EMBO/soda-nel huggingface dataset:
Unloading of homologous recombination factors is required for restoring double-stranded DNA at damage repair loci (2016)
Figure 2

Figure 2. Srs2 is required to restore dsDNA during de novo telomere addition.

A. Schematic of the genetic de novo telomere addition assay used in B. Cells with a galactose-inducible HO-cut are grown on YP agar with raffinose prior to plating appropriate dilutions on YPD (no DSB induction) to score cell titre, and YP with galactose to induce HO expression and DSBs. DSB repair via de novo telomere addition leads to URA3 loss and the ADH4-MNT2 locus becoming part of terminal restriction fragments containing telomeres, which can be assayed by Southern blotting. Triangle represents the HO site.

B. Srs2 requirement in de novo telomere addition in cells with and without functional HR. All strains are pif1-m2. Strains used: NK1264; NK2375, NK2376; NK2014, NK2015; NK2451, NK2452; NK2012, NK2013; NK2457, NK2458; NK2363, NK2364; NK2469; 2369, NK2370; NK2473-2475.

C. Schematic of the qPCR assay used in D to monitor (TG1-3)n addition to DSBs. Triangle represents HO site, blue arrows – qPCR primers.

D. Dynamics of (TG1-3)n addition monitored by qPCR through a time-course experiment (asynchronous populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Strains used: NK3292, NK3293; NK4670, NK4671; NK4112, NK4113; NK4114, NK4115; NK3292 est2∆, NK3293 est2∆; NK4232, NK4233.

E. Schematic of the qPCR assay coupled with PsiI digestion used in F to quantify ssDNA/dsDNA ratio at the de novo telomere addition locus. Numbers indicate positions of PsiI restriction sites and qPCR primer sequences relative to DSBs. Blue arrows represent qPCR primers, dashed lines – telomeres.

F. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronised populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D) while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671.
Unloading of homologous recombination factors is required for restoring double-stranded DNA at damage repair loci (2016)
Figure 3

Figure 3. Analysis of Srs2 requirement in BIR.

A. Schematic of the quantitative BIR assay. Modified chr. VIIL (red) and chr. IIR (blue) share a 6,272 bp homology (grey shadow) used to repair an HO-induced DSB by BIR. Black boxes indicate hybridization probes used in Southern blotting experiments to monitor re-synthesis of resected DNA (RS-probes, red) and BIR (BIR-probes, blue) respectively. Numbers next to the one-ended arrows indicate distances (in bp) from the homology to the distal restriction sites of the DNA fragments analysed by Southern blotting using the corresponding probes. Numbers between the restriction sites indicate the sizes of restriction fragments detected by the corresponding hybridisation probes.

B. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.

C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point.

D. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C – control strain NK3980.

E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.

Strains used: NK4070, NK4079; NK5321, NK5322.
Unloading of homologous recombination factors is required for restoring double-stranded DNA at damage repair loci (2016)
Figure 4

Figure 4. Srs2 is required to relieve Rad51-dependent inhibition of DNA synthesis.

A. Schematic of the genetic system used to analyse inducible DSB repaired by SSA. Chr. V contains ura3-52 and URA3 (at the endogenous URA3 locus) separated by ~ 4 kb of DNA containing KAN (grey box, arrow above indicates the promoter) and an HO site (triangle). Galactose-inducible expression of the HO endonuclease leads to DSB formation at the HO site. After DSB repair via SSA, the majority of cells become Kans Ura- as the 766 bp homology between URA3 and ura3-52 (grey shadows) is predominantly used.

B. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A. Average ± SD (n=4) is shown for each genotype. Strains used in B and D: NK4691-4693; NK4805-4808; NK5081-5084; NK5085-5091.

C. Schematic of the quantitative SSA assay used in panel D. Grey shadow represents the annealing region of 766 bp present on both sides of a DSB. Numbers next to the one-ended arrows indicate distances (in bp) from the homology to the restriction sites, SalI and EcoRI, used to generate DNA fragment L analysed by Southern blotting.

D. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. Also see Figure EV3 for blot images. Average ± SD (n=4) is shown for each time point.

E. A schematic for the basic DNA strand extension assay used in F-G.

F. Rad51 inhibits DNA synthesis of phiX174 ssDNA substrate (0.5 nM) by Polδ. Increasing amount of Rad51 (0.03, 0.08, 0.15, 0.25, 0.5, 0.75, 1.5 µM) was incubated with the pre-loaded replication complex (RFC (17.5 nM), PCNA (10 nM) and DNA) and DNA synthesis was started by the addition of Polδ (10 nM) and nucleotides containing α-32P labelled dATP.

G. Srs2(1-910) suppresses the inhibition of DNA synthesis by Rad51. The reaction was carried out the same as in F except of the increasing amount of Srs2(1-910) (5, 15, 50, 150 nM) was added to indicated reactions before start of DNA synthesis. The relative % of DNA synthesis is indicated.
Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism (2021)
Figure 2

Figure 2. Histopathological effects of 144DG11 and its pharmacokinetics.

(A) Left panel, Mice, treated as indicated, were sacrificed and the indicated tissues were collected and stained for PG (arrows) with PAS following diastase treatment. Scale bars, 200 µm (liver, muscle), 50 µm (cortex, heart, peripheral nerve). Right panel, PAS staining was quantified, as described in (Kakhlon et al., 2018), based on analysis of 4 sections from each tissue in n=3 wt, n=7 Gbeys/ys vehicle-treated, and n=9 144DG11-treated mice.

(B) Total glycogen in the indicated tissues, collected from n=5 nine month old vehicle and 144DG11-treated mice, was quantified as described in (Kakhlon et al., 2018). Repeated-measures 2-way ANOVA tests show that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). *, Significant difference (p<0.05) as determined by two-tailed t-tests.

(C) 144DG11 Pharmacokinetics. Gbeys/ys mice injected with 144DG1 were sacrificed 30, 60, 90, and 210 min post injection and the indicated tissues were removed, as well as 200 uL of serum drawn. Graph shows means (+/- SEM) of 144DG11 levels in the different tissues determined by LC-MS/MS (see Materials & Methods). Results obtained from n = 3 mice at each time point.
Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism (2021)
Figure 3

Figure 3. In vivo metabolic profile of mice treated with 144DG11.

A-F Mice were monitored over a 24 hr period. Effective mass was calculated by ANCOVA (see Materials & Methods). Data are mean±SEM from nine month old mice (n=11, wt vehicle-treated, n=6 Gbeys/ys vehicle-treated, and n=7 Gbeys/ys 144DG11-treated). Vehicle-treated Gbeys/ys mice demonstrate lower respiratory quotient (in the light) (A), total energy expenditure (TEE) (B), and fat oxidation (C) compared to wt controls. 144DG11 treatment increased these parameters (for fat oxidation only in the dark and total time). Carbohydrate oxidation and ambulatory activity, not significantly affected by the diseased state, were increased by 144DG11 even beyond wt control levels (D and E) (note, while 144DG11 increased carbohydrate oxidation in the light (D), p was only <0.06). 144DG11 also reversed the decrease in meal size and water sip volume observed in Gbeys/ys mice as compared to wt control (F).

(G) Blood metabolic panel based on n=3, 9.5 month old mice treated as indicated. Blood glucose was increased and blood triglycerides decreased in Gbeys/ys cells by 144DG11 (p<0.05).

Data information: *p<0.05 v wt controls, #p<0.05 v Gbeys/ys vehicle treated mice. Statistical differences were determined by two-tailed t-tests.
Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism (2021)
Figure 5

Figure 5. 144DG11 interacts with LAMP1 and its associated interactome.

(A) Hetero-assembly forms around 144DG11 and not around endogenous molecules as shown by the liquid crystals formed in experiments 1-3 (see Materials & Methods).

(B) STRING network of targets at the interactome of 144DG11. Targets also show strong protein-protein interactions among themselves, as connectors show (see legend). SLC17A5, Solute Carrier Family 17 Member 5; LAMP-1, Lysosomal Associated Membrane Protein 1; LAMTOR4, Late endosomal/lysosomal adaptor, MAPK and MTOR activator 4; GBA, Glucosylceramidase; SCARB2 (aka LIMP2), Scavenger Receptor Class B Member 2; DPP4, Dipeptidyl peptidase-4; GAA, Lysosomal alpha-glucosidase; HEXA, Beta-Hexosaminidase Subunit Alpha.

(C) Cellular thermal shift assay (CETSA) of different targets of the 144DG11hetero-assembly (B). Only LAMP1 was significantly protected by 144DG11 from heat-mediated denaturation (see right shift in its Tm in the lower panel) suggesting its specific interaction with 144DG11. n=3 biological replicates. Error bars represent S.D.

(D) Surface plasmon resonance assays demonstrate dose and pH-responsive interaction between LAMP1 and 144DG11. Full interaction (but with apparent KD of 6.3mM) was demonstrated only at the lysosomal pH 4.5-5. (E) Upper panel, LAMP1 was deglycoslated as detailed in Materials & Methods. RNase B is a positive control for a glycosylated protein. Glycosylation status is shown after short (24 h) or long (72h) dialysis. The results demonstrate a full deglycosylation of both LAMP1 luminal part and RNase B, with unique bands appearing after a long dialysis. Lower panel, surface plasmon resonance, performed as in (D), showing that the luminal domain of deglycosylated LAMP1 specifically binds 144DG11 with apparent KD of 52.5 nM. (F) Three binding modes of 144DG11 (gray) according to LAMP1 grids predicted by SiteMap (green), fPocket (cyan) and FtSite (magenta). Two out of three binding modes (SiteMap and fPocket) are identical. In FtSite part of the molecule went through a rotation relative to the other two. See Materials & Methodss5.
Spatiotemporally controlled induction of autophagy-mediated lysosome turnover (2013)
f1

(a) Schematic for spatiotemporally controlled light-activated lysosomal damage. Illumination of individual lysosomes labelled with the robust photosensitizer AlPcS2a leads to light-activated damage. (b) AlPcS2a can be specifically targeted onto lysosomes. Using our staining procedure, AlPcS2a signals colocalized with Lysotracker Red (top panels) and lamp1-RFP (middle panels), but not with other cellular organelle markers such as EGFP-LC3B (bottom panels, marker for autophagic structures), in HeLa cells. Scale bars, 10 μm.
Spatiotemporally controlled induction of autophagy-mediated lysosome turnover (2013)
f3

(a) A HeLa cell expressing EGFP-Ub and lamp1-RFP, and labelled with AlPcS2a, was 635-nm illuminated within the white square region. (b) Punctate structures containing ubiquitin appeared within the 635-nm illuminated region (white arrow) 30–50 min after light activation. (c) Ubiquitin puncta colocalized with damaged lysosomes. Magnified view of the 635-nm illuminated cell region showed that ubiquitin signals localized on lysosomes (lamp1-labelled) lacking AlPcS2a fluorescence (indicating that it had been 635-nm illuminated). White arrows indicate examples of Ub on damaged lysosomes. (d) Quantification of ubiquitin signals within three independent cellular regions (A1, A2 and A3) revealed a specific recruitment of ubiquitin to the site of light activation (inset, right, A1). Scale bars, 10 μm (a,b,d); 3 μm (c).
Spatiotemporally controlled induction of autophagy-mediated lysosome turnover (2013)
f4

(a) A HeLa cell expressing EGFP-Ub and TagRFP-p62, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in ubiquitination of and p62 recruitment to the photodamaged lysosomes, as shown in b. (b) Magnified view of the 635-nm illuminated cell region in a. Ubiquitin and p62 initially distributed uniformly within the region (t=0 min, top panels), but formed distinct foci after light activation (bottom panels). (c) A HeLa cell expressing EGFP-LC3B and lamp1-RFP, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in LC3 recruitment onto photodamaged lysosomes, as shown in d. (d) Magnified view of the 635-nm illuminated cell region in c. LC3 is initially distributed away from lysosomes (lamp1, t=0 min, top panels), but appears on lamp1-positive structures 40 min after light activation (bottom panels). Scale bars, 10 μm (a,c); 3 μm (b,d).
Spatiotemporally controlled induction of autophagy-mediated lysosome turnover (2013)
f5

(a) Two HeLa cells expressing RFP-EGFP-LC3 and labelled with AlPcS2a was 635-nm illuminated within the white rectangular region, resulting in local LC3 puncta formation, as shown in b. (b) LC3 puncta were specifically induced within the 635-nm illuminated cell regions in a (LC3 initially distributed uniformly at t=0). (c) The loss of EGFP fluorescence from light-induced LC3 puncta preceded the disappearance of RFP fluorescence (white arrows indicate examples of LC3 puncta that exhibited diminished EGFP fluorescence), indicating their successful transition from autophagosomes to autolysosomes. Scale bars, 10 μm.
Differential Contribution of Steady-State RNA and Active Transcription in
Chromatin Organization (2019)
Figure 1

Figure 1 - Overall genome organization is preserved upon RNase digestion

A) Experimental scheme of RNase A digestion of K562 cells before and after formaldehyde (FA) crosslinking (termed as bXL and aXL, respectively) followed by the Hi-C assay.

B) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells.

C) Experimental scheme of ActD treatment of K562 cells followed by Hi-C analysis.

D) Agarose gel electrophoresis of RNA extracted from control, 4hr and 24hr ActD treated K562 cells.

E) Barplots showing qRT-PCR gene expression levels (mean +/- S.D.) of PTEN, FNDC3B, and STAM genes in control, 4hr and 24hr actinomycin D treated K562 cells. p-value: two-tailed t-test, (*** p < 0.05). The results represent data from 3 technical replicates from 3 independent biological preparations.
Gi-coupled receptor activation potentiates Piezo2 currents via Gβγ (2020)
Figure 3

Figure 3. Activation of other Gi-coupled receptors also potentiates native MA currents in DRG neurons and recombinant Piezo2 currents

(A-B) Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+D2RS-YFP (A, n=13) and HEK293 cells transfected with Piezo2 and GFP (B, n=5) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.

(C) Quantification of Piezo2 current inactivation rate (Tau) in HEK cells transfected with Piezo2+D2RS-YFP. Data are shown as mean ± SEM and scatter plots (n=13)

(D,E) Quantification of normalized MA currents in responding large DRG neurons (D, n=5) versus non-responding DRG neurons (E, n=3) before (black) and after (red) application of sumatriptan 10 µM to activate the Gi-coupled Htr1d receptors. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.

(F) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=5, Paired t-test). Data are shown as mean ± SEM and scatter plots.

(G-H) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng/ml Gβγ (G, n=7) or with 0.0001% Lubrol (vehicle for Gβγ) (H, n=6). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.

(I,J) Quantification of MA current inactivation rate (Tau) measured for large DRG neurons dialyzed with Gβγ (n=7, **P<0.01, Paired t-test) and vehicle (Paired t-test). Data are shown as mean ± SEM and scatter plots.
Gi-coupled receptor activation potentiates Piezo2 currents via Gβγ (2020)
Figure 4

Figure 4. PI3K and MAPK participate in the potentiation of Piezo2 currents after activation of GABAB receptors.

(A-C) Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the PI3K inhibitor Wortmannin 50 nM (B, n=10) or treated with the MAPK inhibitor U0126 10 µM (C, n=7). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data shown as mean ± SEM. Cells were treated with wortmannin or U0126 for 30 min before the experiments, and were present throughout the measurements. D) Putative pathway of regulation of Piezo2 currents by Gi-coupled receptors; activation of Gi-coupled receptors promotes the release of Gβγ activating PI3K and MAPK kinase, thus indirectly potentiating Piezo2 currents.
Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin (2018)
Figure 1

Figure 1. Irc5 prevents DNA damage accumulation during replication stress.

A MMS-induced checkpoint activation is not altered in irc5-Δ1 cells. The level of Rad53 phosphorylation was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody.

B Prolonged checkpoint activation in irc5-Δ1 cells during recovery from MMS treatment. Rad53 phosphorylation status was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody; M, MMS.

C Lack of Irc5 results in an elevated number of cells with Rad52-YFP foci. At indicated time points samples of wild type (W3749-14C) and irc5-Δ1 (TB039) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm.

D Disruption of IRC5 leads to accumulation of ssDNA-containing lesions. ChEC analysis of wild type (BYR52MN) and irc5-Δ1 (TB040) cells cultured in the presence or absence of 0.03% MMS for 2 hours (n=3).

E Quantification of data presented in D (n=3).

F Increased levels of bright Rfa1-YFP foci in cells lacking Irc5. At indicated time points culture samples of wild type (W3775-12C) and irc5-Δ1 (TB041) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm.
Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin (2018)
Figure 2

Figure 2. Irc5 is required for S phase progression in the presence of MMS-induced DNA damage.

A The irc5-Δ1 mutant exhibits S-phase delay in the presence of MMS. DNA content of wild type (W303-1a) and irc5-Δ1 (IL012) cells was measured by FACS and plotted as histograms.

B Completion of DNA replication is delayed in the irc5-Δ1 mutant after MMS treatment. Intact chromosomes were isolated from wild type (W303-1a) and irc5-Δ1 (IL012) cells and processed for PFGE. M, 0.03% MMS.

C Quantification of data presented in B. Error bars represent mean value ± standard deviations of mean for the intensity values of three distinct chromosome bands representing chromosome XII or III (n=3). Values for G1 were set to 100.

D Samples used for PFGE in B were processed for FACS analysis.
Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin (2018)
Figure 3

Figure 3. Translocase activity of Irc5 protects against DNA damage accumulation and enables replication completion.

A Lack of Irc5 translocase activity results in elevated number of cells with Rfa1-YFP foci. At indicated time points samples of wild type (TB042) and irc5DAEA (TB043) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value.

B Increased levels of bright Rad52-YFP foci in cells expressing Irc5DAEA lacking ATPase activity. At indicated time points samples of wild type (TB044) and irc5DAEA (TB045) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value.

C Translocase activity of Irc5 is important for completion of DNA replication after MMS treatment. PFGE was performed as in Fig 2B using wild type (EMD09) and irc5DAEA (EMD10) strains.

D Quantification of data presented in C. Error bars represent mean value ± standard deviations of mean for the intensity values of three distinct chromosome bands representing chromosome XII or III (n=3). Values for G1 were set to 100.

E Samples used for PFGE in C were processed for FACS analysis.
Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin (2018)
Figure 5

Figure 5. Irc5 promotes cohesin accumulation at stalled replication forks.

A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. PK-tagged Scc1 interaction with chromatin was monitored in wild type (JC1513) and irc5Δ-1 (TB064) cells. Error bars represent mean value ± standard deviations of mean (n=3). ARS305 + 7 kb locus was used as a control for cohesin-free chromosomal region. POA1 was used as a control locus for the chromosomal region that is bound by cohesin but is located ~ 30.000 bp from the nearest replication origin.

C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607 as well as late replication origin ARS501. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB065) and irc5Δ-1 (TB066) cells. Error bars represent mean value ± standard deviations of mean (n=3).