EMBO/soda-nel huggingface dataset:
Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1 (2015)
Figure 1

Figure 1. BAK1 disruption sensitizes PEPR signaling toward cell death.

(A and B) Root length (averages ± SD, n ≥ 15) (A) and cell death staining of cotyledons and primary root tips (B) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days, 2 days after germination. BAK1 (D416N) is a kinase-dead variant. (C) Overview of 3-week-old plants. (D) Pep2 induces PEPR1-FLAG association with SERK1-, SERK2-, BKK1-, and BAK1-HA in N. benthamiana. Experiments were repeated at least three times, with the same conclusions. IP and IB denote imunoprecipitation and immunoblotting with the indicated antibodies, respectively. NT indicates non-transformed plants. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants.)
Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1 (2015)
Figure 2

Figure 2. BIK1-dependent sensitization of PEPR signaling in the absence of BAK1.

(A) Immunoblot analysis for BIK1-HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min. Arrowheads indicate phosphorylated BIK1-HA. (B) Immunoblot analysis for Pep2-triggered MAPK activation. Ponceau S-stained loading controls are shown (bottom). (C) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings that were exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bak1-3 plants. Two independent experiments were combined for statistical analysis.)
Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1 (2015)
Figure 3

Figure 3. BAK1 disruption leads to reprogramming of PEPR–mediated defense outputs.

(A) Scatter plots for log2 fold changes of gene expression in WT plants versus bak1-3 plants treated with Pep2 for 2 and 10 h. The regression of these scatter plots is indicated by the red lines. (B) Hierarchical analysis of genes exhibiting a >2-fold change in expression level in bak1-3 plants after Pep2 application for 10 h in a whole-genome microarray analysis. Using Genevestigator v3, these genes were separately cross-referenced to public databases to show their expression responses to salicylate (SA), methyl-jasmonate (JA), or ethylene (ET). (C and D) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding bak1-4 and bak1-3 values in C and D, respectively. Relative cycle threshold (Ct) values of two independent experiments were combined for statistical analysis.)
Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1 (2015)
Figure 4

Figure 4. PROPEP proligand production and PEPR1 accumulation following Pep application are reinforced in the absence of BAK1.

(A) qRT-PCR analysis of PROPEP genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis. (B) Anti-GFP (upper) and anti-PROPEP3 (middle) immunoblot analysis of PROPEP3-Venus in seedlings submerged in liquid medium and treated with (+) or without (-) 0.5 M Pep2 for the indicated times. The positions of the molecular weight markers are shown on the left. Arrowheads and asterisks indicate PROPEP3-Venus and cross-reactive bands, respectively. (C, D and E) Immunoblot analysis of PEPR1-FLAG in 10-day-old seedlings following Pep1 or flg22 application. (C and D) Plants were exposed to 1 M Pep1 or flg22 for the indicated times. (E) Plants were pre-exposed to MG132 (50 µM), Wortmannin (Wm) (30 µM) and Tyrphostin A23 (TyrA23) (100 µM) for 1 h before being exposed to Pep1 for 10 h. The numbers below the immunoblots represent relative intensities of the PROPEP-Venus (B) and PEPR1-FLAG (C-E) bands normalized to the backgrounds in the Ponceau S-stained loading controls (bottom), with the band/background values in WT plant extracts 24 h (upper) and 0 h (middle) after Pep2 application, respectively, set as 1.0.
Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1 (2015)
Figure 5

Figure 5. Reprogramming of PEPR-mediated defense outputs upon disruption of BAK1/BON-mediated cell death suppression.

(A) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.) (B) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.) (C) Fresh weight (averages ± SD, n ≥ 25) of 2-week-old plants grown on soil. (*, p < 0.05 in two-tailed tests compared to the corresponding values of WT plants. ■, p < 0.05 in two-tailed tests. Two independent experiments were combined for statistical analysis.) (D) Immunoblot analysis for BAK1 in 10-day-old seedlings with or without 0.5 M Pep1 for 24 h. The numbers below the upper panel represent relative intensities of the BAK1 band normalized to the backgrounds in the Ponceau S-stained loading controls, with the value in non-elicited WT plants set as 1.0.
Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1 (2015)
Figure 6

Figure 6. PROPEP production and release during bacterial resistance.

(A) qRT-PCR analysis in 4-week-old plant leaves challenged with the indicated P. syringae pv. tomato (Pst) DC3000 strains for the indicated times. (B) Anti-GFP immunoblot analyses in seedlings challenged with Pst DC3000 (DC3000) and Pst DC3000 ∆hrcC (∆hrcC) for the indicated times. (C) Anti-PROPEP3 immunoblot analysis of PROPEP3-Venus (upper) in plant tissues (expressed in plants) and in the extracellular fraction (released into medium), 24 h and 48 h after the inoculation with (+) or without (-) Pst DC3000. Lower panels indicate Ponceau S-stained loading controls. The numbers below the upper panel represent relative intensities of the PROPEP3-Venus band normalized to the backgrounds in the Ponceau S-stained loading controls. NT denotes non-transgenic WT plants. (D) Trypan blue cell death staining of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 (AvrRpm1) for 48 h. (E) Anti-PROPEP3 immunoblot analyses in the seedlings exposed to the indicated Pst DC3000 strains or 1 M Pep1 for 48 h. (F) Anti-GFP immunoblot analysis for the extracellular fraction of the seedlings exposed to Pst DC3000 AvrRpm1 for 48 h. (B, C, E and F) Arrowheads and arrows indicate an apparently full-length and processed form of PROPEP3-Venus, respectively. Asterisks indicate cross-reactive bands. The positions of the molecular weight markers are shown on the left of the immunoblots.
Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1 (2015)
Figure 7

Figure 7. PEPRs are required to mount SA-dependent basal resistance in the absence of BAK1.

(A) Growth of H. arabidopsidis (Hpa) Noco2 in 2-week-old plants. Spores were counted at 6 days post inoculation (dpi). Results are averages ± SE of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding values of Col plants. ■, p < 0.05 in two-tailed tests between the indicated values. Three independent experiments were combined for statistical analysis.) (B) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (*, p < 0.05, **, p < 0.01 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.) (C) Total SA levels were determined in the rosette leaves of 4-week-old plants with or without 1 µM flg22 application for 24 h. Data are averages (±SD) of material from three independent plants. The experiments were repeated twice, with similar results. (▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.) (D) qRT-PCR analysis of the PR1 gene in 4-week-old plants exposed to 1 µM flg22. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of four independent experiments were combined for statistical analysis.) (E) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (**, p < 0.01, ***, p < 1.0e-10 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)
The Hedgehog signalling pathway regulates autophagy (2012)
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(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h. (a) Endogenous LC3 was detected by immunoblot in total cell lysates, quantified by densitometric analysis and normalized to actin. Relative levels were expressed in percentage with control cells set to 100 and shown in graph. (b) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels. Graph shows mRNA levels quantified by densitometric analysis and normalized to actin. (c) 8xGli-luciferase construct was transfected in HeLa cells for 24 h, followed by treatment with 10 μM purmorphamine, or DMSO as a control, for another 24 h. Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control. (d) Endogenous LC3-II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h, either in the absence or presence of trehalose (100 mM). Where indicated, bafA1 (400 nM) was added for the last 4 h. Quantification by densitometric analysis relative to actin is shown in the graph. (e) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions, either in the presence of bafA1 or DMSO as a control, and LC3-II and actin levels were detected. (f) HA-HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h. The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph. P-values were calculated by odds ratio. (g) U20S cells stably expressing HaloTag-p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine. After 48 h, cells were lysed, run on a SDS–polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager. Fluorescence was normalized to total protein levels, detected on the same gels by Kryton Fluorescence protein stain. A representative gel and its quantitated normalized levels are shown. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student’s t-test unless indicated: ***P0.001; **P0.01; *P0.05. SDS–PAGE, SDS–polyacrylamide gel electrophoresis.
The Hedgehog signalling pathway regulates autophagy (2012)
f2

(a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h. Where indicated, cells were treated with bafA1 for the last 4 h. LC3-II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph. (b) HeLa were transfected with Ptch1 or Ptch2 together with HA-HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence. P-values were calculated by odds ratio. (c) Atg5+/+ or Atg5−/− MEFs were transfected with GFP-HttQ74 for 48 h, cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence. (d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h (d) or 400 nM bafA1 for 4 h (e). Quantification and statistical analysis is shown in graphs. (f) siRNA-targeting Ptch2 was transfected into HeLa cells stably expressing mRFP-GFP-LC3 and representative confocal microscopy images are shown. Scale bars represent 26 μm. Percentage of GFP-positive vesicles (autophagosomes), RFP-positive vesicles (total number of vesicles) or percentage of autolysosomes (calculated by subtracting numbers of GFP from RFP vesicles), quantified using a Cellomics array scan, was calculated relative to control siRNA-transfected cells. Although an increase in autophagosome biogenesis generally increases both the number of GFP+ vesicles and the number of GFP-/RFP+ vesicles as a result of an enhanced autophagy flux, defects in autophagosome degradation, such as in bafA1-treated cells, increase GFP+ vesicles but not GFP−/RFP+ structures, as GFP remains intact. Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100. In all panels, unless indicated, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student’s t-test: **P0.01; *P0.05; NS, not significant.
The Hedgehog signalling pathway regulates autophagy (2012)
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(a) Smo siRNA-treated cells were transfected with HA-HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA-positive cells by immunofluorescence. Graphs show mean values and error bars represent s.d. of a representative experiment performed in triplicate. P-values were calculated by odds ratio. (b) siRNA-targeting Smo was transfected into HeLa cells stably expressing mRFP-GFP-LC3. Representative confocal microscopy images are shown. Bars represent 26 μm. The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA-transfected cells. Statistical tests were performed on data from three independent experiments in triplicate. *P0.05; ***P0.001.
The Hedgehog signalling pathway regulates autophagy (2012)
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(a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti–actin antibodies. (b) Gli2−/− MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a. (c) LC3-II levels were detected by western blotting of total lysates from wild-type and Gli2−/− mice embryos (E12.5). For quantification, LC3-II levels from a total of five wild-type and five Gli2−/− embryos were normalized to actin, wild-type was set to 100. Graphs show mean values and error bars represent s.d. Statistical analysis were performed by two-tail Student’s t-test **P0.01. Pmph, purmorphamine.
The Hedgehog signalling pathway regulates autophagy (2012)
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(a,b) Control (a) or hh[ts2] (b) larvae were incubated at permissive temperature (18 °C) to the third instar stage, then cultured at non-permissive temperature (29 °C) in well-fed conditions for 24 h. Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed. (c) Lysotracker Red-labelled fat body containing single-cell flipout clones (GFP positive) overexpressing patched. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed. (d) Confocal image of a GFP-positive patched-expressing clone, with mCherry-Atg8a expressed in all cells. Fat body was dissected from well-fed larvae and imaged after formaldehyde fixation. (e) GFP-marked flipout clones of CiCell-expressing cells. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed. (f) Confocal image of fixed mCherry-Atg8a expressing fat body containing a GFP-marked clone of CiCell-expressing cells. (g) Cells homozygous for the null costal2[2] allele (marked by lack of GFP) were generated by flp/FRT-mediated recombination in mCh-Atg8a expressing fat body. Larvae were starved 4 h before dissection and fixation. Scale bar represents 25 μm in a, b, c and e, and 10 μm in d and f. Genotypes: (a) y,w; (b) hh[ts2/ts2]; (c) hs-flp; UAS-ptc/+; Act>CD2>GAL4 UAS-GFPnls/+; (d) hs-flp; UAS-ptc/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/+; (e) hs-flp; UAS-CiCell/+; Act>CD2>GAL4 UAS-GFPnls/+;(f) hs-flp; UAS-CiCell/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/+; (g) hs-flp; FRT42D cos2[2]/Cg-GAL4 UAS-mChAtg8a[2L] FRT42D UAS-GFPnls.
The Hedgehog signalling pathway regulates autophagy (2012)
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(a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells. Each circle represents an autophagy gene; those with largest fold-changes are indicated in red. The central line represents no changes in expression; above the central line, genes whose expression is increased: below, those with reduced levels. Grey lines indicate 2-fold increase or decrease. (b) Table showing the 10 genes with higher fold changes in expression and the values obtained. (c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control. (d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager. (e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. LC3-II and actin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.
Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions (2019)
Figure 1

Figure 1: Knock-in cell lines

A Scheme of the knock-in alleles of the SOX1-BRA-Reporter-OCT4-HALO-SOX2-SNAP (SBROS) cell line. Red hexagons: STOP codons. Yellow triangles: loxP sites. B Example images showing the localization of SOX2-SNAP and OCT4-HALO by fluorescence microscopy of SBROS cells. Scale bar: 50µm. C Correlation between OCT4-HALO and total OCT4 levels determined by immunofluorescence and HALO labelling. R is Pearson's correlation coefficient. D Distributions of OCT4 (n=2682), SOX2 (n=1236) and NANOG (n=2416) levels in WT E14 and SBROS cell lines as determined by quantitative immunofluorescence. Dashed lines: mean protein levels; Dotted lines: median protein levels. E Protein half-lives of OCT4-HALO and SOX2-SNAP in SBROS cells. Whiskers: minimum and maximum values; Box: lower and upper quartiles; Solid line: median. Black dots: outliers. F Cell cycle duration of E14 WT (n=6) and SBROS (n=8) cells. Error bars: SE. p-value: two-sided t-test with unequal variance.
Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions (2019)
Figure 2

Figure 2: Cross-regulation and single-cell fluctuations of SOX2 and OCT4

A Endogenous SOX2 levels in single cells (turquoise) normalized to the value at t=0 in SNSF cells upon induction of SOX2-SNAP (n=37, left) or a truncated SOX2 version missing the DNA binding domain (YPET-SOX2-delDBD, n=47, right). Black line: average SOX2-SNAP levels after induction normalized to the maximum level (n=22); Yellow line: average YPET-SOX2-delDBD levels after induction, normalized to the maximum level (n=20). B Sox2 and Oct4 mRNA levels upon overexpression of YPet-SOX2 or YPET-SOX2-delDBD (n=3). p-values: two-sided t-test with unequal variance. C Changes of OCT4 half-life upon SOX2-SNAP or YPET-SOX2-delDBD overexpression (n=20 cells each). p-values: Mann-Whitney U test. SBROS cells harbouring inducible SOX2-SNAP or YPET-SOX2-delDBD were treated with 0 or 500 ng/ml dox for 6-8 hours followed by pulse-labelling of OCT4-HALO and live-cell imaging. D Sorting strategy to evaluate the impact of endogenous fluctuations of SOX2 and OCT4 on OCT4 and SOX2 protein levels, respectively. E Changes of SOX2 and OCT4 levels after sorting for high and low SOX2 levels in cells expressing intermediate OCT4 levels (n=4). Error bars: SE; p-values: one-sided t-test with unequal variance. F Changes of SOX2 and OCT4 levels after sorting for high and low OCT4 levels in cells expressing intermediate SOX2 levels (n=4). p-values: one-sided t-test with unequal variance. G Representative traces of the absolute number of SOX2-NLUC molecules (top) and inferred concentration in nM (bottom) in in-silico synchronized cells (n=10). H Single cells were ranked according to their SOX2 expression at t=0 and assigned a color code for their initial rank from low (blue) to high (red) levels, and changes in ranks over time are shown (n=59). I Rank-based autocorrelation function of the SOX2 ranks (n=59). Error bars: SE estimated by bootstrapping. J Representative single cell traces of the integrated intensity of OCT4-HALO in single cells (top) and the corresponding inferred concentration (bottom) in in-silico synchronized cells (n=10). K Representation of the ranks as in G. L Rank-based autocorrelation function as in I (n=47). Error bars: SE estimated by bootstrapping.