EMBO/soda-nel huggingface dataset:
Attenuation of pattern recognition receptor signaling is mediated by a MAP 2 kinase kinase kinase (2015)
Figure 3

Figure 3. Flg22-induced MAPK phosphorylation is enhanced in the mkkk7 mutant. A) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col-0 and in mkkk7. Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t=0, 10 and 30 min post induction. The p44/42 antibody was used to detect phosphorylated MAPKs. Position of the individual phosphorylated MAPKs is indicated at the right. Equal loading of proteins is shown with an α-Actin antibody as a loading control (bottom panel). Three biological replicates were done with identical results B) MPK6 phosphorylation is specifically enhanced in mkkk7. Comparison of phosphopeptide abundances from selected MAP kinases in Col-0 (blue) and mkkk7 (red) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring (SRM) mass spectrometry. Phosphopeptide corresponding to MKKK7 are only detectable in Col-0 seedlings and are non-detectable (ND) in mkkk7. Bars represent means of measured peptide areas (sum of all transition areas) for three biological replicates, with error bars ± SEM (n=3). Asterisks indicate significant difference between Col-0 and mkkk7 at individual time points (student t-test, *>0.05, **>0.01 and ***>0.001). ND indicates integration of an area without transitions significantly above background. Above each graph the protein name and the phosphorylated residue (in brackets) is indicated as well as the corresponding phosphopeptide sequence. Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation is indicated by ”[+80]”.
Attenuation of pattern recognition receptor signaling is mediated by a MAP 2 kinase kinase kinase (2015)
Figure 4

Figure 4. Flg22-induced defense gene expression is enhanced in mkkk7. A) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment. Protoplasts were isolated from 4 week old plants and transfected with pWKRY29:fLUC (WRKY29) or pFRK1:fLUC(FRK1) constructs together with 35S:rLUC, as indicated in the graph. Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated. The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample, as fold induction. All measurements were normalized to the rLUC activity. Bars represent means ± STDEV (n=2). Experiment was repeated 6 times with similar results. B) WRKY29 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 for t=0, 1, 2, and 4h. WRKY29 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test). C) FRK1 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 fort=0, 1, 2, and 4h. FRK1 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test). For each qRT-PCR experiments shown in B) and C) at least 2 biological replicates were done showing the same trend.
Attenuation of pattern recognition receptor signaling is mediated by a MAP 2 kinase kinase kinase (2015)
Figure 5

Figure 5. Phosphorylation of MKKK7 on specific Serine residues is required for negative regulation of flg22-induced WRKY29 gene expression. A) Transient co-expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22-induced WRKY29 gene expression. Protoplasts were transfected with pWRKY29:fLUC, 35S:rLUC and indicated overexpression constructs of MKKK7 (OE-MKKK7, OE-MKKK7AA or OE-MKKK7DD) as indicated on the horizontal axis. Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs. All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample, shown as fold induction on the vertical axis. Results shown are means ± STDEV (n=2). At least two biological replicates were done with similar results B) Protein structure of MKKK7 and mutated versions of MKKK7 with the protein kinase domain shown in yellow and an ARM/HEAT repeat domain shown in blue. The position of the phosphorylated Serine residues is indicated with triangles and bold S below the protein structure. The red triangles indicate phosphorylated Serines that were targeted for mutagenesis or the corresponding phospho-mimic Aspartic acid. Blue triangles indicate the substitution with the non-phosphorylatable amino acid Alanine. Amino acid substitute versions of MKKK7 are shown below the wild-type. S, Serine; A, Alanine; D, Aspartic acid.
Attenuation of pattern recognition receptor signaling is mediated by a MAP 2 kinase kinase kinase (2015)
Figure 6

Figure 6. MKKK7 negatively regulates basal resistance to virulent bacterial infection. A) Four-week-old seedlings were dipped into a suspension containing virulent Pst DC3000 and 72 h later the disease symptoms were scored. Data represents mean values ± SEM (n=20;***, p<0.001; paired t-test). Three biological experiments were done showing similar results. B) Quantification of bacterial growth in Arabidopsis lines Col-0, mkkk7 and p35S:MKKK7-GFP in the mkkk7 background. Four to five-week-old plants were pressure-infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re-isolated on selective media. The number of colony forming units (cfu/cm2) was determined at t=0, 2 and 3days post inoculation (dpi). Data represents mean values ± SEM (n=6; **, p<0.01; paired t-test). Experiments were done at least twice with similar results. C) Disease symptom development in Pst-infected lines with estradiol inducible constructs of ind-MKKK7AA L8, ind-MKKK7AA L10, ind-MKKK7DD L1 and ind-MKKK7DD L3. Two independent transgenic lines for each construct were grown under short-day conditions and disease symptoms were scored 3 dpi. Data represents mean values ± SEM (n=20; *, p<0.05; **, p<0.01; paired t-test). The vertical axis represents the percentage disease symptoms. Experiments were done at least twice with similar results.
Attenuation of pattern recognition receptor signaling is mediated by a MAP 2 kinase kinase kinase (2015)
Figure 7

Figure 7. Overexpression of MKKK7DD reduces flg22-induced ROS burst in leaves. Analysis of reactive oxygen species (ROS) production after treatment with flg22. A) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col-0 and mkkk7. B) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines. C) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col-0 and two independent inducible MKKK7DD transgenic lines. Graphs represent means with error bars ± SEM (n=24). The vertical axis represents the relative increase in ROS production (photon counts) after PAMP treatment. At least three biological replicate experiments were done with similar results.
Regeneration in starved planarians depends on TRiC/CCT subunits modulating the unfolded protein response (2021)
Figure 2

Figure 2. cct3A RNAi in starved conditions shows increased numbers of mitoses after both mitotic peaks and stem cell depletion at late time points of regeneration. (A) The graph shows the mitotic numbers of trunks during different time points of regeneration in the starved condition for cct3A RNAi and controls. Error bars are s.d. from the mean and asterisks indicate P < 0.001 (three asterisks), P < 0.01 (two asterisks) and n.s. indicates not significant using two-tailed Student's test with equal variance. n ≥ 9 planarians per time point. Also shown are maximum projections of representative trunks labelled with anti-H3P at different time points of regeneration in the starved condition. On the bottom, the number of planarians with the phenotype shown from the total is displayed. Note that at 50dR planarians show few (a4) or no mitoses (a5). The dashed line delimits the body of the planarian.

(B) The graph shows the mitotic numbers during 72 hours of regeneration in feeding conditions for cct3A RNAi and controls. Error bars are s.d. from the mean and n.s. indicates not significant using two-tailed Student's test with equal sample variance; n ≥ 8 planarian per time point.

(C) Maximum projections of representative trunks after FISH for smedwi-1 at different time points of regeneration under starved and feeding conditions. On the bottom, the number of planarians with the phenotype shown from the total is displayed. At 15dR the expression levels are similar. At 30dR some planarians have almost no expression (c5) and at 50dR almost all planarians show no expression (c10 and c11) in starved conditions. Under the feeding condition the expression levels at 30dS are similar. On the bottom, the number of planarians with the phenotype shown from the total is displayed. The dashed line delimits the body of the planarian.

(D) Relative expression of smedwi-1 at 13 and 50 days of regeneration after either cct3A or gfp RNAi under feeding and starved conditions. Error bars are s.d. from the mean and asterisks indicate P < 0.001 (three asterisks), P < 0.05 (one asterisk) and n.s. indicates not significant using two-tailed Student's test with equal variance. n = 3 replicates (5 planarians each) per time point.

Data information: hR, hours of regeneration; dR, days of regeneration. Scale bars, 1 mm (a1-a2) and 250 µm (a3-a5, c1-c3 and c6-c9) and 500 µm (c4-c5).

Source data are available online for this figure.
Regeneration in starved planarians depends on TRiC/CCT subunits modulating the unfolded protein response (2021)
Figure 6

Figure 6. NanoString analysis of stress response genes on normal and low glucose cultured LSK cells (hematopoietic stem cells and multipotent hematopoietic progenitors).

(A) Volcano plots displaying shRNA-Cct3- and shRNA-luciferase-infected LSK cells cultured either in normal glucose (upper graph) or low glucose conditions (lower graph). Dots represent all the NanoString analyzed transcripts. Red dots are UPR and ER stress related genes. The UPR and ER stress genes significantly down-regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph. For better visualization two dots with q ≤ 0.001 have been removed from the volcano plot (see them in Dataset EV4). Y-axis indicates the false discovery rate (FDR) (q- value), and the X-axis indicates the log2 fold changes. The FDR-based method of p-value adjustment was conducted to calculate the q-values by nSolver software using Benjamini-Hochberg methods. Significance is established by q-value < 0.05 and indicated by the dotted horizontal line. n= 3 mice.

(B) "UPR and ER stress" genes are significantly enriched (only down-regulated) in shCct3 cells compared to controls under low glucose conditions (**P < 0.01 using two-sided Chi-square test).

(C) The graph shows that the ratio between Xpb1(spliced) / Xpb1(unspliced) is lower (i.e., lower levels of UPR) in LSK which have Cct-3 down-regulated compared to controls only when cultured under low glucose conditions. Error bars are s.d. from the mean. P < 0.001 (three asterisks) and n.s. indicates not significant using two-tailed Student's test with equal sample variance. n = 3 mice.

Source data are available online for this figure.
SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade (2021)
Figure 1

Figure 1. SNX10 involves in OMV-induced downregulation of E-cadherin in intestinal epithelial cells

A-C WT and SNX10 KO Caco-2 cells were treated with indicated doses of OMVs for 24h. Protein expression of E-cadherin and SNX10 was determined by immunoblots (A) and quantified by ImageJ software (B) (n = 3 independent experiments). The fold changes in mRNA levels of CDH1 (encoding E-cadherin) and SNX10 in Caco-2 cells were determined by RT-qPCR (C) (n = 6 independent experiments).

D-F WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/mL) for the indicated time. Protein expression of E-cadherin and SNX10 was determined by immunoblots (D) and quantified by ImageJ software (E) (n = 3 independent experiments). The fold changes in mRNA levels of CDH1 and SNX10 were determined by RT-qPCR (F) (n = 6 independent experiments).

G, H Confocal images of E-cadherin staining in Caco-2 cells treated with OMVs (100 μg/mL) for the indicated time were captured (G), and the fluorescence intensity of E-cadherin staining was quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed). Scale bar: 20 μm.

I TEER value of Caco-2 cell monolayers incubated with or without OMVs (100 μg/mL) for 24 h was analyzed (n = 9 independent experiments).

J Caco-2 cell monolayers on transwell membranes were treated with or without OMVs (100 μg/mL) for 24h. FITC-dextran was added to these cells (top of the membrane). After 2h, FITC-dextran levels in the bottom chamber wells were measured (n = 9 independent experiments).

Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.
SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade (2021)
Figure 2

Figure 2. Blockage of OMV-induced E-cadherin reduction in SNX10 deficient cells is due to the inhibited nuclear localization of Snail/Slug proteins

A, B WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. The fold changes in mRNA levels of SNAI1 (encoding Snail) (A) and SNAI2 (encoding Slug) (B) in Caco-2 cells were determined by RT-qPCR. n = 6 independent experiments. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ns, not significant.

C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. Cytoplasmic and nuclear proteins were extracted for Snail and Slug determination by immunoblots.

D-F WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. Snail and Slug was assessed by immunoﬂuorescence staining (D) and quantiﬁed as the percentage of Snail (E) or Slug (F)-positive nuclei respectively. Scale bar: 20 μm. n = 3 independent experiments, n = 18 fields analysed. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.

G Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/mL OMVs for 24 h. Proteins from nucleus, cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies.

H WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/mL OMVs for 24 h. Proteins from nucleus, cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies.
SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade (2021)
Figure 3

Figure 3. SNX10 deficiency impairs Lyn-mediated nuclear localization of Snail/Slug

A Lysates from Caco-2 cells transfected with SNX10-Flag were subjected to pull down assay with anti-Flag antibody conjugated agarose, followed by immunoblots with the indicated antibodies.

B Representative images of co-staining of SNX10-Flag and Lyn in Caco-2 cells expressing SNX10-Flag. Scale bar: 20 μm.

C, D Levels of p-Lyn, Lyn, E-cadherin and SNX10 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24h were determined by immunoblots (C) and quantified by ImageJ software (D) (n = 3 independent experiments).

E, F Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24h. Lysates were extracted and subjected to immunoblots with the indicated antibodies (E) and quantified by ImageJ software (F) (n = 3 independent experiments).

G, H WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24h. Total cell lysates were subjected to immunoblots with the indicated antibodies (G) and quantified by ImageJ software (H) (n = 3 independent experiments).

I Cytoplasmic and nuclear proteins were extracted from WT and SNX10 KO Caco-2 cells treated with or without Tolimidone (10 μM), and the indicated proteins were analyzed by immunoblots.

J TEER value of Caco-2 cell monolayers after incubation with or without Tolimidone (10 μM) for 24 h was analyzed (n = 9 independent experiments).

K Caco-2 cell monolayers on transwell membranes were treated with or without Tolimidone (10 μM) for 24h. FITC dextran was added to these cells (top of the membrane). After 2h, FITC-dextran levels in the bottom chamber wells were measured (n = 9 independent experiments).

Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.
SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade (2021)
Figure 4

Figure 4. Interaction of SNX10 and caspase-5 is essential for OMV-induced Lyn phosphorylation

A Lysates from WT and SNX10 KO Caco-2 cells treated with or without 100 μg/mL OMVs for 24 h were analyzed by immunoblots with the indicated antibodies.

B Lysates from Caco-2 cells transfected with SNX10-Flag were subjected to pull-down assay with anti-Flag antibody conjugated agarose, followed by immunoblots with the indicated antibodies.

C Representative images of co-staining of SNX10-Flag and caspase-5 in Caco-2 cells expressing SNX10-Flag. Scale bar: 20 μm.

D WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h and then subjected to IP with anti-caspase-5 antibody.

E Representative images of co-staining of Lyn and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h. Scale bar: 20 μm.

F The co-localization of Lyn and caspase-5 was quantified by ImageJ software. n = 3 independent experiments, n = 18 fields analysed. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.

G Cytoplasmic and nuclear proteins were extracted from Caco-2 cells transfected with control siRNA, CASP4 siRNA or CASP5 siRNA and the indicated proteins were detected by immunoblots.

H Lysates of Flag-tagged full-length SNX10 or its different truncated mutants transfected Caco-2 cells were subjected to immunoprecipitation.

I Caco-2 cells were transfected with Flag-tagged full-length SNX10 or its indicated truncated mutant, followed by treatment with or without OMVs (100 μg/mL) for 24h. Lysates were extracted and subjected to immunoblots with the indicated antibodies.
SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade (2021)
Figure 5

Figure 5. SNX10 deficiency blocks LPS release from early endosomes into the cytosol

A Co-staining of SNX10-Flag and LPS in Caco-2 cells transfected with SNX10-Flag after treatment with OMVs (100 μg/mL) for 24 h. Scale bar: 20 μm.

B LPS levels in the cytosolic and residual fractions of Caco-2 cells treated with OMVs (100 μg/mL) for the indicated time were detected by LAL assay (n = 6 independent experiments).

C Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24h. LPS levels in the cytosolic and residual fractions of Caco-2 cells were evaluated (n = 6 independent experiments).

D WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24 h. LPS levels in the cytosolic and residual fractions of Caco-2 cells were detected (n = 6 independent experiments).

E, F WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/mL) for the indicated time and stained with antibodies against LPS and Rab5 (E). Scale bar: 20 μm. Colocalized LPS and Rab5 were quantified by ImageJ software (F) (n = 3 independent experiments, n = 18 fields analysed).

G LPS levels in the cell culture supernatants of WT and SNX10 KO Caco-2 cells stimulated by OMVs (100 μg/mL) for the indicated time were detected by LAL assay (n = 6 independent experiments).

H LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without chloroquine (CQ) (200 nM) or bafilomycin A1 (Baf A1) (50 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments).

Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade (2021)
Figure 6

Figure 6. Recruitment of caspase-5 to PIKfyve by SNX10 triggers LPS release from early endosomes into the cytosol

A Lysates from WT and SNX10 KO Caco-2 cells treated with or without 100 μg/mL OMVs for 24 h were analyzed by immunoblots with the indicated antibodies.

B Co-staining of SNX10-Flag and PIKfyve in Caco-2 cells transfected with SNX10-Flag. Scale bar: 20 μm.

C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24h and then subjected to IP with anti-PIKfyve antibody.

D, E Co-staining of PIKfyve and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (D). Anti-PIKfyve and anti-caspase-5 antibodies were used to detect endogenous PIKfyve and caspase-5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (E) (n = 3 independent experiments, n = 18 fields analysed).

F Caco-2 cells were stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for 24h. Proteins of lysates extracted from Caco-2 cells were subjected to immunoblots with the indicated antibodies.

G, H Co-staining of PIKfyve and Rab5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (G). Anti-PIKfyve and anti-Rab5 antibodies were used to detect endogenous PIKfyve and Rab5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed).

I LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments).

J Caco-2 cells transfected with control siRNA or CASP5 siRNA were treated with OMVs (100 μg/mL) for the indicated time. LPS levels in the cytosolic and residual fractions of Caco-2 cells were detected by LAL assay (n = 6 independent experiments).

K LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100μg/mL) with or without bafetinib (1μM) for the indicated time were detected by LAL assay (n = 6 independent experiments).

Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.