EMBO/soda-nel huggingface dataset:
Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways (2018)
Figure 1

Fig 1. Hel2-mediated K63-linked polyubiquitination is crucial for NGD and RQC.

(A) Schematic drawing of the R(CGN)12 reporter mRNA including the two quality control pathways induced by the R(CGN)12 translation arrest sequence. Ribosome stalling occurs during translation of the R(CGN)12 arrest-sequence (shown in orange) and induces RQC and NGD. In the RQC pathway, the stalled ribosome is dissociated into subunits, peptidyl-tRNA remaining on the 60S subunit is ubiquitinated by Ltn1 (shown in pink) and degraded by the proteasome. In the NGD pathway, an endonucleolytic cleavage produces two fragments, the 5'NGD-intermediate (5'NGD-IM) and 3' NGD-intermediate (3'NGD-IM). The green and thin grey lines indicate GFP and HIS3 open reading frames (ORFs), and the black line indicates an untranslated region (UTR). (B) Schematic drawing of the truncated Hel2 mutant proteins. Activities in RQC or NGD induced by the R(CGN)12 sequence are indicated. (C) Western blot showing that Hel2(1-315) is defective in RQC but not in NGD. The arrest products derived from the R(CGN)12 reporter in ltn1∆ cells expressing truncated Hel2 mutant protein were detected with an anti-GFP antibody. (D) Northern blot showing the 5' NGD-IM derived from the R(CGN)12 reporter in ski2∆ cells expressing the indicated Hel2 mutant proteins. 5' NGD-IMs were detected with a DIG-labelled GFP probe. (E) Primer extension mapping of 5' ends of 3' NGD-intermediates in Hel2-WT or Hel2(1-315) mutant cells at nucleotide resolution. . The primer extension samples were analysed using 5% TBE-Urea-PAGE and detected by fluorescence. Non-specific reverse transcription (ReTr) products are indicated by asterisks. (F) Dissection of NGDRQC+ and NGDRQC-: The carboxyl-terminal region of Hel2 is required for both NGD and RQC which is likely triggered on a disome unit (pale yellow). It contains the primarily stalled, leading ribosome followed the colliding ribosome. For NGDRQC+ cleavages occur on mRNA covered by the disome, whereby the leading ribosome undergoes RQC. In the mutant Hel2 lacking the C-terminus, an alternative NGD pathway takes place (NGDRQC-). Here, cleavages occur on mRNA covered by the ribosomes following the disome unit (blue) and the leading ribosome is not affected by RQC (grey).
Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways (2018)
Figure 2

Fig 2. Both uS10 ubiquitination and Rqt2 are required for NGDRQC+

(A) Northern blot analysis demonstrating that K63-linked ubiquitination was required for an endonucleolytic cleavage by R(CGN)12. The ski2∆UB-WT and ski2∆ub-K63R cells were transformed with the R(CGN)12 reporter, and total RNA samples were separated by 2% agarose/MOPS gel. 5'NGD-IMs in the cells were detected as in Fig 1D. (B) Western blot showing that K63-linked ubiquitination was required for RQC. Protein samples from UB-WT, UB-WT ltn1∆, ub-K63R, ub-K63R ltn1∆ cells expressing the GFP-R(CGN)12-HIS3 reporter were subjected to Western blot analysis using an anti-GFP antibody to detect the arrest products. Note the accumulation of RQC-specific CAT-tails in lane 2. (C) Western blot analysis demonstrating polyubiquitination of uS10: Affinity-tagged Hel2-Flag-TEV-ProteinA (FTP) was co-purified with ribosomes harbouring HA-tagged wild type uS10 (uS10-3HA ribosomes) or uS10 mutated in its ubiquitination sites (K6/8R). Western blots of whole protein extracts were performed using an anti-HA antibody to detect polyubiquitinated uS10-3HA. (D) Western blot analysis showing that uS10-polyubiquitination in Hel2-bound ribosomes was mainly K63-linked: Affinity tagged Hel2(Hel2-ProteinA-TEV-His6; PTH-Hel2) was co-purified with ribosomes from a strain expressing wild type ubiquitin (UB-WT) or K63R mutant ubiquitin (ub-K63R) and HA-tagged uS10 (uS10-3HA). Western blot analysis of whole protein extracts was performed using anti-HA antibody. (E) Western blot of in vitro polyubiquitination assays of uS10. Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants. Polyubiquitinated HA-tagged uS10 (uS10-3HA) was detected by Western blot analysis using an anti-HA antibody. (F-H) Northern blot analysis of NGD-cleavage sites in the absence of uS10 ubiquitination or RQT complex components: The full-length GFP-R(CGN)12-FLAG-HIS3 mRNA and 5' NGD-intermediates (5' NGD-IM) or 3' NGD-intermediates (3' NGD-IM) were detected in the indicated mutant cells by Northern blotting with DIG-labelled probes. 5' NGD-intermediates were detected by DIG-labelled GFP probe and 3' NGD-intermediates were detected by the DIG-labelled HIS3 probe. SCR1 was used as loading control. FL = full-length. Note the upstream shift of NGD cleavage sites in lanes F4, G4, H6, and H8. (I) Mapping of 5' ends of 3' NGD-intermediates as demonstrated in Fig 1E. Non-specific reverse transcription (ReTr) products are indicated by asterisks. Note that both uS10 ubiquitination and Slh1/Rqt2 were required for an endonucleolytic cleavage within the road-blocked ribosome (X1-X4). (J) Model for RQC-coupled (NGDRQC+) and -uncoupled (NGDRQC-) endonucleolytic cleavages in the R(CGN)12 reporter mRNA. Endonucleolytic cleavage sites are depicted corresponding to panel I.
Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways (2018)
Figure 3

Fig 3. uS10 in colliding ribosome is efficiently ubiquitinated

(A) Top: Schematic drawing of the (CGA-CCG) dicodon reporter mRNA used for in vitro translation experiments. Bottom: Scheme outlining the principle of ribosome nascent chain (RNC) purification. Stalled and colliding ribosomes are pulled down via an affinity tag on the nascent chain. (B-C) Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (B) Western blot showing the arrest products derived from the GFP-X-FLAG-HIS3 reporter. Translation products were detected by western blot using an anti-GFP antibody. (C) Northern blot for the 5' NGD-IM derived from the (CGA-CCG) reporter in ski2∆ cells. 5'-NGD-IMs were detected with a DIG-labelled GFP probe. SCR1 was used as a load control. (D) Western blot of test translations using the (CGA-CCG) dicodon stalling mRNA reporter shown in (A). The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2∆uS10-3HA strain. Expression of the translation products (free His- and V5 tagged truncated uL4 protein and the same protein attached to tRNA) was visualized with an anti-V5 antibody. (E) Sucrose gradient fractions (top) and western blot analysis (bottom) of the (CGA-CCG) dicodon-stalled RNC samples. After the translation reaction, the RNCs were affinity-purified as indicated in (C). The eluate was loaded on a 10-50 % sucrose gradient and fractionated. Each collected fraction was analysed using anti-HA antibody to detect uS10-HA. Note that disomes are preferentially polyubiquitinated over monosomes.
Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways (2018)
Figure 6

Fig 6. Hel2 polyubiquitinates the monoubiquitinated eS7A by Not4.

(A) Western blot analysis after overexpression of Hel2 in strains expressing HA-tagged ribosomal proteins using an anti-HA antibody. Note the increase of the polyubiquitinated eS7 as well as uS10 and uS3. (B) Structural overview of the ubiquitination sites on eS7. The general overview on the disome interface is depicted according to Fig 4A. Individual ubiquitination sites are highlighted as red spheres in the detailed cartoon model representation. (C) Western blot analysis showing the role of Hel2 and Not4 in eS7A ubiquitination: Both copies of eS7 (eS7A and eS7B) were deleted and transformed with a plasmid containing HA-tagged eS7A (eS7-WT) or eS7A mutated in the four potential ubiquitination sites (4KR). Whole protein extracts we obtained from eS7AΔeS7BΔ cells or cells with an additional deletion in E3 ligases Hel2 and Not4 (hel2Δ and not4Δ). Note that the four mutated lysine residues were responsible for Not4-dependent monoubiquitination. (D) Western blot showing that Not4 was required for Hel2-mediated polyubiquitination of eS7 in Hel2-bound ribosomal complexes: Cells expressing HA-tagged eS7A or the eS7-4KR mutant, as well as PTH-tagged Hel2 in eS7AΔeS7BΔ and eS7AΔeS7BΔnot4Δ background were probed for eS7-ubiquitination. Either cell lysates or affinity-purified Hel2-ribosome complexes were used. The levels of the ubiquitinated eS7A in the lysates and in the affinity-purified samples with PTH-Hel2 were determined by Western blot analysis using an anti-HA antibody to detect eS7. (E) Western blotting of in vitro ubiquitination assays of eS7A. The reactions were performed with the indicated components including His-tagged ubiquitin. Ribosomes were purified from Hel2Δ cells expressing HA-tagged eS7A. Polyubiquitinated HA-tagged eS7A (eS7A-3HA) was detected by Western blot analysis using an anti-HA antibody. We observed Hel2-mediated polyubiquitination of the monoubiquitinated eS7A. (F) Western blot analysis of eS7A in vitro ubiquitination assays showing that Hel2-mediated polyubiquitination of eS7 was mainly K63-linked and required Not4-dependent monoubiquitination: These assays were performed similarly as described in (E) except yeast strains were lacking Not4 (not4Δ). Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants and eS7A-ubiquitination was monitored using an anti-HA antibody.
Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways (2018)
Figure 7

Fig 7. Not4-dependent ubiquitination of eS7 is crucial for NGDRQC-.

(A-C) Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (A) 5' NGD-IM detection in not4∆ski2∆, hel2∆ski2∆, slh1∆ski2∆, not4∆ slh1∆ski2∆. Quantification of full length and the 5'NGD-IM relative to the loading control (SCR1) is given below. Note the size difference of 5'-NGD IMs resulting from NGDRQC+ and NGDRQC- and that the shorter 5'-NGD intermediate (representing intermediates of the NGDRQC- pathway) was reduced in not4∆slh1∆ski2∆ mutant cells. (B) Northern blot as in (A) except uS10-WTski2∆ or uS10-K6/8Rski2∆ mutant cells with or without a deletion of Not4 (not4∆) were used. Note that the shorter 5'-NGD intermediates are also reduced in ski2∆not4∆uS10-K6/8R cells. (C) Cells expressing HA-tagged eS7A or the eS7A-4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5'-NGD intermediates by Northern blotting. Note that the four mutated lysine residues in eS7 were responsible for NGDRQC- in the R(CGN)12 reporter. (D) Western blot analysis showing that K83 and K84 of eS7A are main target sites for Hel2 and Not4-mediated ubiquitination: Similarly, as in Fig 6C, eS7AΔeS7BΔ mutant cells were used and wild type and single mutants of HA-tagged eS7A were expressed. Ubiquitinated eS7-HA was monitored using an anti-HA antibody. (E) Northern Blot analysis probing for the levels of 5' NGD-IM derived from the R(CGN)12 reporter expressed in eS7AΔeS7BΔski2Δ cells and HA-tagged eS7 or eS7 mutants. We observed, that ubiquitination at K83 or K84 of eS7A is mainly responsible for NGDRQC-. (F) The levels of polyubiquitinated eS7A were increased by the overproduction of wild-type Hel2 (FL) but neither Hel2ΔRING nor Hel2(1-315) mutants. eS7A-3HAhel2Δ mutant cells harbouring the indicated plasmids expressing wild-type Hel2 (FL), Hel2ΔRING or Hel2(1-315) mutant proteins were harvested. Protein samples were analysed by Western blotting with an anti-HA (Top panel) or anti-FLAG antibody (Bottom panel). (G) Hel2(1-315) polyubiquitinates eS7A but not uS10. Western blotting after an in vitro ubiquitination assay using the Hel2-(1-315) mutant and purified ribosomes containing HA-tagged uS10. (H) Model for quality control pathways induced by R(CGN)12-mediated translation arrest. If the RQC pathway is intact, the leading ribosome that is stalled by the arrest sequence undergoes RQC. The uS10 ubiquitination and ATPase activity of Slh1/Rqt2-dependent subunit dissociation induce the endonucleolytic cleavages in the sequence covered by the first and the second ribosome (disome). In the absence of uS10 ubiquitination and Rqt2, RQC in the first ribosome, as well as NGD in the disome, are eliminated. Instead, RQC-uncoupled NGDRQC- takes place upstream of the disome. The carboxyl-terminal region of Hel2 is crucial to induce RQC thereby NGD in the disome (NGDRQC+), but dispensable for RQC-uncoupled NGD (NGDRQC-).
Phosphorylation of myosin A regulates gliding motility and is essential for Plasmodium transmission (2022)
Figure 2

Figure 2. Effect of 3'UTR replacement of myoA by the 3'UTR of dhfs on life cycle progression and myoA expression

A. Scheme showing simplified myoA loci in transgenic parasite lines.

B. Blood stage growth rate of mutated parasite lines. A single parasite was injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9 for each mouse (black dots). Bars represent the mean of 4-7 mice.

C. Infected midgut showing oocysts stained with mercurochrome; scale bar, 100 μm. The graph shows oocyst numbers per infected mosquito. Numbers of mosquitoes that were analyzed are indicated above the graph. Data derived from at least two independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers).

D. Ratio of salivary gland to midgut sporozoites of the indicated parasite lines. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean.

E. Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging. Arrow indicates direction of a motile sporozoite. The graph shows the fraction of motile hemolymph sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean.

F. qRT-PCR analysis of schizonts and oocysts of the PbMyoA 3'dhfs parasite line. Individual data points correspond to the mean of three technical replicates.

Significance for (B), (D) and (E) determined by One-way analysis of variance with Tukeys Multiple Comparison test. Significance for (C) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.
The human host response to monkeypox infection: a proteomic case series study (2022)
Figure 1

Figure 1. The human host response to monkeypox virus infection determined at the level of the plasma proteome.

a) Schematic overview of the workflow using discovery proteomics (Zeno SWATH MS (Wang et al, 2022b)) in parallel to a targeted proteomic assay that quantifies COVID-19 severity biomarkers ((Wang et al, 2022a)) to characterize the plasma proteome in an MPX case series, and comparison the proteomes to those of healthy volunteers and COVID-19 patients.

b) Volcano plot of contrast MPX vs healthy controls; α <= 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins.

c) Gene set analysis (GSEA) of REACTOME (Croft et al, 2014) terms enrichment for contrast MPX vs control. Y-axis shows -log10 of adjusted p-value (fdr) for Normalized Enrichment Score (x-axis) for each term. Terms with fdr <= 0.3 are labeled.

d) Boxplots illustrating key proteins that differ between patients with MPX and controls (p-values and fdr for corresponding contrast MPX vs Control are provided in brackets): TTR (p-value = 9E-10, fdr = 2E-7), LBP (p-value = 2E-4, fdr = 2E-3), APOC1 (p-value = 5E-8, fdr = 3E-6), and C9 (p-value = 2E-8, fdr = 2E-6). Here, as usual, the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.

e) Correlation between MPX severity (NSkin lesions) and protein expression (y-axis). One MPX patient had an unclear additional skin condition (not a pure case of MPX) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome; however, the proteome of this patient was largely in agreement with those of the other MPX cases (FigureEV 3). As a measure of MPX severity, the log2(1 + NLesions / 15) was used. Here NLesions is the number of lesions. R2 shows squared correlation coefficient. MPX patients are colored orange, control patients green.
LIF is a mitogen for choroidal EC and protects  choriocapillaris from  injury: implication for  AMD (2021)
Figure 1

Figure 1. Identification of LIF as the EC mitogen in LN-229 conditioned medium (CM) .

A) LN-229 CM stimulated growth of BCE cells, n=3.

B) VEGF neutralizing antibody (B20) failed to suppress BCE cell growth induced by LN-229 CM, n=3.

C) Reverse-phase fractions of LN-229 CM induced BCE cell growth. BCE cells were incubated with fractions (2 μl/well) as indicated in the Figure, n=3.

D) Candidate proteins generated from Mass-spectrometry analysis of LN-229 CM reverse-phase fractions. Candidates were identified by excluding intracellular proteins and proteins showing higher abundance in inactive fractions compared to those in mitogenic factions. Proteins were ranked for relative abundance as described in Materials and Methods.

E) The anti-LIF neutralizing antibody abolished BCE cell growth induced by reverse-phase fractions, n=3.

F) Recombinant human LIF proteins stimulated growth of BCE cells in a dose-dependent manner. BCE cells were cultured in the presence of vehicle, VEGF (10 ng/ml) and the indicated concentrations of recombinant human LIF (rhLIF, Sigma), n=3.

G) LIF and VEGF synergistically stimulated BCE cell growth. Cell proliferation was analyzed after 6 days using alamar blue as described in Materials and Methods, n=3.

Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant.
LIF is a mitogen for choroidal EC and protects  choriocapillaris from  injury: implication for  AMD (2021)
Figure 2

Figure 2. LIF promotes BCE cell growth via the JAK-STAT3 pathway.

A) The JAK inhibitor baricitinib (Ba) blocked LIF-induced STAT3 phosphorylation. BCE cells were pre-incubated with DMSO, baricitinib (2 μM), cobimetinib (Co) (150 nM) or BEZ235 (BE) (5 nM) for 1 hour and were then treated with vehicle or LIF (10 ng/ml, Sigma) for 15 minutes. Ctrl, no pre-incubation with inhibitors.

B) Baricitinib suppressed LIF-induced BCE cell growth. BCE cells were pre-incubated with DMSO, baricitinib, cobimetinib, or BEZ235 for 1 hour and then treated with vehicle, LIF (10 ng/ml) or VEGF (10 ng/ml). Cell proliferation was analyzed after 6 days, n=3.

C) and D) STAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNAs targeting STAT3. qRT-PCR were performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and are presented in C. In D, cells transfected with siRNAs were treated with LIF (10 ng/ml) or vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with the indicated antibodies.

E) LIF-induced BCE cell growth was abolished by STAT3 knockdown. BCE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) or vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading at 590 nm for each vehicle group was set as 1, n=3. siNegative, negative control siRNA not targeting any known genes.

Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.
LIF is a mitogen for choroidal EC and protects  choriocapillaris from  injury: implication for  AMD (2021)
Figure 3

Figure 3. LIF inhibits BAE cell growth through the JAK-STAT3 pathway.

A). Recombinant human LIF inhibited growth of BAE cells in a dose-dependent manner. BAE cells were cultured in the presence of vehicle and indicated concentrations of recombinant human LIF (rhLIF). Cell proliferation was analyzed after 6 days, n=3.

B). JAK inhibitor baricitinib blocked activation of STAT3 by LIF. BAE cells pre-incubated with DMSO and inhibitors for 1 hour were treated with vehicle and LIF (10 ng/ml) for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no pre-incubation with inhibitors; Ba, baricitinib (2 μM); Co, cobimetinib (150 nM); BE, BEZ235 (5 nM).

C). The JAK inhibitor baricitinib reversed LIF-induced BAE growth inhibition. BAE cells pre-incubated with inhibitors for 1 hour were treated with vehicle and LIF (10 ng/ml, Sigma). Cell proliferation was analyzed after 6 days using alamar blue, n=3.

D) and E). Knockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and shown in D. In E, cells transfected with siRNAs were treated with LIF (10 ng/ml, Sigma) and vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies.

F). LIF-induced BAE cell growth inhibition was abolished by knockdown of STAT3. BAE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) and vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading for each vehicle group was set as 1, n=3.

Data information: Bars and error bars represent mean±SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test.
LIF is a mitogen for choroidal EC and protects  choriocapillaris from  injury: implication for  AMD (2021)
Figure 4

Figure 4. LIF promotes angiogenesis in in vivo models.

A, B) Intravitreal injection of LIF increases blood vessel density in the mouse eyes. Adult mice were intravitreally injected with VEGF (10 ng) or LIF (10 ng to 100 ng, Sigma). Seven days after injection, PFA-fixed choroid-sclera complexes and retina were subjected to CD31 IF. Representative images of CD31-positive vessels are shown in A. Scale bar = 100 µm.Vascular density, determined with ImageJ software, is shown in B), n=5-8.

C, D) OCT-A imaging of LIF-treated mouse retina. Adult mice were intravitreally injected with 1 μl of LIF (50 ng, Sigma) or vehicle solution (PBS). Retinal OCT-A images were obtained 7 days after the injection and representative images are shown. Blood vessel density was determined as percentage of vessel-covered area/total area surface using ImageJ software and shown in D), n=7-8.

E). STAT3 was phosphorylated in retinal ECs after LIF treatment. 50 ng of our in-house LIF with low-endotoxin levels was injected. Phospho-Stat3 was examined on cryosection of retina after 2 hour by IHC. Magnification in the dashed box is shown in the lower panel. n = 5. Scale bar = 100 µm.

F). Five-day old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days, retinas were subjected to IF staining with Dyight-488-labeled lectin. Representative images for similar ocular loci and quantification of lectin-labeled area using ImageJ software were shown, n=4. Scale bar = 100 µm.

Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.