EMBO/soda-nel huggingface dataset:
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages (2009)
figf6

(a) Double knockdown of Rubicon and Atg14L. A549 cells stably expressing GFP–LC3 were infected with adenovirus expressing either shRNA against Rubicon and Atg14L or control shRNA (luciferase). The image is available in Supplementary Information, Fig. S5a. The GFP–LC3 puncta were counted in nutrient-rich (N) or starvation (S) conditions. The data are mean ± s.d. of three independent experiments; at least 23 cells were counted for each experiment. (b) A549 cells were infected with adenovirus harbouring shRNA for Rubicon or control (luciferase). The cells were cultured in N or S medium in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) for 1–4 h. Cell lysates were subjected to western blotting with the antibodies indicated. (c) Quantification of Atg16L puncta in Rubicon knockdown cells. The images are available in Supplementary Information, Fig. S5c. The data are mean ± s.d., n = 60 (60 cells, sample of Medium: N, shRNA: Control), n = 68 (68 cells, sample of Medium: S, shRNA: Control), n = 78 (78 cells, sample of Medium: N, shRNA: Rubicon), n = 83 (83 cells sample of Medium: S, shRNA: Rubicon). d) Bulk degradation activity of Rubicon knockdown cells. The bulk degradation activity in control and Rubicon knockdown cells in nutrient-rich medium was measured. Wortmannin (W, 100 nM) was added as a negative control. The data are mean ± s.d. of three independent experiments. (e) A549 cells infected with adenovirus harbouring shRNA for Rubicon and control (luciferase) were cultured in N or S medium for 4 h and cell lysates were subjected to immunoblotting with the antibodies indicated. Full scans of the blots in b and e are available in Supplementary Information, Fig. S6.
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages (2009)
figf7

(a) A549 cells transfected with a GFP–Rubicon expression plasmid and were subjected to immunofluorescence with various organelle marker antibodies. Scale bar, 10 μm. (b) A549 cells stably expressing GFP–LC3 were subjected to Rubicon knockdown (KD) and then transfected with mStrawberry, mStrawberry–Rubicon or mStrawberry–Rubicon mutant lacking the Beclin 1 binding region (Δ393849). Cells with strong expression of mStrawberry–Rubicon showed an overexpression-dependent dominant-negative phenotype; therefore, we selected cells with modest expression. The number of GFP–LC3 puncta was counted and mean ± s.d. are shown. mStrawberry, n = 12 (12 cells); mStrawberry-Mm-Rubicon, n = 14 (14 cells); mStrawberry-Mm-RubiconΔ393–849, n = 13 (13 cells).
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages (2009)
figf8

(a) The maturation of autophagosomes is enhanced in Rubicon knockdown cells. A549 cells stably expressing GFP–LC3, with Rubicon or control shRNA, were incubated in nutrient-rich medium with or without the protease inhibitors (PI) E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) treatment for 4 h. The cells were stained with an anti-Lamp1 antibody (upper panels) and colocalization efficiency between GFP–LC3 and Lamp1 was counted (lower panel). The data are mean ± s.d. of 30 cells. (b). Electron microscopy analysis of Rubicon knockdown cells. Control (luciferase) or Rubicon shRNA A549 cells grown in nutrient-rich medium was subjected to electron microscopy analysis (left panels). The arrows indicate autophagosomes and the arrowheads indicate autolysosomes. Scale bar, 1 μm. Autophagosomes and autolysosomes were counted in 21 cells, and the mean ± s.d. are shown (right panel). (c) EGFR degradation in Rubicon knockdown cells. Control or Rubicon shRNA A549 cells were treated with EGF (200 ng ml−1) for the indicated periods and the lysates were subjected to western blotting with the antibodies indicated (left panel). The band intensity was measured in three independent experiments and the mean ± s.d. are shown (right panel). (d) The effect of Rubicon overexpression in the turnover of EGF receptor. A549 cells were transfected with advenovirus-based GFP or GFP–Rubicon. After 24 h, EGF was added to cells expressing GFP–Rubicon or control GFP. At the times indicated, the cells were lysed and subjected to western blotting with the indicated antibodies. (e) Model of the function of three Beclin 1 containing complexes. The Atg14L complex functions in autophagosome formation. The UVRAG complex functions in autophagosome and endosome maturation. The Rubicon–UVRAG complex suppresses autophagosome and endosome maturation. Enhanced autophagosome maturation and/or endocytosis may lead to enhanced autophagosome formation. Full scans of the blots in c and d are available in Supplementary Information, Fig. S6.
DNA asymmetry promotes SUMO modification of the single-stranded DNA binding protein RPA (2021)
Figure 1

Figure 1 - RPA is SUMO modified on two major sites.

A Diagrams showing the domain organization of SUMO (Smt3), Siz2, Rfa1, Rfa2 and Rfa3. Orange circle indicates known (Rfa1 and Smt3) or predicted (Siz2) SUMO modification sites. Dash lines represent truncation mutants used in this study in addition to the full-length proteins.

B SYPRO-stained SDS-PAGE showing purified recombinant SUMO, E1∆CT, E2, Siz2 and RPA proteins. Mass in kilodaltons is annotated for all bands for the molecular weight marker.

C SUMO conjugation assays performed under multiple turnover conditions using wild-type or mutant forms of RPA, SUMO and Siz2. Only the 80-minute time points are presented to highlight the formation of Rfa1-SUMO and Siz2-SUMO species. The complete time series are presented in Appendix Fig S1. Bands for Mw marker are fully annotated in Figure 1B.
DNA asymmetry promotes SUMO modification of the single-stranded DNA binding protein RPA (2021)
Figure 3

Figure 3 - DNA with a 3' overhang increases SUMO conjugation to RPA.

A Plot showing the initial rate of SUMO conjugation versus RPA-DNA concentration as obtained from single-turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA-DNA complexes. Representative gels are shown in Appendix Fig S4.

B Histograms presenting Kd, k2 and specificity (k2/Kd) constant for SUMO conjugation to RPA-DNA.

Data information: In (A) data show mean ± s.d. of three technical replicates. In (B) Histograms show mean ± s.e.m. of the three technical replicates presented in (A).
DNA asymmetry promotes SUMO modification of the single-stranded DNA binding protein RPA (2021)
Figure 4

Figure 4 - SUMO conjugation to RPA is dependent on Siz2 SAP domain and partially dependent on Rfa2 WH domain.

A, B SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2∆CT lacking (A) or including (B) its SAP domain. In both cases, reactions were done in technical triplicate and SDS-PAGE gels were stained with SYPRO. Only one representative gel is shown. Bands for Mw marker are fully annotated in Figure 1B.

C Histograms derived from the gels presented in (B) and presenting Rfa1 SUMO conjugation rates for RPA complexes with or without the WH domain of Rfa2.

D Histograms derived from the curves presented in Appendix Fig S5 and presenting apparent dissociation constants (Kd) of Siz2∆CT with DNA and its complexes with RPA or RPA∆WH.

Data information: In (C) and in (D), data show mean ± s.d. for three technical replicates.
DNA asymmetry promotes SUMO modification of the single-stranded DNA binding protein RPA (2021)
Figure 6

Figure 6 - Siz2 DNA binding activity is correlated with SUMO conjugation to RPA in vitro.

A Fluorescence polarization assays performed with 50 nM 6-FAM-ds20, a 6-FAM-labeled 20 nucleotide double-stranded DNA, and serially diluted Siz2∆CT or Siz2∆CT mutant.

B Histograms derived from the curves presented in (A) and presenting apparent dissociation constants (Kd) for each Siz2∆CT variant.

C Histograms derived from the gels presented in (D) and presenting Rfa1 SUMO conjugation rates for RPA complexes in the presence of different Siz2∆CT variants.

D SUMO conjugation assays performed under multiple turnover conditions as in Fig 4. Experiments were performed in technical triplicates (six times for WT). The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.

Data information: In (A) data show mean ± s.d. for three technical replicates. Data were fitted to a single-site binding model accounting for ligand depletion and no detectable binding was measured for Siz2∆CT K65E K66E. In (B) data show mean ± s.e.m. for three technical replicates presented in (A). In (C) data show mean ± s.d. for three technical replicates (six for WT).
DNA asymmetry promotes SUMO modification of the single-stranded DNA binding protein RPA (2021)
Figure 7

Figure 7 - Some residues that differ between Siz2 and Siz1 contribute to SUMO conjugation of RPA.

A Fluorescence polarization assays performed with 50 nM 6-FAM-ds20 and serially diluted Siz2∆CT or Siz2∆CT mutant.

B Histograms derived from the curves presented in (A) and presenting apparent dissociation constants (Kd) for each Siz2∆CT variant.

C Histograms derived from the gels presented in (D) and presenting Rfa1 SUMO conjugation rates for RPA complexes in the presence of different Siz2∆CT variants.

D SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2∆CT variants. The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.

Data information : In (A) data show mean ± s.d. for three technical replicates. Data were fitted to a single-site binding model accounting for ligand depletion. In (B) data show mean ± s.e.m. for three technical replicates presented in (A). In (C) data show mean ± s.d. for three technical replicates except for WT. The values for WT (six technical replicates) are the same as in Fig 6 (these experiments were all performed at the same time).
DNA asymmetry promotes SUMO modification of the single-stranded DNA binding protein RPA (2021)
Figure 9

Figure 9 - Binding of full-length Siz2 to ds20ss32 DNA or ds20ss32-RPA complexes.

A Electrophoretic mobility shift assays (EMSA) performed with 10 nM 6-FAM-ds20ss32, a 6-FAM-labeled 20-mer dsDNA with a 32 nucleotide 3' overhang and its complexes with RPA, RPA with K170-SUMO modified Rfa1 or RPA reconstituted with ∆WH-Rfa2 and serially diluted Siz2. All titrations were done in technical triplicates. One representative gel is shown.

B Binding curves derived from the gels presented in (A) and presenting Siz2 binding to DNA and DNA-RPA complexes with K170-SUMO modified Rfa1 or without the WH domain of Rfa2.

C Histograms derived from the curves presented in (B) and presenting apparent dissociation constants (Kd) for Siz2 binding to DNA and DNA-RPA complexes with RPA with K170-SUMO modified Rfa1 or RPA reconstituted with ∆WH-Rfa2.

Data information: In (B) and (C) data show mean ± s.d. for three technical replicates.
Condensin minimizes topoisomerase II-mediated entanglements of DNA in vivo (2020)
Figure 2

Figure 2. Topoisomerase II does not minimize the knotting probability of chromatin.

(A) Experimental layout to test the DNA topology simplification activity of cellular topo II upon the addition of ICRF-193.

(B) Lanes 1 and 2: negatively supercoiled plasmid (YEp24) and its equilibrium distribution of Lk topoisomers upon its relaxation with Topo I. Lanes 3 and 4: distribution of Lk topoisomers of YEp24 upon its relaxation in lysates of ∆top1 TOP2 yeast cells in absence and after the addition of ICRF-193. Plots compare the relative intensity of individual topoisomers of the Lk distributions in lanes 2, 3 and 4.

(C) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome present in the lysates of ∆top1 TOP2 yeast cells before and after the addition of ICRF-193 (see details in Fig EV3). Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots (see details in Fig EV4). The graph shows Pkn of YEp13 (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.

(D) Experimental layout to compare the activities of TOP2 and Top2-∆83 on DNA and chromatin. Arrowheads indicate the extrachromosomal expression of TOP2 and Top2-∆83 under the inducible pGAL1 promoter.

(E) Lk distributions of the control plasmid (YEp24) relaxed by lysates of ∆top1 top2-4 yeast cells that expressed TOP2 or Top2-∆83. Plots compare the relative intensity of individual Lk topoisomers.

(F) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome produced in the presence of TOP2 or Top2-∆83. Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots. Graph: Pkn of YEp13 (mean ± SD from three independent experiments).

Gel signals are: N, nicked DNA circles; S, supercoiled DNA; L, diagonal of linear DNA fragments; Lk, distribution of Lk topoisomers; 0, unknotted DNA (nicked); Kn, ladder of knotted forms (nicked). P-values (Student's t test): ns, p > 0.05.
Condensin minimizes topoisomerase II-mediated entanglements of DNA in vivo (2020)
Figure 3

Figure 3. Condensin inactivation boosts the occurrence of chromatin knots.

(A) Top, DNA content (n/2n) of exponentially growing (OD600 = 0.6-0.8) smc2-8 yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). Graph: Pkn of YEp13 before and after the inactivation of condensin.

(B) Experiments conducted as in (A) but in scc1-73 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of cohesin.

(C) Experiments conducted as in (A) but in smc6-9 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of the Smc5/6 complex.

(D, E) Experiments conducted as in (A), but in cells arrested in G1 with alfa-factor (D) or in metaphase with nocodazole (E) for 2 h at 26oC and for one additional hour at 26oC or 35oC.

(F, G) Experiments conducted as in (B), but in cells arrested in G1 with alfa-factor for 2 h at 26oC (F) or in metaphase with nocodazole (G) and for one additional hour at 26oC or 35oC.

(H, I) Experiments conducted as in (C), but in cells arrested in G1 with alfa-factor (H) or in metaphase with nocodazole (I) for 2 h at 26oC and for one additional hour at 26oC or 35oC.

Data information: Gel signals (N, Lk, 0, Kn) are as described in Fig 2. Graphs show mean ± SD from three independent experiments in A, B, D, E, F, G; and from two independent experiments in C, H, I. P-values (Student's t test): ns, p > 0.05; **p < 0.01; ***p < 0.001.
Condensin minimizes topoisomerase II-mediated entanglements of DNA in vivo (2020)
Figure 6

Figure 6. DNA length dependence of the topological effects of Smc5/6 complex inactivation.

(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of Smc5/6 complex (35oC) in smc6-9 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of Smc5/6 complex (mean ± SD from two independent experiments).

(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of Smc5/6 complex.
A combination of two human monoclonal antibodies cures symptomatic rabies (2020)
Figure 2

Figure 2 - Intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail cures symptomatic Tha-RABV-infected mice.

A. Cumulative Kaplan-Meier survival curves of mice treated at different time-points post-infection. Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. **P<0.01, ***P<0.001. Treatments description= infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).

B. Cumulative Kaplan-Meier survival curves of all the treated animals that presented with clinical signs (n=31, all treatments combined). Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. ****P<0.0001.

C. Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups.

D. Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi (LOD: limit of detection = 0.02 µg/mL).

E. Viral load in the brain of mice from different experimental groups. The samples were either collected at the time of death, or at 100 dpi for the survivors.

F-N. Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: (F) IFN-β1, (G) ISG15, (H) Mx1, (I) IFN-γ, (J) CXCL10, (K) CCL5, (L) IL-6, (M) TNF-α and (N) IL-1β, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.

Data information: Horizontal lines indicate the median. The orange crosshatched areas correspond to the 95%CI of the median from the infected and non-treated mice, and the blue crosshatched areas correspond to the 95%CI of the median from the non-infected mice. nd: not detected.

Treatments description= ni: non-infected (n=5); nt: infected, non-treated (n=5); 6 (2+2): infected, treated at 6 dpi (2+2 mg/kg) (n=4); 7 (2+2): infected, treated at 7 dpi (2+2 mg/kg) (n=9); 8 (2+2): infected, treated at 8 dpi (2+2 mg/kg) (n=15); 8 (10+10): infected, treated at 8 dpi (10+10 mg/kg) (n=4). Exact P values are shown in Appendix Table S2.
A combination of two human monoclonal antibodies cures symptomatic rabies (2020)
Figure 3

Figure 3 - Brain histopathological analysis in the brain of representative mice from different experimental groups.

A-R. Histopathological analysis of the brain in Tha-RABV-infected non-treated mice (at 11 dpi), versus surviving mice (at 100 dpi) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail. The detection of Tha-RABV, using immunohistochemistry (IHC) on brain sections, revealed a heterogeneous invasion between the non-treated mice, ranging from extensive (A) to multifocal (B). Higher magnification of extensive invasion in the cortex (F), hippocampus (G), brain stem (H) and cerebellum (I). (N,O) Microglial cells were in general not reactive, but (P) small foci of reactive microglia (hyperplasia with thicker cell processes) could randomly be detected. (C,J,K,L,M) In contrast, even if a non-specific IHC signal was sometimes observed, no virus was detected on the brain sections of treated mice at 100 days post-infection. Only rare signs of neuro-inflammation were observed, as perivascular cuffing (D) (2/7 mice) and minimal meningitis (E) (3/7 mice). As for control non-treated mice, reactive microglial cells (Q) (2/7 mice) or resting microglia (R) were observed in treated mice. Representative images.

Data information: A-C and F-M: Tha-RABV IHC; N-R: Iba1 IHC to detect microglial cells; D-E: HE staining. F-I are high magnifications of A; J-M are high magnifications of C; O-P are high magnifications of N.
The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2 (2014)
Figure 1

MPK4 and PAT1 interact in Arabidopsis

MPK4‐HA is detected in PAT1‐Myc immunoprecipitates from double transgenic Arabidopsis plants. Immunoblots of input and anti‐Myc IPs probed with anti‐HA and anti‐Myc antibodies. Left panel, input; right panel, anti‐Myc IP.
Yeast pat1Δ mutants are unable to grow at 37°C while wild‐type (B4742) strain grows at 30°C (left panel) and 37°C (right). Growth at 37°C is restored in pat1Δ expressing AtPAT1. Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures.
Co‐IP between PAT1‐HA and LSM1‐GFP. Proteins were transiently co‐expressed in N. benthamiana and tissue harvested 3 days post‐infiltration. Immunoblots of GFP IPs (top panel) and inputs (20 μg each, bottom panel). Immunoblots were cut in half and probed with anti‐HA antibodies and anti‐GFP antibodies.
Agarose gel electrophoresis of 5′ RACE PCR to detect capped transcripts of EIF4A1, UGT87A2 and EXPL1 in 14‐day‐old Col‐0 and pat1‐1.

Source data are available online for this figure.
The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2 (2014)
Figure 3

pat1 mutants have a serrated leaf, semi‐dwarf phenotype

Plants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. Col‐0 and eds1‐2 plants and pat1‐1 and pat1‐1/eds1‐2, respectively, were placed in the middle of the same pots before the pictures were taken.
The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2 (2014)
Figure 4

pat1 mutants display EDS1‐dependent, constitutive defense responses

PR gene expression is elevated in mpk4‐2 and pat1‐1 mutants. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test, P‐values are only shown for mean values significantly different from Col‐0.
pat1 is more resistant to colonization by P. syringae pv. tomato DC3000. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are shown as log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher′s LSD test.
The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2 (2014)
Figure 5

PAT1‐GFP is present in P‐bodies after PAMP treatment

Confocal microscopy with root elongation zones. Five‐day‐old Col‐0/PAT1‐GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min (second panel) followed by treatment with 100 μg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 μm.
Confocal microscopy with 5‐day‐old VCS‐GFP seedlings treated as in (A). The scale bar corresponds to 10 μm.
PAT1 protein is induced by PAMP treatment. Immunoblot detection of equal amounts of protein from Col‐0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water (180 min after infiltration). Immunoblots were probed with anti‐PAT1 antibodies. Negative control pat1‐1 was loaded for comparison. Coomassie brilliant blue protein loading control is indicated by CBB.
Anti‐GFP immunoblotting of proteins extracted from Col‐0/PAT1‐GFP seedlings treated over a time course with flg22 (top panel) and Coomassie brilliant blue (CBB)‐stained PVDF loading control is shown (bottom panel). No PAT1‐GFP was detected in negative control Col‐0.

Source data are available online for this figure.