EMBO/soda-nel huggingface dataset:
Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis (2019)
Figure 6

Figure 6 - The caspase-4/11 inhibitor, VX-765, suppresses DSS-induced colitis in GPx8-/- mice.

A. The caspase-4/11 and -1 inhibitor, VX-765, inhibited noncanonical inflammasome activation. BMDMs isolated from mice were primed with LPS for 5-6 h, treated with 20 μM VX-765, and transfected with LPS for 12 h.

B-F. Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model as shown in Fig 1A and analyzed by a Kaplan-Meier survival plot (B), the percentage of body weight (C) and clinical scores (D) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). (E-F) Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Full-length and cleavage forms of caspase-11, caspase-1, and GSDMD (E) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment. Production of IL-1β and IL-6 (F) in colon tissue lysates was assessed on day 14 after treatment.

Data information: In A, data are presented as the mean ± SD, n=5, technical repeats. In B-F, data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).
Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis (2019)
Figure 7

Figure 7 - Lower GPx8 and higher caspase-4 expression in colon tissue of ulcerative colitis patients.

A. GPx8 was predominantly expressed in macrophages of UC patients. Colon biopsies of non-inflamed regions from UC patients were stained with the pan-macrophage marker, CD68, or the monocyte/macrophage activating marker, CD163, combined with anti-GPx8. Quantification of double-positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double-positive cells. The total cell numbers are the sum of double- and single-positive cells.

B. Relative expression levels of GPx8, GPx7 and caspase-4 in non-inflamed biopsy specimens from healthy controls (Ctrl) (n = 14) and UC patients (n = 25) are demonstrated by immunoblots.

C. Data are normalized to the mean of healthy controls and presented as the mean ± SEM. Mann Whitney test for GPx8 and Student's two‐tailed t‐test for GPx7 and caspase-4 expression analysis.

D. Paired GPx8 and caspase-4 expression from each individual are linked by lines and presented as the mean ± SEM. The p-value was analyzed by the Fisher's exact test.

Data information: NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001.

Expanded View Figure legends
Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli (2020)
Figure 1

Figure 1. RapZ binds GlcN6P and is required for regulation of the GlmY/GlmZ/glmS cascade by GlcN6P.

A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆rapZ mutant Z28 under normal growth and GlcN6P starvation conditions. Both strains, which also carried a chromosomal glmS'-lacZ fusion, were treated with 60 μg/ml Nva-FMDP or H2O ("mock"). Samples were harvested hourly for Northern analysis and determination of β-galactosidase activity. Growth curves are shown in Appendix Fig S1. Blots were re-probed using a 5S rRNA specific probe to provide loading controls.

B The purification profile of Strep-RapZ from the ∆glmS strain Z904 under GlcN6P replete and depletion conditions is shown (top). The cleared lysate (CL), flow through (FT), washing steps (W) and the elution fractions (E1-3) from StrepTactin affinity chromatography were separated on 12.5 % SDS-PAA gels and stained with Coomassie blue. Metabolites were extracted from E2 and analyzed by HILIC-MS/MS. The extracted ion chromatograms of the LC-MS analysis targeting GlcN6P (retention time 16.6 minutes) are shown below. The samples derived from purification of Strep-RapZ (panels i and ii) or Strep-KdpE (panels iii and iv) were analyzed with the SRM transition m/z 258.1 to m/z 97 in the negative ion mode. The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg/μl (panel v).

C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites. The Strep-tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite (i.e. 100, 500, 1000, 2500 and 5000 nM) were injected using a single cycle kinetics approach.

Data information: In (A), β-galactosidase activities are presented as mean ± SD. n = 2.
Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli (2020)
Figure 2

Figure 2. RapZ is required for full activity of the glmY σ54 promoter and upregulates glmY expression under GlcN6P starvation conditions.

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. In (A) to (D) strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined.

A Strains Z197 and Z360 were used, which harbor glmY'-lacZ and glmZ'-lacZ fusions, respectively.

B Expression of glmY'-lacZ in strain Z197 and the ∆rapZ mutant Z225 is compared.

C Strains Z190 and Z201 were addressed, which transcribe glmY'-lacZ either from the σ54-promoter or the σ70 promoter, respectively.

D Strains Z190 and the ∆qseF mutant Z196 are compared, both of which transcribe the glmY'-lacZ fusion solely from the σ54 promoter.

E Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures. Strains Z197 and the ∆rapZ mutant Z225 were used. Tested plasmids were pFDX4291 (vector control for pFDX4324 and pYG82 = VC1), pFDX4324 (rapZ), pYG82 (rapZquad), pBGG237 (vector control for pBGG164 = VC2) and pBGG164 (strep-rapZ).

Data information: Note that experiments (A) and (B) as well as (C) and (D) were performed in parallel, respectively. Therefore, the same values are presented for strains Z197 and Z190. In (A-E), β-galactosidase activities are presented as mean ± SD. (A-D): n = 3; (E): n = 4.
Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli (2020)
Figure 3

Figure 3. RapZ stimulates phosphorylation of the TCS QseE/QseF by interaction and GlcN6P counters this process.

A BACTH assay addressing interaction of RapZ with QseF and QseE. The following plasmid combinations were tested using reporter strain RH785 (columns 1-6): pBGG352/pBGG349, pBGG353/pBGG348, pYG199/pBGG349, pYG246/pBGG348, pKT25/pUT18C (negative control), pKT25-zip/pUT18C-zip (positive control).

B SPR spectroscopy experiments addressing interaction of RapZ with the cytoplasmic part (aa 196-475) of kinase QseE and response regulator QseF. Strep-RapZ was captured onto a sensor chip and increasing concentrations of QseE'-His10 or QseF-His10 were injected.

C To assess the role of RapZ for QseF activity, β-galactosidase activities were determined from strains Z196 (∆qseF) and Z1110 (∆qseF ∆rapZ) at indicated times during growth. Strains harbored the glmY'-lacZ fusion that is solely expressed from the σ54 promoter and the following plasmids: pKESK23 (VC = vector control; black and grey; note that activities are too low for display), pYG89 (qseF, green), pYG90 (qseF-D56E, purple), pYG93 (qseF-D56A, red).

D In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10-tagged QseE' (aa 196-475) in presence of various concentrations of RapZ. Samples were removed following [γ-32P]-ATP addition at indicated times and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.

E Analysis of QseE' autophosphorylation (lanes 1-6) and phosphoryl group transfer to QseF (lanes 7-12) in absence or presence of 5 μM Strep-RapZ. To assess phosphoryl group transfer, 1 μM QseF-His10 was added to the assay.

F To analyse the role of GlcN6P, 5 μM RapZ or the equivalent volume of buffer was pre-incubated with the indicated GlcN6P concentration for 5 min and subsequently 1 μM QseE'-His10 was added. Following an additional incubation for 5 min, [γ-32P]-ATP was added and the reactions were stopped after 1 min.

Data information: In (A) and (C), β-galactosidase activities are presented as mean ± SD. (A): n = 3; (C): n = 4.
Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli (2020)
Figure 4

Figure 4. sRNAs GlmY and GlmZ counteract activation of QseE/QseF by RapZ.

A β-Galactosidase activities of strains Z197 (wild type) and Z1118 (∆glmY ∆glmZ), which carry the chromosomal glmY'-lacZ fusion, were determined during growth.

B Strains Z197 (wild type) and Z225 (∆rapZ) were transformed with the following plasmids expressing the mentioned sRNAs: pBR-plac (vector control = VC), pYG83 (glmY), pYG84 (glmZ) and pSD69 (gcvB). sRNA expression was induced with 1 mM IPTG and β-galactosidase activities were determined in the exponential growth phase.

C To assess the impact of GlmY* and GlmZ on stimulation of QseE' autophosphorylation by RapZ, 1 μM QseE'-His10 was incubated with [γ-32P]-ATP in absence or presence of 5 μM Strep-RapZ and/or the sRNAs GlmY*, GlmZ and GcvB. In lanes 3-11 QseE' was co-incubated with RapZ as well as the indicated sRNAs provided at 0.5 μM, 1.25 μM and 2.5 μM. In lanes 14-16, QseE' was incubated with 2.5 μM of each sRNA without RapZ. Samples were removed 1 min after addition of [γ-32P]-ATP and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.

Data information: In (A) and (B), β-galactosidase activities are presented as mean ± SD. (A): n = 4; (B): n = 3.
Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli (2020)
Figure 5

Figure 5. GlmY* half-life increases under GlcN6P starvation conditions and RapZ is required for this effect.

Northern blot experiments assessing the half-lives of GlmY* and GlmZ under normal growth and GlcN6P starvation conditions. Bacterial cultures were either treated with 100 μg/ml Nva-FMDP or H2O (mock). Transcription was stopped by rifampicin addition when cultures attained OD600 = 1.0 and samples were removed at indicated times for Northern analysis.

A Analysis of GlmY* and GlmZ decay in the wild type strain Z8.

B Analysis of GlmY* and GlmZ decay in the ∆rapZ mutant Z28.

C Semi-logarithmic plots of GlmY* and full-length GlmZ decay for half-life determination.

Data information: In (C) data are presented as mean, n = 2. Half-lives are presented as mean ± SD where applicable.
Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli (2020)
Figure 6

Figure 6. GlcN6P releases GlmY* from RapZ leading to rapid degradation of the sRNA.

Northern blot experiments (A-C) assessing GlmY* half-life under GlcN6P replete (+GlcN) and depletion conditions (-GlcN). Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis.

A Analysis of the ∆glmS strain Z1126. The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub-cultures was resupplied with GlcN (indicated by arrow).

B The ∆glmS ∆rapZ double mutant Z1127 was tested.

C Semi-logarithmic plots of GlmY* decay for half-life determination.

D EMSA experiments addressing the role of GlcN6P for GlmY*/RapZ interaction. Radiolabelled GlmY* was incubated with incremental concentrations of RapZ (left panel) or RapZ-CTD (right panel) in absence or presence of 7.5 mM GlcN6P. Binding reactions were separated on native PAA gels and analyzed by phospho-imaging. The RapZ/GlmY* complex is indicated by an arrow.

E EMSA following incubation of GlmY* with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 (lane 2) to 8 mM (lane 10). The fraction of GlmY* remaining in the gel pocket is marked with an asterisk. The RapZ/GlmY* complex is indicated by an arrow.

F EMSA following incubation of GlmY* with 1200 nM RapZ in absence or presence of 7.5 mM of the indicated metabolite. The RapZ/GlmY* complex is indicated by an arrow.

Data information: In (C) data are presented as mean, n = 2.
Increased Alu RNA processing in Alzheimer brains is linked to gene expression changes (2021)
Figure 5

Figure 5. Alu RNA processing ratio is associated with clinical, pathology, genetic and molecular markers of AD in MAP patients.

(A) Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients with regard to final clinical diagnosis (p < 0.01 for the comparison between NCI and MCI, between NCI and AD, and between MCI and AD, unpaired, non directional t-test). NCI=no cognitive impairment (n=71), MCI=mild cognitive impairment (n=67), AD=Alzheimer's disease (n=87). Boxplot representation as in subfigure 2D.

(B) Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients (n=241) with regard to Braak stage classified into three categories (p = 0.004 for the comparison between 0-2 and 3-4, and p < 0.001 for the comparisons between 3-4 and 5-6 and between 0-2 and 5-6, unpaired, non directional t-test, n=39[0-2],n=148[3-4] and n=54[5-6]). Boxplot representation as in subfigure 2D.

(C) Scatterplot depicting the positive correlation between TP53 expression values and Alu RNA processing ratio in AD patients (r=0.65, p < 0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats.

(D) Scatterplot depicting no correlation between TP53 expression values and full length Alu RNA levels in AD patients (r=0.06, no correlation, ns=non-significant). Statistical test as in subfigure 5C.
Increased Alu RNA processing in Alzheimer brains is linked to gene expression changes (2021)
Figure 7

Figure 7. Alu RNA destabilization leads to increase in expression of Alu RNA processing correlated genes

(A) Correlation between gene expression and Alu RNA processing ratio for AD upregulated genes (Dataset EV3) (upper panel) and a random set of non-differentially expressed genes (Dataset EV5) (lower panel). For every gene a correlation coefficient was calculated (Pearson correlation) together with the respective p-value. Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Based on the r and p value genes were classified into four categories: i)NS, non-significant for p value > 0.05, ii) no correlation or weak correlation (r≤0.05), iii) significant (p < 0.05) and strong correlation (0.5<r≤0.75) and iv) significant (p < 0.05) and very strong correlation (0.75<r). The bar graphs represent the percentage of each category for each set of genes. The exact r and p values of each gene are listed in Dataset EV6 for the upper panel and Dataset EV7 for the lower panel.

(B) Experimental design for targeting Alu RNAs in a cell culture assay employing SHSY5Y neural cells.

(C) Expression levels of full length Alu RNA (RT-qPCR) in the Alu RNA KD experiment. Statistical significance (p value threshold 0.05) for the comparison between anti-Alu LNA treated samples (anti-Alu) and samples treated with a scramble control LNA (ctrl) with p=0.049 and n=3, unpaired non-directional t-test. Error bars represent standard deviation from the mean.

(D) Expression levels of P53 (RT-qPCR) in the Alu RNA KD experiment. Statistical significance (p value threshold 0.05) for levels in anti-Alu LNA treated samples (anti-Alu) greater than samples treated with a scramble control LNA (ctrl) with p=0.046 and n=3, unpaired directional t-test. Error bars represent standard deviation from the mean.

(E) Expression levels (RT-qPCR)_of selected genes from panel A that are either strongly correlated with Alu RNA processing (left, r> 0.5) or only weakly/not correlated (right, r <0.5). Statistical significance (p value threshold 0.05) for anti-Alu greater than control (depicted as asterisk, n=3/group, unpaired directional t-test, error bars represent standard deviation from the mean). Pearson correlation co-efficient for each gene is depicted below the name of each gene.
Increased Alu RNA processing in Alzheimer brains is linked to gene expression changes (2021)
Figure 8

Figure 8. HSF1 accelerates Alu RNA processing in vitro.

(A) In vitro incubation of one of the Alu RNA consensus sequences for different incubation periods. In vitro transcribed Alu RNA (67nM) incubated at 37oC with 250nM HSF1 in the course of 6 hours with time intervals of 1 hour.

(B) Comparison among Hsf1 (~60KDa), denatured HSF1, Poly A polymerase (~56KDa) and no protein (just TAP buffer) with regard to Alu RNA processing (estimating relative full length RNA remaining) (two replicates). The full gels are available as Source Data For Figure 8. Relative full length RNA remaining was calculated using ImageJ area under the curve software over time. Error bars represent standard deviation from the mean.

(C) Comparison among Alu RNA (three replicates), and two control RNAs (two replicates) regarding the full-length RNA levels remaining after in vitro incubation for 90 minutes at 37C with HSF1. Sizes of control RNAs are control for RNA #1, 143nt and for control RNA #2, 432nt. Incubation in the absence of HSF1 but presence of the same buffer (TAP) was used as control to take into account any non HSF1 specific RNA destabilization due to non-specific degradation. Threshold for statistical significance was a p value of 0.05 with p=0.03 (unpaired, non-directional t-test) for the comparison between HSF1 and no protein incubation (n=3, for Alu RNA). The full gels are available as Source Data For Figure 8. Error bars represent standard deviation from the mean.

(D) Plotting of the position of the first base (5' end) of Alu RNA fragments across the Alu consensus sequence produced by Alu RNA that has been processed in vitro for 90 minutes at 37C in the presence of HSF1 (upper panel) and compared with one of the in vivo samples of Figure 1 (lower panel). The x axis represents an Alu RNA metagene aligned at the start site of the Alu consensus sequence and the y axis shows the 5' end count for Alu RNA fragments aligning to any position downstream of position +1. The in vivo sample x axis depicts a 11nt sift compared to the in vitro one.

(E) Scatterplot depicting the positive correlation between HSF1 mRNA expression values and Alu RNA processing ratio in MAP AD patients (r=0.72, p<0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats.

(F) Scatterplot depicting lack of correlation between HSF1 mRNA expression values and full length Alu RNA levels in MAP AD patients (ns=non-significant, with p value 0.05 as the significance threshold). Statistical test as in subfigure 8E.
Wip1 regulates Smad4 phosphorylation and inhibits TGF-β signaling (2020)
Figure 1

Figure 1. Overexpression of Wip1 inhibits TGF-β signaling.

(A-C) Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), Xnr1 (50 pg) and BMP4 (100 pg).

(D, E) wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. FAST1, Smad1 and Smad4 were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.

(F-Q) Dorsal (F-N) or ventral (O-Q) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10.25 as analyzed by in situ hybridization. Embryos are shown in vegetal views with dorsal to the top. Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos. Control, an embryo injected with LacZ only. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng) and LacZ (100 pg). Scale bar, 150 μm.
Wip1 regulates Smad4 phosphorylation and inhibits TGF-β signaling (2020)
Figure 2

Figure 2. Depletion of Wip1 up-regulates TGF-β signaling.

(A, B) Activin/Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps in (B) were treated with Activin protein (5 ng/ml). (-), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Xnr1 (5 pg), Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).

(C) Knockdown of Wip1 up-regulates the activity of Activin/Nodal-responsive promoter. HEK293T cells transfected with Co siRNA (50 nM) or Wip1 siRNA (50 nM) were stimulated with Activin protein (10 ng/ml) for 10 h and then subjected to luciferase assays. Data are expressed as the mean ± SEM (n=3 biological replicates). ***P<0.001 by unpaired Student's t-test.

(D-J) Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. (-), no injection of Noggin mRNA. Embryos in (E-J) are shown in dorsal views with anterior to the top. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.

(K, L) Activin or TGF-β induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with Activin (10 ng/ml) for 3 to 12 h as indicated or with increasing concentration of TGF-β1 (10, 20 ng/ml) for 3 h. t=0 in (K) is 48 h after siRNA transfection. P-Smad2 serves as an indicator of active Activin/Nodal signaling. Arrows indicate non-specific bands.
Wip1 regulates Smad4 phosphorylation and inhibits TGF-β signaling (2020)
Figure 3

Figure 3. Silencing of Wip1 promotes the biological effects of TGF-β.

(A, B) Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with wt Wip1, Wip1(D277A), Co siRNA or Wip1 siRNA as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.

(C-F) Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20-μL pipette tip and then incubated in the absence or presence of TGF-β1 for the indicated times (C, D) or subjected to transwell assay with or without TGF-β1 for 20 h (E, F). Data in (D, F) were quantified from three biological replicates and are represented as mean ± SEM. **P<0.01, ***P<0.001 by unpaired Student's t-test. Scale bar, 200 μm.
Wip1 regulates Smad4 phosphorylation and inhibits TGF-β signaling (2020)
Figure 4

Figure 4. Mesoderm expansion in Wip1-depleted embryos.

(A-D) Knockdown of Wip1 expands the expression of dorsal mesodermal markers. Embryos injected dorsally with Co MO (40 ng) or Wip1 MO (40 ng) were subjected to in situ hybridization at stage 10.25. Embryos are shown in dorso-vegetal views with dorsal to the top. Scale bar, 150 μm.

(E-I) Morphological phenotypes of Wip1 morphants. Embryos were injected radially in the marginal zone with Co MO (80 ng), Wip1 MO (80 ng), wt Wip1 (1 ng) and Wip1(D277A) mRNA as indicated and cultured to tadpole stages. Embryos are shown in lateral views with anterior to the left. Un Co, uninjected control. Scale bar, 1 mm.

(J) Quantification of the phenotypes shown in (E-I). Severe defects include microcephaly, shortened and kinked body axis, and no eye. Mild defects indicate normal body axis with malformed eyes.