p53  (Bio-Rad)

Journal: The FASEB Journal

Article Title: Adenosine A2A receptor activation reduces chondrocyte senescence

doi: 10.1096/fj.202201212RR

Figure Lengend Snippet: A2AR activation enhances the formation of an N‐terminally truncated anti‐senescence p53 variant, Δ133p53α. (A, B) These are the 12 currently known human p53 variants with the full‐length p53 (FLp53) at the top and the various N‐terminal and/or C‐terminal truncated variants below shown to the top right in figure B. In the top left figure (A) note the western blot image from a TC28a2 chondrocyte cell lysate we collected and immunostained for K382Ac‐p53 (green). This identified a larger protein of approximately the same MW as FLp53, also immunostained with an N‐terminus antibody corresponding to amino acids surrounding Ser‐20 in human p53 (red, with overlap appearing white/yellow). The K382Ac‐p53 antibody also recognized a smaller protein of around 35 kDa (green) as compared to known GAPDH with MW of approximately 36–37 kDa located just slightly above. Of the p53 variants, this most closely corresponds to Δ133p53α based on MW and the fact that K382 is only present in FLp53 and the α C‐terminal p53 variants. (C) RT‐qPCR from RNA collected from TC28a2 cells treated with or without 1 μM CGS21680 demonstrates relative increase in the RNA using primers that would recognize the Δ133p53 variants as compared to the primers for FLp53. (D) TC28a2 cells treated with or without 1 μM CGS21680 for an hour display a relative increase at the protein level using an antibody to K382Ac p53 of the Δ133p53α isoform to FLp53 (3.0 ± 0.8 vs. 1.0 ± 0.06, p = .005, n = 3–4). Below shows an immunofluorescence time course demonstrating change in p53 fluorescence as detected by this C‐terminal acetylated lysine 382 antibody in TC28a2 cells treated with CGS. (E) The WB findings from figure D were repeated using two alternate non‐acetylation‐dependent antibodies with mid‐p53 epitopes that recognize all known isoforms. Both antibodies demonstrate significantly increased levels of Δ133p53α in TC28a2 chondrocytes treated with CGS21680 that is reversed with pre‐treatment using A2AR antagonist ZM241385 (1 μM) followed by addition of agonist CGS21680 (labeled ZM241385 in the figure) (F) Western blot with antibody ab26 was performed on lysates from primary human chondrocytes treated with or without 1 μM CGS21680 for 1 h display a significant increase in the amount of Δ133p53α as assessed by western blotting of cell lysates treated with CGS21680 as compared to untreated chondrocytes (1.52 ± 0.051 vs. 1.00 ± 0.084, p < .0001, n = 4, t ‐test). MW, molecular weight markers; CTRL, untreated control chondrocytes; CGS, CGS21680.

Article Snippet: Ms‐p53/DO‐11 (MCA1704) was purchased from Bio‐Rad (Hercules, CA, USA).

Techniques: Activation Assay, Variant Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence, Fluorescence, Labeling, Molecular Weight, Control

Journal: Cell Death & Disease

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

doi: 10.1038/s41419-021-04224-3

Figure Lengend Snippet: K562 ( HR-EGFP/3 ′ -EGFP ) cells were transfected with 10 µg expression plasmid for p53α and corresponding amounts for p53β, p53γ, Δ40p53α, Δ133p53α, Δ160p53α or empty vector (EV) in controls (ctrl) as indicated in the graphs. Seventy-two hours after transfection FACS analysis (left panels of B , C ) and protein harvesting (right panels of B , C ) were performed. Recombination (rec.) fold changes were analyzed flow cytometrically by quantification of EGFP-positive cells among living cells. Mean values from p53α-expressing cells were set to 1 (absolute mean frequencies: 4*10 −5 ). Data were collected from ≥18 individual measurements each. For graphic presentation, calculation of SEM and statistically significant differences via Kruskal-Wallis test followed by two-tailed Mann−Whitney U test GraphPadPrism8.4 software was used. # indicates a statistically significant difference between ctrl and the respective p53-isoform data. ****(# # # #) P < 0.0001. Quantification of protein levels was carried out using ImageLab software, normalized to β-actin, and indicated above the representative immunoblot. A Schematic overview of different domains of p53 isoforms. p53α (highlighted in black) consists of transactivation domain I (TAD I), transactivation domain II (TAD II), proline-rich domain (PRD), DNA-binding domain, hinge domain (HD), oligomerization domain (OD), C-terminal domain (CTD) while parts of the other p53-isoforms are replaced by other sequences or missing. Note that the color code in the scheme was used in the subsequent graphs. B Role of p53β and p53γ in replication-associated recombination. Left panel shows Recombination (Rec.) fold changes. Right panel shows representative Western Blot analysis of the indicated p53 isoforms. β-Actin served as a loading control. C Role of p53α, Δ40p53α, Δ133p53α, and Δ160p53α in replication-associated recombination. Left panel shows Rec. fold changes. Right panel shows representative Western Blot analysis of the indicated p53 isoforms. β-Actin served as loading control.

Article Snippet: Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Two Tailed Test, MANN-WHITNEY, Software, Western Blot, Binding Assay, Control

Journal: Cell Death & Disease

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

doi: 10.1038/s41419-021-04224-3

Figure Lengend Snippet: K562 cells were transfected with expression plasmids for p53α, alternative p53 isoforms [ A , B p53β, p53γ or C , D Δ40p53α, Δ133p53α, Δ160p53α] or EV in controls (ctrl). Forty-eight hours after transfection DNA fiber spreading assay was performed. Graphic overviews in the left panel show the experimental outline and representative fibers. Cells were subsequently incubated with 5-chloro-2′-deoxyuridine (CldU, 20 µM) and 5-iodo-2′-deoxyuridine (IdU, 200 µM) for 20 min. During IdU-incorporation cells were either mock-treated ( A , C ) or treated with 3 µM MMC ( B , D ). Both, CldU- and IdU-tracks were measured but for clarity graphic presentations in the middle panel focus on IdU-tracks in ongoing forks (≥361 to ≥423 fibers in two independent biological experiments). Right panels show the graphic presentation of FA with the respective schematic overview on top. DNA fibers were reanalyzed comparing track lengths of IdU incorporation (red) originating from the same CldU track (green). Graphs represent ≥66 to ≥121 fibers from two independent biological experiments. For graphic presentation, calculation of SEM, and statistically significant differences GraphPadPrism8.4 software was used. Statistically significant differences among groups were calculated by Dunn’s multiple comparisons test. # indicates a statistically significant difference between ctrl and the respective p53 isoform. (#) P < 0.05, *** P < 0.001, ****(# # # #), P < 0.0001 (scale bar: 5 µm).

Article Snippet: Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Techniques: Transfection, Expressing, Incubation, Software

Journal: Cell Death & Disease

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

doi: 10.1038/s41419-021-04224-3

Figure Lengend Snippet: K562 cells were transfected with expression plasmids for p53α, alternative p53 isoforms [ A − C p53β, p53γ, D − F Δ40p53α, Δ133p53α, Δ160p53α] or EV in controls (ctrl). Forty-eight hours after transfection, cells were mock- or MMC-treated (3 μM, 45 min, 3 h release) and processed for immunostaining to visualize POLι and PCNA foci as well as their co-localization. At least 100 nuclei were scored in ≥ two independent experiments. Mean values for p53α expressing cells after MMC-treatment were set to 1 [on average 103 POLι ( A , D ), 133 PCNA ( B , E ), and 15 POLι-PCNA co-localized ( C , F ) foci/nucleus]. For graphic presentation, calculation of SEM and statistically significant differences via Dunn’s multiple comparisons test GraphPadPrism8.4 software was used. Representative images of MMC-treated samples are shown in ( G ). The experiments shown in ( A ) and ( D ), ( B ) and ( E ) as well as ( C ) and ( F ) were performed together (values of ctrl and p53α are identical but separated in different panels for clarity) and with the experiments presented in Fig. . *(#) P < 0.05, **(# #) P < 0.01, ***(# # #) P < 0.001, ****(# # # #), P < 0.0001 (scale bar: 5 µm).

Article Snippet: Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Techniques: Transfection, Expressing, Immunostaining, Software

Journal: Cell Death & Disease

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

doi: 10.1038/s41419-021-04224-3

Figure Lengend Snippet: K562 cells were transfected with a total amount of 10 μg plasmid DNA, containing either EV only in controls (ctrl) or expression plasmid for p53α plus EV (p53α + EV) or for p53α plus p53α (p53α + p53α) or for p53α plus one of the alternative isoforms (p53α + p53β, p53α + p53γ, p53α + Δ40p53α, p53α + Δ133p53α, and p53α + Δ160p53α). For graphic presentation, calculation of SEM and statistically significant differences via Dunn’s multiple comparison test GraphPadPrism8.4 software was used. # indicates a statistically significant difference between ctrl and the respective p53-isoform values. *(#) P < 0.05, **(# #) P < 0.01, ***(# # #) P < 0.001, **** P (# # # #) < 0.0001. A, B : DNA fiber-spreading assays were performed 48 h after transfection. The experimental design was as described in the legend to Fig. . Both mock-treated CldU- and IdU-tracks were measured but for clarity graphic presentations focus on IdU-tracks in ongoing forks (≥421 fibers ( A ) and ≥320 fibers ( B ) in two independent biological experiments). POLι ( C ), PCNA ( D ), POLι-PCNA colocalization ( E ) foci fold changes in K562 cells revealed by immunofluorescence microscopy. Mean values of p53α + p53α expressing and MMC-treated samples were set to 1 [on average 103 POLι ( C ), 133 PCNA ( D ), 15 POLι-PCNA colocalization ( E ) foci per nucleus]. Experiments were performed together with the ones shown in Fig. . ( F ) Western blots for co-expression of p53 isoforms. Co-expression of p53α and C-terminal isoforms were detected by anti-p53 antibody DO-1 (mouse, 554293, BD Biosciences, Franklin Lakes, New Jersey, USA). Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Article Snippet: Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Comparison, Software, Immunofluorescence, Microscopy, Western Blot

Journal: Cell Death & Disease

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

doi: 10.1038/s41419-021-04224-3

Figure Lengend Snippet: K562 cells were transfected with expression plasmids for p53α, alternative p53 isoforms [ A p53β, p53γ, B Δ40p53α, Δ133p53α, Δ160p53α] or EV in controls (ctrl). Forty-eight hours after transfection, cells were harvested for immunoprecipitations (IP) after preparation of crosslinked chromatin ( A ) or without crosslinked chromatin preparation ( B ). For the pull-downs of PCNA ( A ) anti-PCNA antibody (mouse, ab29, abcam, Cambridge, UK) or control Mouse IgG (Santa Cruz, Dallas, Texas, USA), for the pull-downs of POLι ( A ) POLι-antibody (rabbit, A301-304A, Bethyl, Montgomery, USA) or Rabbit IgG were used. For the pull-down of p53 ( B ) a mix of anti-p53(DO11) (mouse, MCA1704, BioRad Laboratories, München, Germany) and anti-p53(Pab421) (mouse, OP03, Calbiochem, Darmstadt, Germany) or control Mouse IgG were used. Subsequent immunoblotting relied on anti-POLι (rabbit, A301-304A, Bethyl, Montgomery, USA), anti-p53(DO1) (mouse, mAb, 554293, BD, Biosciences, Franklin Lakes, New Jersey, USA) in ( A ), anti-p53(DO11) (mouse, GTX75258, Genetex, Irvine California, USA), anti-p53 (PAb421, rabbit, ab245685, Abcam Cambridge, UK), anti-PCNA (mouse, ab29, abcam, Cambridge, UK) in ( B ) and light chain-specific peroxidase-coupled secondary antibody. *, asterisks mark bands possibly stemming from proteolytic cleavage of the p53-isoform. Quantification of band intensities of co-precipitated proteins relative to their input is indicated. Representative Western blots from three to four experiments are shown.

Article Snippet: Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Techniques: Transfection, Expressing, Control, Western Blot

Journal: Cell Death & Disease

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

doi: 10.1038/s41419-021-04224-3

Figure Lengend Snippet: K562 cells were transfected with expression plasmids for p53α, alternative p53 isoforms [ A p53β, p53γ, B Δ40p53α, Δ133p53α and Δ160p53α] or EV in controls (ctrl). Forty-eight hours after transfection, cells were either mock- or MMC-treated (3 μM, 45 min, 3 h release), subsequently cells were lysed with either lysis buffer for protein extraction (50 mM Tris [pH7.4], 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 25 mM Sodium fluoride, 25 mM β-Glycerol phosphate, 0.1 mM Sodium vanadate, 0.2% Triton X-100, 0.3 % Nonidet P40, complete protease inhibitor [Roche]) or IP lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP40, complete protease inhibitor [Roche]) and then processed for immunoblotting using ubiquityl PCNA (Lys164, D5C7P, Cell Signaling, Massachusetts, USA) antibody, recognizing PCNA protein only when ubiquitinated at Lys164, as well as antibodies against PCNA and p53. β-Actin was used as loading control. “ub” indicates ubiquitination. Quantification of respective protein expression level was carried out using ImageLab software. Levels of PCNA mono-/polyubiquitination were corrected for PCNA and normalized to ctrl ( C , D ) which was set to 1 on each blot. Statistically significant differences among groups were calculated by Friedman test followed by Wilcoxon-signed ranks test in case of statistical significance. Representative Western Blots from cells expressing ctrl, p53α, and C-terminal isoforms ( A ) or cells expressing ctrl, p53α, and ΔN-isoforms ( B ). Heatmap of mean values from ≥ 5 independent experiments ( C ) or ≥ 4 independent experiments ( D ).

Article Snippet: Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Techniques: Transfection, Expressing, Lysis, Protein Extraction, Protease Inhibitor, Western Blot, Control, Ubiquitin Proteomics, Software

Journal: Cell Death & Disease

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

doi: 10.1038/s41419-021-04224-3

Figure Lengend Snippet: U2OS cells ( A ) or HSPCs ( B ) were transfected with nonsense siRNA (sictrl), siRNA targeting all p53-isoforms (sip53) or siRNA targeting Δ133p53/Δ160p53 isoforms (siΔ133p53). A, B : 24 h after transfection, cells were sequentially incubated with CldU (20 µM) and IdU (200 µM) for 20 min. During IdU-incorporation, cells were either mock-treated or treated with 3 µM MMC. Both CldU- and IdU-tracks were measured while only IdU-tracks in ongoing forks (≥257 fibers from two independent biological experiments) are graphically presented for clarity. Knockdown of p53-isoforms was verified by immunoblot staining using anti-p53 (DO-11, MCA1704, Biorad) shown in the right panel of ( A, B ) each. Quantification of Δ133p53α relative to α-Tubulin levels was achieved by Imagelab and is indicated above the blots. The dashed frame in ( A ) marks an unspecific band stained by DO-11 in U2OS cells that had to be cut out, followed by antibody reincubation and a long exposure for 300 s to permit immunodetection of the Δ133p53α-band. Statistically significant differences between groups were calculated by Dunn’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Techniques: Transfection, Incubation, Knockdown, Western Blot, Staining, Immunodetection

Journal: Cell Death & Disease

Article Title: p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

doi: 10.1038/s41419-021-04224-3

Figure Lengend Snippet: A Hetero-oligomerization or aggregation of p53α with alternative p53 isoforms, particularly with p53α + Δ133p53α or p53α + Δ160p53α, can antagonize p53α functions in the POLι-dependent DDT. B C-terminal p53 isoforms still interact with PCNA [ , ] but are compromised in binding to three-stranded DNA junctions like replication forks due to shortened OD . Intact DBD, OD, and RPA-interaction sites , but compromised interactions with POLι and PCNA due to lack of the N-terminal end in Δ40p53α permit limited replication fork recognition only. Δ133p53α or Δ160p53α are compromised in DNA-binding due to the N-terminally truncated DBD and in RPA-, POLι and PCNA-binding due to the missing N-terminus. However, all N-terminal p53 isoforms retain the OD, enabling hetero-oligomerization with p53α and therefore dominant-negative interference with p53α functions in performing idling events in complex with POLι and PCNA, giving time for PCNA ubiquitination and FR events by HLTF and ZRANB3 . Black arrows, DNA-binding; blue arrows, PCNA-binding; stippled arrow, compromised interaction.

Article Snippet: Co-expression of p53α and ΔN-terminal isoforms were detected by anti-p53 antibody DO-11 (mouse, GTX75258, Genetex, Irvine California, USA).

Techniques: Binding Assay, Dominant Negative Mutation, Ubiquitin Proteomics

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: Antibodies 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: Antibodies 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: Antibodies 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: Antibodies 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: Antibodies 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: Antibodies 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