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