EMBO/soda-nel huggingface dataset:
Promyelocytic leukemia protein targets MK2 to promote cytotoxicity (2021)
Figure 6

Figure 6. MK2 deficiency confers sensitivity of PML-null cells to necroptosis.

(A, B) WT and Mk2-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) (A) or with zVAD (20 μM) + AT-406 (1 μM) (B), and then the contents of p-RIPK1[S321], RIPK1, p-p38, p38 was determined at the indicated time-points.

(C) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM), AT-406 (1 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method.

(D) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method.

(E) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM) and AT-406 (1 μM) for the indicated timeframes. The levels of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates were analysed by immunoblots. Results are representative of three biological replicates.

(F) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice were injected i.v. with TNF (1.5 mg/kg), and survival was monitored for 24 h. Data represent a combination of two independent experiments (n=10). *P < 0.05 for Log-rank (Mantel-Cox) test.

(G, H) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice (n = 5) were injected i.v. with TNF (1.5 mg/kg), before recording body temperature for the next 6 h (G), and collecting serum samples at 3 h to quantitate LDH release (H). *P < 0.05 as determined by two-way ANOVA (G), or P values calculated by unpaired t-test with Welch's correction (H).
Promyelocytic leukemia protein targets MK2 to promote cytotoxicity (2021)
Figure 7

Figure 7. PML interacts with MK2 and inhibits p38 MAPK-MK2 association.

(A, C) WT BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. Cell lysates were immunoprecipitated with anti-MK2, and levels of PML were determined. Mk2-/- BMDMs served as a negative control.

(B) WT BMDMs were treated with TNF (100 ng/ml) for the indicated times, before immunoprecipitating p38 MAPK and determining the levels of PML, p38, MK2 in the precipitates and total cell lysates.

(C) WT BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. Cell lysates were immunoprecipitated with anti-PML, and levels of p38 and MKK3 were determined. Pml-/- BMDMs served as a negative control.

(D) WT and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. p38 MAPK was immunoprecipitated, and its association with MK2 was then analysed.

(E) Recombinant Flag-PML-I, GST-MK2 (60 ng) and His-p38 (60 ng) were incubated, as indicated, at 4 °C for 2 h. p38 was pulled down by anti-p38 and then PML, MK2 and p38 contents in the precipitate and reaction mixture (input) were detected by immunoblotting.

(F) Recombinant Flag-PML-I, GST-MK2 (100 ng) and His-p38 (200 ng) were incubated, as indicated, at 30 °C in in vitro kinase assay buffer (20 mM HEPES, 10 mM MgCl2, 100 nM ATP, 1 mM DTT) for 20 min. The levels of p-MK2, MK2, p38, and PML (FLAG) in the reaction mixture were then determined.

Data information: Experiments (A-F) were independently repeated three times (biological replicates) and generated similar results.
Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance (2020)
Figure 1

Figure 1. Persistence of MMS-induced Rad52 foci in TS deficient cells

(A) Scheme for the two major mechanisms of DDT: HR and TLS. An asterisk represents a blocking DNA lesion for the replicative polymerase.

(B) Representative image of wild-type cells with MMS-induced Rad52-YFP foci (bright field and fluorescence signal)

(C-G) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.
Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance (2020)
Figure 2

Figure 2. Persistence of MMS-induced Rad52 foci in TLS deficient cells

(A) ssDNA repair efficiency of cells expressing RAD18 from the GAL1 promoter as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panel). Cells were synchronized in G1 from galactose-containing medium and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. Cells were shifted to glucose-containing medium 1 hour after MMS inactivation. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panel). See Figure EV1A for control cells released in galactose-containing medium during the entire time course.

(B) ssDNA repair efficiency of cells expressing MMS2 from the GAL1 promoter as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panel). Cells were synchronized in G1 from galactose-containing medium and maintained in galactose (a, c) or shifted to glucose (b) for 1 hour in G1; subsequently, cells were released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. Cells were maintained either in galactose (a), glucose (b) or shifted to glucose 2 hour after MMS inactivation (c). The percentage of cells with foci at each point was normalized to the highest value in galactose, taken as 100 (left panel). Wild type and mms2∆ controls were analyzed in parallel (Figure EV1B).

(C-E) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (D and E, right panel). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (C and E, left panel).

Data information: The mean and SEM of 4 (A), 3 (B, E) and 3-12 (C-D; 12 (wild type), 6 (rev1∆, rev3∆, rad30∆, rev1∆ rad30∆, rev3∆ rad30∆), and 3 (rev1∆ rev3∆ and rev1∆ rev3∆ rad30∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.
Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance (2020)
Figure 3

Figure 3. Persistence of Rad52 foci in TLS mutants is not due to a demand for TLS polymerases during HR

(A) ssDNA repair efficiency of the rev1∆ mutant treated with MMS only in G1 as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panel). Cells were synchronized in G1 and treated with 0.033% MMS for 1 hour; after that the MMS was inactivated with 2.5% sodium thiosulfate and cells were released into fresh medium in the absence of DNA damage. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (middle panel). A scheme on the left shows the experimental set up followed in this (MMS in G1 only) and the other experiments (MMS in S/G2). An asterisk represents a blocking DNA lesion for the replicative polymerase.

(B) Effect of the rev1∆ and rev3∆ mutations in spontaneous and MMS-induced uSCE. The frequency of spontaneous and MMS-induced recombinants was determined from colonies grown in the absence or presence of 0.01% MMS. The mean and SEM of 4 independent fluctuation tests are shown.

(C-D) ssDNA repair efficiency of the rad5∆ and rev1∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels).

Data information: The mean and SEM of 4 (A) and 3 (C, D) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.
Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance (2020)
Figure 4

Figure 4. Rad52 is required for post-replicative ssDNA gap filling through a non-recombinogenic process

(A) Effect of the rad54∆ and rad52∆ mutations in spontaneous and MMS-induced uSCE. The frequency of spontaneous and MMS-induced recombinants was determined in mid-log phase cell cultures before (spontaneous uSCE) and after treatment with 0.02% MMS for 4 hours (DNA damage-induced uSCE). Thus, the frequency of DNA damage-induced uSCE includes the recombinants that arose spontaneously before MMS addition. The frequency of uSCE was determined at 0.02% MMS because at higher MMS concentrations the loss of viability of rad52∆ and rad54∆ mutants did not allow us to select for His+ recombinants. The mean and SEM of 5 independent fluctuation tests are shown; n.a., not applicable.

(B) ssDNA repair efficiency of the indicated mutants as determined from the maximal and final percentages of cells with RPA foci during the time course (See Figures EV2D-E for the time course analyses; cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times).

(C-E) Number of foci per cell in cells with foci (C), fluorescence signal per focus (D) and fluorescence signal of RPA foci per cell (E) at times 1 or 2 (peak) and 7 hours from the time course shown in (B). Counting and quantification were done with the MetaMorph software. Representative images of wild-type and mutant cells with MMS-induced RPA foci (bright field and fluorescence signal) are shown (D; left panels).

(F) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1-YFP at the ARS305 replication origin. Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium. The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release. RPA enrichment was calculated as the ratio between immunoprecipitate and input values. All values were normalized to the wild type at 7 hours, taken as 1.

Data information: (B-F) The mean and SEM of 3 independent experiments are shown. (A-F) Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. In (F) all values were significant relative to the untagged controls (not shown for clarity).
Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance (2020)
Figure 5

Figure 5. Rad52 is required for MMS-induced mutagenesis.

Effect of the lack of HR factors in spontaneous and MMS-induced mutagenesis. The frequency of spontaneous and MMS-induced forward mutagenesis at the CAN1 locus was determined in mid-log phase cell cultures before (spontaneous mutagenesis) and after treatment with 0.01% MMS for 4 hours (DNA damage-induced). Thus, the frequency of DNA damage-induced mutagenesis includes the mutants that arose spontaneously before MMS addition. The frequency of mutagenesis was determined at 0.01% MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine-resistant cells. Survival (%) after 4 hours in MMS relative to the wild type (taken as 100) is shown below the genotypes. The mean and SEM of 6 (wild type), 9 (rad54∆) and 3 (rest) independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test (one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively). Only the statistical analysis of the effect of rad52∆, rad57∆ and rad51∆ on rad54∆-mediated mutagenesis is included for clarity. All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS.
Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance (2020)
Figure 6

Figure 6. Rad52 is required for efficient UV-induced TLS

(A) Effect of the rad54∆ and rad52∆ mutations in spontaneous and UV-induced uSCE. The frequency of spontaneous and UV-induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM without histidine to calculate total and recombinant cells, respectively). The mean and SEM of 3-5 independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. Two and three asterisks represent p-values <0.01 and <0.001, respectively.

(B, C) ssDNA repair efficiency (B) and fluorescence signal at RPA foci per cell (C) of the indicated mutants. ssDNA repair efficiency was determined from the maximal and final percentages of cells with RPA foci during the time course (B, right panel). Cells were synchronized in G1, irradiated with 20 J/m2 and released into S phase for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (B, middle panel). Cell cycle progression was determined by cell sorting (B, left panel). Counting and quantification were done with the MetaMorph software. The mean and SEM of 4 independent experiments are shown. Statistically significant differences according to a paired two-tailed Student's t-test are shown, where two and three asterisks represent p-values <0.01 and <0.001, respectively.

(D) Effect of the rad54∆ and rad52∆ mutations in spontaneous and UV-induced mutagenesis. The frequency of spontaneous and UV-induced forward mutagenesis at the CAN1 locus was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM with canavanine to calculate total and mutant cells, respectively). Cell viability (right panel) was calculated from total irradiated cells relative to non-irradiated cells. The mean and SEM of 6 independent fluctuation tests are shown. Two asterisks represent a p-value <0.01, according to a two-way ANOVA followed by a Bonferroni test.
Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance (2020)
Figure 7

Figure 7. Rad52, Rad51 and Rad57, but not Rad54, are required for efficient Rad6/Rad18 binding to chromatin.

(A) Scheme with the rationale of the ChEC approach for non-DSB lesions. Rad6 fused to MNaseI will induce a detectable cut only if it is bound to a DNA lesion other than a DSB.

(B) Rad6 binds to DNA in the absence and presence of MMS. ChEC analysis of G1-synchronyzed cells released in the absence or the presence of 0.033% MMS for the indicated times.

(C-F) Rad6 binding to DNA depends on Rad18, Rad52 (C), Rad51 (D), Rad57 (E) but not Rad54 (F) as determined by ChEC analyses of asynchronous cell cultures without or with 0.05% MMS for 2 hours.

Data information: (B-F) DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.
Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance (2020)
Figure 8

Figure 8. Rad52, Rad51 and Rad57, but not Rad54, are required for efficient PCNA ubiquitylation.

(A-D) Effect of rad52∆, rad51∆ (A), rad57∆ (B) and rad54∆ (C) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0.025% MMS. Cell cycle progression was followed by cell sorting analysis and western blot against Clb2. Pgk1 was used as loading control. The amounts of mono- and polyubiquitylated PCNA were normalized to the total amount of PCNA. For each time point, rad mutant values were relativized to those of the wild type, taken as 1. The mean and SEM of 3 independent experiments are shown (D). An asterisk indicates a mean significantly different than 1, according to a One-sample t-test.

(E) Model for Rad52-mediated tolerance during DDT. The recombination factors Rad52, Rad51 and Rad57 promote efficient Rad6/Rad18 binding to chromatin under unperturbed conditions. In response to blocking lesions that impair replication fork advance the Rad6/Rad18 complex is mobilized to ssDNA lesions where, together with Mms2/Ubc13/Rad5, ubiquitylates PCNA and promotes TS. The ssDNA lesions that escape from this error-free pathway during S phase accumulate in G2/M, where they are filled in by two additional, less accurate mechanisms regulated by Rad52: UbPCNA-independent HR (recombinogenic) and TLS (mutagenic). Apart from facilitating Rad6/Rad18 chromatin recruitment, Rad52 plays an additional uncharacterized Rad51/Rad57-independent role in TLS.
Aneuploid embryonic stem cells exhibit impaired differentiation and increased neoplastic potential (2016)
Figure 3

Figure 3. Limited differentiation abilities of trisomic ES cells in vitro.

A. The coloning-formation rates of the trisomic and wild-type ES cells. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.

B. Alkaline phosphatase (AP) staining of trisomic and wild-type ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm.

C. Evaluation of AP positive and negative colonies after LIF withdrawal. Black bars, undifferentiated colonies; orange bars, partially differentiated colonies; green bars, totally differentiated colonies. Error bars, ±S.D. n=3. **, P < 0.01.

D. Boxplots of EB diameter at the indicated days of EB formation. Each boxplot was plotted for more than 20 EBs. * P < 0.05, ** P < 0.01.

E. Time course analysis of marker genes expression during EB formation by RT-qPCR. Error bars, ±S.D. n=3.

F. Quantification of Oct4, Nanog and Rex1 expression in trisomic and wild-type ES cell derived EBs at day 8 of differentiation. Error bars, ±S.D. n=3. ** P < 0.01.
Aneuploid embryonic stem cells exhibit impaired differentiation and increased neoplastic potential (2016)
Figure 4

Figure 4. Enhanced teratoma formation capacities of trisomic ES cells.

A. Teratoma growth curves of trisomic and wild-type ES cells after subcutaneous injection into SCID mice. 1×106 cells were injected per site (6 sites/per group). Error bars, ±S.D. * P < 0.05.

B. Four-week teratomas derived from wild-type and trisomic (Ts8 or Ts11) ES cell lines. 5×105 cells were injected per site (8 sites/per group).

C. Representative images of undifferentiated regions in Ts8 ES cell derived teratomas (middle, top and bottom) and the primitive neural rosettes in the teratomas derived from Ts15 ES cells (right, top and bottom). The images of mature neuroectoderm from wild-type ES cell formed teratomas (left, top and bottom) were shown. The bottom panels were the enlarged views of the white dotted boxes of the top panels. Hematoxylin and eosin staining. Scale bars, 50 μm.

D. Immunohistochemical analysis of Oct4 expression in wild-type, Homo-11, Ts6, Ts8, Ts11, or Ts15 ES cell teratomas produced via the injection of 5×105 ES cells. Scale bar, 100 μm.

E. AP staining of the primary cultured teratoma cells derived from trisomic and wild-type ES cells. Ts6, Ts8, or Ts11 ESC teratoma cells grew ES-like colonies that were stained positive for AP. Scale bar, 200 μm.

F. The expression of marker genes in cultured teratoma cells (p3). Error bars, ±S.D. n=3. ** P < 0.01.
Aneuploid embryonic stem cells exhibit impaired differentiation and increased neoplastic potential (2016)
Figure 5

Figure 5. Trisomy correction rescued differentiation defects of trisomic ES cells.

A. Schematic overview for correcting trisomic cells.

B. Karyograms of Di8, Di11 and Di15 ES cell lines. Di8 was the corrected ES cell line from Ts8, showing diploid karyotype and Y chromosome loss. Di11 and Di15 were from Ts11 and Ts15 ES cells respectively.

C. AP staining of trisomic and the corrected diploid ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm.

D. Teratoma growth curves of trisomic and the corrected diploid ES cells. The injected cell number was 5×105/site (8 sites/per group). Error bars, ±S.D. ** P < 0.01.

E. Scatterplot of four-week teratoma weight. The teratomas were formed by the corrected diploid ES cells and their paternal trisomic ES cells. * P < 0.05.
Aneuploid embryonic stem cells exhibit impaired differentiation and increased neoplastic potential (2016)
Figure 7

Figure 7. Rescue of differentiation defects of trisomic ES cells by extracellular factors.

A. Morphology of EBs grew for 8 days derived from individual or mixed ES cell lines. Hematoxylin and eosin staining. Scale bar, 100 μm.

B. Quantification of Nanog expression in trisomic, wild-type, and mixed ES cell-derived EBs cultured for 8 days. Error bars, ±S.D. n=3. ** P < 0.01.

C. The morphology of EBs at day 10 of differentiation. M15 was the normal EB formation condition and was used as control. Black arrows indicate the cystic structures.

D. Boxplots of EBs diameter at day 10 of EB formation under M15, CM, and M15 supplemented with Bmp4 conditions. Each boxplot was plotted for more than 10 EBs. EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition. * P < 0.05, ** P < 0.01.