti-sod1 (Abcam, Cambridge, MA, ab16831, 1:1000), anti-bcl2 (Santa cruz, sc-7382, 1:1000), anti-cleaved caspase3 (Cell Signaling, Danvers, MA, #9664, 1:1000), anti-parp (Cell Signaling, #9532, 1:1000), and anti-β-actin antibody (Sigma, AC-15, 1:2000) was used.

Techniques: In Silico, Binding Assay, Clone Assay, Luciferase, Control, Mutagenesis, Transfection, Activity Assay, Negative Control, Standard Deviation

Journal: Cell Death and Differentiation

Article Title: Functional interplay between p53 and Δ133p53 in adaptive stress response

doi: 10.1038/s41418-019-0445-z

Figure Lengend Snippet: Moderate stimulation induced ME-Δ123p53 protects multiple organs from acute stress in vivo. a C57BL mice (n = 12 per group) were treated with 0.1 Gy X-ray (LDR, once per day) or low dose arsenic (LDA, IP with 0.4 mg/kg arsenic) for 5 consecutive days, and then subjected with 8 Gy X-ray. b Mice (n = 12 per group) were injected with LDA for 5 consecutive days, and subjected to low dose ActD treatment (IP with 60 μg/kg ActD, once per day). Survival rate was recorded and plotted as indicated. c Mice were treated with LDR or LDA as in a and then subjected with 4 Gy X-ray. Mice were sacrificed and indicated organs were collected at 12hpi. TUNEL assay was performed with slices of spleen and thymus as shown. d In thymus, spleen, duodenum, and pancreas, Bcl2 mRNA expression level was measured by qRT-PCR. e Mice (n = 12 per group) were injected with empty adenovirus (ADV-Empty) or adenovirus expressing ME-Δ123p53 (ADV-ME-Δ123p53) (IP with 6 × 109 pfu adenovirus). Five days later, mice were subjected to 8 Gy X-ray irradiation. (Left) Survival rate was recorded and plotted as indicated. (Right) Mice were injected with indicated agents, and then subjected with 4 Gy X-ray. Mice were sacrificed and indicated organs were collected at 12hpi. Protein level of p53, ME-Δ123p53 and f Cleved-caspase3 was measured. g Bcl2 and h indicated glycolytic genes mRNA expression level was measured by qRT-PCR. I Mice (n = 12 per group) were injected with adenovirus expressing nonsense shRNA (ADV-STDsh) or shRNA targeting ME-Δ123p53 (ADV-ME-Δ123p53sh), and then injected with LDA for 5 consecutive days. Next day, mice were subjected with 8 Gy X-ray. Survival rate was recorded and plotted as indicated. j Mice were injected with indicated agents as in i, but then subjected with 4 Gy X-ray. Mice were sacrificed and indicated organs were collected at 12hpi. Bcl2 mRNA expression level was measured by qRT-PCR. Data are presented as mean ± standard deviation, *P < 0.05, **P < 0.01, Anova test

Article Snippet: For western blot, anti-mouse-p53 (F-8, Santa cruz, Santa Cruz, CA, sc-374087, 1:1000, for p53 and ME-Δ123p53), anti-human-p53 antibody DO-1 (Santa Cruz, sc-126, 1:2000), anti-human-p53 (for isoforms) antibody DO-11 (GeneTex, Irvine, CA, GTX5258, 1:1000), anti-mouse/human-p53 antibody Ab-1 (Millipore, Burlington, MA, OP03, 1:500), anti-hif1α (Novusbio, Centennial, CO, NB100-479, 1:1000), anti-sod1 (Abcam, Cambridge, MA, ab16831, 1:1000), anti-bcl2 (Santa cruz, sc-7382, 1:1000), anti-cleaved caspase3 (Cell Signaling, Danvers, MA, #9664, 1:1000), anti-parp (Cell Signaling, #9532, 1:1000), and anti-β-actin antibody (Sigma, AC-15, 1:2000) was used.

Techniques: In Vivo, Injection, TUNEL Assay, Expressing, Quantitative RT-PCR, Irradiation, shRNA, Standard Deviation