EMBO/soda-nel huggingface dataset:
Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis (2020)
Figure 2

Figure 2. HY5 is a potential regulator of PAP8 expression.

A PAP8 promoter deletion strategy. ERI, EcoRI site; HIII, HindIII site; indicated positions are given relative to the transcription start noted as +1; red boxes represent untranslated regions; pink boxes, ORF of an upstream gene; nearly palindromic element is given in blue. -97m3: mutated promoter as described in C with three mutations (m3) indicated with '***'.

B Two or six (-97m3) representative primary transformants expressing GUS under the given PAP8 promoter version; FC+, the corresponding promoters were tested positive in functional complementation of the mutant pap8-1. Scale bar equals 3 mm.

C Proximal PAP8 promoter region; m3, 3-bp substitutions within the -97-bp promoter; 5'-UTR in red; ATG, start codon of PAP8.

D Dual luciferase reporter assay; Renilla luciferase (Rluc) used as internal control and GFPer used as control for the transfected area; the promoters driving Firefly luciferase (Fluc) were transfected in onion epidermis cells without or with constitutively expressed HY5. The Fluc/Rluc activity was set to 1 for the minus-HY5 control; mean ± standard error corresponding to 3 replicates; photon counts are given in source data. *, ε-test = 3,43 > 1.96 corresponding to p-value < 0,001.

E Electro-mobility shift assay of a probe corresponding to the near palindromic PAP8 element (GAcGCTC) with recombinant HY5 protein; a probe containing a canonical G-box element (CACGTG) recognized by HY5 was used as cold competitor.

F Integrative genomics viewer (IGV) images of the Chromatin immunoprecipitation (ChIP) sequencing data30 at the PAP8 locus; TAIR10, annotation according to the Arabidopsis thaliana information resource orange box indicates the PAP8 locus. ChIP on hy5-ks50; 35S:HY5-YFP exposed to blue light or red light using GFP antibody and compared to mock corresponding to ChIP control experiment done without antibody. Each treatment is presented as track overlay of triplicates: the read count is given within the "group autoscale" range in brackets. Close up on the 5'-UTR region centred on the -95-promoter element in yellow.
Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis (2020)
Figure 3

Figure 3. PAP8 is dually localized in plastids and nucleus.

A Schematic illustration of domain structure of Arabidopsis PAP8 fused to GFP. cTP, chloroplast transit peptide; NLS, nuclear localization signal.

B Transiently expressed PAP8FL-GFP (full-length coding sequence of PAP8 fused to GFP) in onion epidermal cells displays a dual localization in the nucleus (white arrowhead) and in plastids (yellow arrowheads).

C Immuno-blots for the detection of PAP8 in total protein extracts (Col-0 Tot) the nuclear fraction (N) and the plastidic fractions (EP, etioplast; CP, chloroplast) of etiolated (Dark) or photomorphogenic (Light) Arabidopsis seedlings by immuno-Western blotting using a PAP8 antiserum (PAP8). As control, a mixture of antisera raised against Histone 3 (H3) and the large subunit of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RbcL) was used to evaluate reciprocal contaminations. The lanes are extracted from the same blots provided in the Source Data file.

D-G Co-expression analysis of PAP8FL-GFP (D) with PAP8ΔcTP-RFP (E) in onion epidermal cells; (F) green and red channels merged; yellow arrowheads show plastids; white arrowheads show nuclei as observed with DIC, differential interference contrast in (G); FL, PAP8 full length ORF; ΔcTP, deletion of the cTP; scale bars equal 20 µm.

H Confocal imaging on Arabidopsis cotyledons expressing PAP8ΔcTP-GFP, details of a few palisade cells (see Fig. S7B for a view in a cross section between the two cotyledons); magenta, auto-fluorescence of the chloroplast; the white arrowhead shows a nucleus; scale bar equals 5 µm.

I,J Confocal imaging on Arabidopsis cotyledons stably expressing pP8::PAP8ΔNLS-GFP; ΔNLS, deletion of the NLS (I) during skotomorphogenesis and (J) after 24h light; Yellow arrowheads show the GFP signal; the picture is a merge of different channels: GFP in green, Pchl,: protochlorophyllide, or Chl: chlorophyll in magenta marked with arrowheads, and propidium iodide, showing the waxy cuticle in red, the empty space correspond to the layer of highly vacuolated epidermal cells.
Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis (2020)
Figure 4

Figure 4. PAP8 is functional in both the chloroplast and the nucleus.

(A,B) (A) Transient expression of PAP8NLSm5-GFP mutated in the NLS as described in Fig. EV4A. The circle marks the position of the nucleus (N) as observed with DIC in (B); yellow arrowheads show stromules (str). Scale bars equal 20 µm.

C-G Pictures of representative genotypes obtained in the functional complementation test of pap8-1 using pP8::PAP8-NLSm5 (C, F) or pP8::PAP8ΔcTP (G) both constructions without GFP tags; ⌀ indicates pap8-1 without any PAP8 transgene. (D-G) Pictures using Keyence technology of plants with genotypes as labelled; transgenes expressed using the 1.1-kb PAP8 promoter. Scale bars equal 1 mm.
Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis (2020)
Figure 5

Figure 5: PAP8 is essential for the PHYB-mediated light induction of photomorphogenesis.

A Phenotypes of given genotypes subjected to 5 days of illumination at 8 µmol.m-2.s-1 660-nm red light. PBG, pCaMV35S::PHYB-GFP transformed in pap8-1/+ (among 25 lines selected for GFP expression see data source; 2 doubly heterozygous pap8-1/+; PBG/- lines #6 and #7 segregated the photobodies alteration with the albinism).

B Hypocotyl length, given as the mean ± SD, of plants grown as in (A) (R8, grey bars) or at 30 µmol.m-2.s-1 660-nm red light (R30, pink bars) showing partial insensitivity of pap8-1 to the PBG overexpression. Measurements are given in source data; in the order of the graph n equals (50, 133, 58, 43, 36, 112, 25, 60) δ-test (comparison of the mean) R8: δPBG/PBGp8-1= 20.38 corresponding to p-value < 10-31 δwt/p8-1= 0.5 < 1.96 not significant at α set to 0.05).

C-G Nuclear accumulation of PBG observed under GFP excitation in the given genotypes. (C,D) Epi-fluorescence microscopy. (E,F) Confocal microscopy showing the size of the nuclear bodies. Scale bars equal 5 µm. (G) Box plot (Min, 1st quartile, median as the central band, 3rd quartile, max) on the diameter of the nuclear bodies (NBs); n equals the number of records.

H Immuno-blots using a GFP antibody or a PAP8 antibody showing respectively the levels of PHYB-GFP and PAP8 in the given genotypes grown in the dark for 3 days or in light; n.a., not applicable as the pap8-1 mutant can only be visually distinguished from wild type after light exposure; 2 lines (L#06 and L#07) for PBG/pap8-1 were tested. Coomassie blue staining presented as loading; signals were quantified using ImageJ.

I RT-qPCR analysis on wild type, pap8-1, PBG and PBG pap8-1. Seedlings were grown in the dark (D) or under white light (L, 30 µmol.m-2.s-1); levels of transcripts are given relative to EF1α; error bars correspond to standard errors on technical triplicates and the dark sample is the wild-type PBG line. δ-test (comparison of the mean) ***, P < 10-72.

J Immuno-blots showing the levels of PIF1, PIF3, HY5 in given genotypes grown in the Dark or Light condition as noted: p5/+, mix of an heterozygous pap5-2 siblings progeny undistinguishable from wild type; p5-2, pap5-2 and pifq, quadruple pif1-1 pif3-3 pif4-2 pif5-3 mutant; Histone H3 (H3), RbcL and PAP8 were used as controls; n.a., not applicable.
Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis (2020)
Figure 6

Figure 6. PAP8 interacts with pTAC12/HMR/PAP5.

A Bimolecular fluorescence complementation tests using in combination PAP8ΔcTP-NY (P8Δc-NY) with PAP5ΔcTP-YC (P5Δc-YC) or PAP8ΔcTP-YC (P8Δc-YC) with PAP5ΔcTP-NY (P5Δc-NY); PAP10-RFP (P10-RFP) was used as internal positive control for transfection; the ratio in grey depicts the number of green-fluorescent cells over red-fluorescent cells. Arrowheads indicate nuclei. See Appendix Fig. S6 for control experiments; transgenes expressed under CaMV35S promoter.

B Overlay of 1H-15N correlation 2D NMR spectra of free 15N-labelled PAP8 alone (blue) or in complex with PAP5 (red). Grey areas depict changes of signals in the PAP8 spectrum.

C 15N-Filtered Diffusion Ordered Spectroscopy-NMR measurements to PAP8. Exponential decay curves of PAP8 in absence or in presence of MBP-PAP5 are shown in red and black respectively. The units on the y-axis are normalized values of the integrals of the signal measured in the amide proton region.
Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis (2020)
Figure 7

Figure 7: Temporal resolution of protein content in seedlings during the dark-to-light transition.

A Immuno-blots showing the levels of PAP8, PAP5, Histone H3 (H3), Rubisco (RbcL), and HY5 ; D, Dark; Ph, photomorphogenic growth conditions from germination on. *, HY5 is detected as a modified form (+ 6 kDa); the arrows indicate the two different post-translational modified forms of HY5 for which accumulation occurs upon light exposure.

B Relative protein contents normalized to histone H3 during the transition from skotomorphogenesis to photomorphogenesis, we propose a phase of photo-initiation corresponding to the 5 minutes of light allowing PAP8 and PAP5 to rise to nearly 100% of their maximum. The first macroscopic signs of greening are indicated around 3 hours while the photosynthetic apparatus bursts at 8 hours to be fully accumulated at 48 hours.
Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks (2021)
Figure 1

Figure 1. Role of Rad59 and DNA binding domain of Rad52 in suppressing BIR at two-ended DSBs.

Models of DSB repair by SDSA and BIR.

Schematic of the H-150 assay. Blue boxes depict homologous sequence on one end of the DSB and green boxes depict homology on the other end. A DSB is induced at modified MATa locus (Chr. III). Strand invasion occurs within the "Z" sequence (blue box) and after copying Yα-inc (dashed line) and X sequence (150 bp), the X sequence is used to capture the second end during SDSA. When copying continues to the end of chromosome via BIR (dashed line), an acentric chromosome forms and an unrepaired chromosome segment remains, leading to cell death.

Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel B). A detailed map of restriction cut sites and expected size for different repair products are illustrated in Appendix Figure S1.

Percentage of BIR products among repair outcomes by 6 hr are shown. (mean ± SD; n = 3).

Viability of indicated strains is shown (mean ± SD; n = 3).

Graph shows repair efficiency among all cells by 6 hrs (left) and represents the sum of DSB repair by BIR and SDSA compared to the uncut parental band at 0 hr (mean ± SD; n = 3). Graph shows repair by BIR and SDSA at 6 hrs after break induction in mutant cells compared to repair products in WT, which are set to 100% (right).

Schematic of H-150 no-gap assay, where 150 bp of Yα homology on the second end of the break is immediately adjacent to the break site.

Representative Southern blots showing DSB repair products in indicated mutants in H-150 no-gap assay.

Percentage of BIR product among all repair products by 6 hr are shown. (mean ± SD; n = 3).

Viability of indicated strains (mean ± SD; n = 3).

Graphs showing repair efficiency compared to uncut parental band (left) (mean ± SD; n ≥ 3) or compared to the repair products in WT (right) at 6 hrs after DSB induction. See Fig 1F legend for details.

Data information: Welch's unpaired t-test was used to determine the p-value in all panels.
Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks (2021)
Figure 2

Figure 2. Rad52 and Rad59 suppress BIR during MAT switching.

Schematic of the H-1400 assay. A DSB is induced at MATa and repaired by recombination with HMLα-inc. There is ~1400 bp homology between MATa and HMLα-inc sequences on the second end (green box).

Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box). A detailed map of restriction cut sites and expected size for different repair products are illustrated in Appendix Figure S1.

Percentage of BIR among all repair products by 6 hr (mean ± SD; n = 3).

Viability of indicated strains (mean ± SD; n = 3).

Graphs show analysis of repair efficiency compared to uncut parental band (left) (mean ± SD; n = 3) and repair efficiency compared to WT (right) at 6 hrs after DSB induction (right). See Fig 1F for details.

Schematic of the H-2100 no-gap assay. A DSB is induced at MATα and repaired by recombination with HMLα-inc. There is ~2100 bp homology between MATα and HMLα-inc sequences on the second end (green box).

Representative Southern blots showing DSB repair products in WT, rad59∆, and rad52-R70A. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box). A detailed map of restriction cut site and expected size for different repair products are illustrated in Appendix Figure S1.

Percentage of BIR product among repair products by 6 hr are shown. (mean ± SD; n = 3).

Viability of indicated strains (mean ± SD; n = 3).

Graphs show analysis of repair efficiency compared to uncut parental band (left) (mean ± SD; n = 3) and repair efficiency compared to WT (right) at 6 hrs after DSB induction (right). See Fig 1F for details.

Data information: Welch's unpaired t-test was used to determine the p-value in all panels.
Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks (2021)
Figure 3

Figure 3. Longer homology mitigates the loss of Rad52 DNA binding domain and Rad59.

Schematic of allelic recombination between two chromosomes III. A DSB is induced at MATa and repaired by recombination with the homologous chromosome carrying MATα-inc. Chromosome ends are marked with TRP1, LEU2, and KANMX to distinguish different repair products or chromosome loss. Trp+, Leu-, G418R colonies are scored as BIR products. Reciprocal sectored colonies (Trp+, Leu-/+ G418R/S) represent gene conversion with crossover.

Percentage of BIR product among all cells are shown.

Percentage of chromosome loss of indicated mutants.

Comparison of BIR percentage among the products in different assays shown in figures 1-3 in WT and rad52-R70A mutant cells. Percentage of repair products are measured at 6 hrs after break induction (H-150, H-1400, and H-2100 assays) or scored among plated cells (allelic assay) were pooled together.

Schematic of mutation analysis during allelic recombination (left). Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site. Mutation rate (right) of WT, rad59∆, rad52-R70A was calculated as previously described (Saini et. al., 2013).

Data information: χ2 test is used to determine the p-value in figure 3B and C, number of colonies tested per mutant is indicated in Appendix Table S1. Mutation rates in figure 3E are reported as the median value, and statistical comparisons between median mutation rates were performed using the Mann-Whitney U-test.
Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks (2021)
Figure 5

Figure 5. Role of Sir2 in suppressing BIR during repair of two-ended breaks.

(A-C) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of WT and sir2∆ in the H-150 assay (A), H--150 no-gap assay (B) or native mating type switching H-1400 assay (C). (mean ± SD; n = 3). Welch's unpaired t-test was used to determine the p-value in panels A-C.
Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks (2021)
Figure 6

Figure 6. Role of the MRX complex in suppressing BIR during repair of two-ended breaks.

(A-B) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of indicated mutants in the H-150 no-gap assay (A) or H-150 assay (B). (mean ± SD; n ≥3). Welch's unpaired t-test was used to determine the p-value in panels A and B.

(C) Schematic of resection assay with sites of restriction enzyme BfaI cleavage and location of probe (top), representative of Southern blots, and graph showing kinetics of resection two break end (bottom) (mean ± SD; n =3) in WT, mre11∆, yku70∆, and yku70∆ mre11∆.
NKG2D discriminates diverse ligands through selectively mechano-regulated ligand conformational changes (2021)
Figure 1

Figure 1. Characterization of in-situ force-free kinetics of NKG2D interacting with different ligands and comparison with in-solution affinities.

A Diagrammatic sketch of in-situ binding kinetic assay and functionalization of RBC.

B Representative adhesion frequency (Pa) vs. contact duration (tc) curves forNKG2D expressing NK cells (n ≥ 3) in contact with RBCs (n ≥ 3) coated with a ligand (MICA in red, MICB in orange, ULBP1 in green, or ULBP3 in blue) at different contact durations, fitted by non-linear in-situ binding kinetics model (Huang et al., 2010). Site densities of NKG2D (mr) and its ligands (ml) are indicated.

C-E In-situ force-free affinities (C), on-rates (D) and off-rates (E) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B).

F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in-situ and in-solution assay. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1.

Data information: In-solution affinities of proteins purified from E. coli were from previous study (McFarland & Strong, 2003). In-solution affinities of proteins purified from mammalian cells were measured by BLI (Fig EV2A-G). Every dot in (C-E) represents one independent binding experiment. Error bars in (B-E) are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).

Source data are available online for this figure.
NKG2D discriminates diverse ligands through selectively mechano-regulated ligand conformational changes (2021)
Figure 2

Figure 2. Correlation analysis of NK cell functions with in-situ force-free kinetics of NKG2D binding with ligands.

A-C Schematic diagram (A) of IFN-γ release assay and IFN-γ production (B) by human peripheral NK cells stimulated with plate-coated NKG2D ligands (MICA in red, MICB in green and ULBP3 in blue) assessed by Cytometric Bead Array, of which are their 50% effective concentration (EC50) (C).

D-F Plots of reciprocals of EC50 vs. the effective in-situ affinities (AcKa) (D), the effective in-situ on-rate (AcKon) (E) and in-situ off-rate (koff) (F).

Data information: The IFN-γ release assay in (B) was one representative experiment of three total independent experiments. Data points in (B) and bars in (C) represent mean values. Error bars in (B) and (C) represent mean ± SEM. ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).

Source data are available online for this figure.
NKG2D discriminates diverse ligands through selectively mechano-regulated ligand conformational changes (2021)
Figure 3

Figure 3. Mechanical force differentially regulates NKG2D's ligand dissociation and discrimination.

A, B Schematic diagram (A) and experimental setup (B) of BFP assay to characterize force-dependent dissociation kinetics of NKG2D binding with different ligands.

C Verification of the binding specificity of NKG2D with different ligands.

D Representative force vs. time curve for measuring single NKG2D-ligand bond lifetime.

E, F Force-dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN and 15 pN (E) and full force spectrum (F).

G Ratios of average bond lifetimes for NKG2D/MICA to that of NKG2D and other ligands.

Data information: Every dot in (C) represents the adhesion frequency of one cell-bead pair and the experimental data came from at least two to three repeated trials. Bond lifetimes in (E) and (F) (in which n = 1505 for MICA, n = 1852 for MICB, n = 531 for ULBP1, n = 1241 for ULBP3) of all ligands with NKG2D came from at least 19 NK cell-bead pairs of at least four independent biological experiments. Horizontal lines in (C), bars in (E), and data points in (F) represent mean values. Error bars in (C), (E) and (F) represent mean ± SEM.

Source data are available online for this figure.
NKG2D discriminates diverse ligands through selectively mechano-regulated ligand conformational changes (2021)
Figure 4

Figure 4. Mechanical force induced allosteric regulation of NKG2D's ligand dissociation and discrimination.

A, B SMD snapshots of NKG2D dissociation with MICA (A) and ULBP3 (B) in the presence of force (directions are indicated by black arrows).

C The force vs. extension trajectories for the simulations are shown in (A) and (B). Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in (A) and (B) are marked.

D, E Zoomed-in binding interfaces of NKG2D with MICA (D) or ULBP3 (E), corresponding with the configuration (shown as gray dashed box in (A) and (B), respectively).

F Distance vs. time curves for force-induced H-bond formation between indicated residues within NKG2D-MICA binding interfaces. The dashed red lines represent H-bonds whose distances are less than 3.5 Å.Source data are available online for this figure.
NKG2D discriminates diverse ligands through selectively mechano-regulated ligand conformational changes (2021)
Figure 5

Figure 5. Abolishments of force-induced binding residues impair bond lifetime and function of MICA and NKG2D interaction.

A, B Abolishments of force-induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n= 580) impairs NKG2D's catch bond with MICA WT (n = 1505) (A) and IFN-γ release (B).

C-E 50% effective concentration (EC50) (C) and IFN-γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM (D) and 22nM (E).

Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN-γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points in (A) and (B), horizontal lines in (C), bars in (E), and data point in (F) represent mean values. Error bars in (A-E) represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).

Source data are available online for this figure
NKG2D discriminates diverse ligands through selectively mechano-regulated ligand conformational changes (2021)
Figure 6

Figure 6. Correlation analysis of NK cell functions with in-situ force-dependent kinetics of NKG2D binding with ligands.

A-C Plots and Pearson correlation analysis of reciprocals of EC50 vs. the force-dependent in-situ koff at 5 pN (A), 10 pN (B) 15pN (C).

D Heatmap of force-dependent in-situ affinities AcKa of NKG2D binding with different ligands.

E-G Plots and Pearson correlation analysis of reciprocals of EC50 vs. the force-dependent effective in-situ affinities AcKa at 5 pN (E), 10 pN (F) and 15 pN (G).

H Comparison of corresponding Pearson coefficients between all NKG2D-ligands in-situ binding kinetics and ligand-induced NK functions.

I Detection range comparison in force-dependent affinity of NKG2D and indicated ligands under difference force. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1.

Source data are available online for this figure.