EMBO/soda-nel huggingface dataset:
Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2 (2020)
Figure 3

Figure 3. The S2 site in SARS2 PLpro

A) A previous structure of SARS PLpro bound to a non-hydrolysable, Lys48-linked diubiquitin probe (pdb 5e6j, (Békés et al, 2016)) explained the noted preference of PLpro for longer Lys48-linked chains. While the proximal ubiquitin unit occupies the S1 site in a highly similar fashion in SARS~Ub and SARS2~Ub structures (see B, Fig 2A, EV2), the second, distal, ubiquitin unit binds to the α2 helix of PLpro, through a common binding mode involving the ubiquitin Ile44 patch. On helix α2, a central Phe70 in SARS PLpro residue is flanked by residues involved in polar contacts.

B) Structure of the SARS2 PLpro~Ub complex. The S2 site consisting of α2 helix with Phe69 residue, is fully conserved in SARS2 PLpro (Fig EV1).

C) Calculated enzymatic efficiencies for K48-diUb-TAMRA cleavage, followed by fluorescence polarisation with PLpro wild-type (reproduced from Appendix Fig S1A), and PLpro F69S. (Left) Using the same concentration range of SARS2 PLpro, the F69S mutant activity could not be fitted; (middle) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin-TAMRA (45 vs. 86 M-1s-1, compare Fig 1D, Appendix Fig S1A). (Right) A ~3-fold lower efficiency for PLpro F69S cleaving ISG15-TAMRA yields values similar to cleavage of ISG15CTD-TAMRA (Appendix Fig S1A), suggesting that the S2 site contributes the difference in binding for the N-terminal Ubl-fold. Experiments for F69S were performed in technical triplicate and biological duplicate; wild-type data is reproduced from experiments shown in Fig 1D, Appendix Fig S1A.

D-F) Gel based analysis showing hydrolysis time course of triubiquitin (D), proISG15CTD (E) and proISG15 (F) using wild-type PLpro (left, wild-type gels reproduced from Appendix Fig S1B-D to enable direct comparison) or PLpro F69S (right). PLpro F69S has a strong effect on triubiquitin hydrolysis (D), no marked effect on hydrolysis of proISG15CTD (E) and reduces proISG15 cleavage to the same levels as proISG15CTD (compare E, F), Experiments were performed in duplicate, see Source Data for uncropped gels.
Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2 (2020)
Figure 4

Figure 4. High throughput screen of SARS2 PLpro against known drugs.

A) High throughput screening of SARS2 PLpro was performed against 5576 approved drugs and late-stage clinical compounds, in 1536-well format using Ub-Rhodamine (see Methods). Two replicates out of three are shown; hit compounds were those that inhibited PLpro activity by more than 40% in all three replicates. Correlation (R2) between all screens exceeded 0.8­9. See Fig EV4 for assay design and quality control, and Methods.

B) Hit compounds and compound rac5c (see Fig 5), were further assessed in 10-point IC50 titrations using the Ub-Rhodamine assay, using a starting concentration of 100 µM serially diluted in 1:3 steps. Degree of inhibition is shown as a titration heat-map from dark (full inhibition) to light blue (low/no inhibition). The catalytic domain of human USP21 (Ye et al, 2011) was used as a counterscreen. Each PLpro hit compound showed either no activity in the titration analysis, or an identical inhibition profile against PLpro and USP21, suggesting assay interference. Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM. IC50 assays were performed in technical triplicate in two independent experiments.
PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in 
P. falciparum (2021)
Figure 3

Figure 3. PfCDPK7 interacts with PMT and EK in the parasite and regulates PMT protein expression.

A-B. 3D7 (A) or parasites overexpressing EK-GFP (B) were used to prepare protein lysates followed by immunoprecipitation using anti-PfCDPK7 or anti-GFP antibody, respectively. Subsequently, protein lysate or indicated IP were subjected to Western blotting using anti-PMT (A), anti-GFP (B). PMT was found to co-IP with PfCDPK7 (A, lane 2) and EK-GFP with PfCDPK7 (B, lane 3).

C. 3D7 and PfCDPK7-KO parasites at schizont stage (~40 h) were used for Western blotting using anti-PMT antisera. Actin served as a loading control. Right panel: Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin. Fold change in PMT was determined with respect to 3D7 (mean± SEM, n=4; biological replicates, **p<0.01, t-test).
PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in 
P. falciparum (2021)
Figure 4

Figure 4. Phosphorylation of EK S37 is important for parasite development.

A. Parasites overexpressing EK-GFP or its S37A mutant were synchronized and parasitemia was determined at the end of first (48 h.p.i) and second cycle (96 h.p.i). Fold change in parasitemia of EK_S37A parasites with respect to WT EK overexpressing parasites was compared, which was significantly reduced (mean± SEM, n=3; biological replicates, *p<0.05, ANOVA).

B. Giemsa stained thin blood smears prepared at indicated hours post-infection (h.p.i) from assay. Arrows indicate parasites exhibiting abnormal morphology, which were observed at the beginning of each replication cycle.

C. IFA was performed on parasites expressing WT EK-GFP or its S37A mutant 6-12h post-invasion to detect (i) RAP1, which is transferred to PV post invasion, and (ii) MSP1 which is located at the PVM. Parasite expressing WT EK-GFP exhibited typical round ring like shape. In contrast, several parasites expressing the S37A mutant exhibited irregular morphology with much smaller vacuolar space and in some parasites RAP1 was found outside the PV (Scale bar-1 μm).

D. Quantitation of abnormal parasites in IFA described in panel C (mean± SEM, n=3; biological replicates, ***p<0.001, t-test).

E. Parasites EK-GFP or its S37A mutant were synchronized and parasites were allowed to mature to schizonts/segmentors. The graph shows average number of merozoites per schizont from three biological replicates. Each replicate represents an average of merozoites from 30-40 schizonts. (mean± SEM, n=3; biological replicates, *p<0.05, t-test).
PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in 
P. falciparum (2021)
Figure 5

Figure 5. PfPI4K is involved in the localization of PfCDPK7 and parasite maturation.

A. 3D7 parasites were treated with DMSO or 50nM MMV048 for 24h. Subsequently, parasites were fixed and IFA was performed to detect ER marker BiP and PfCDPK7. While PfCDPK7 was present in puncta proximal to ER (left panel), inhibitor treatment caused a loss in this vesicular localization and resulted in a more diffuse distribution of the enzyme (right panel) (Scale bar-1 μm).

B. Western blotting was performed on parasites treated with DMSO, 10nM UCT943 or 50nM MMV048 as described above using anti-PfCDPK7 or anti-Actin (loading control) antibody. Please note that this protein lysate was also used for Western blots in Fig. 6C with other antibodies. Therefore, this loading control blot for actin shown here pertains to the loading control in figure 6C as well.

C. 3D7 parasites were treated with DMSO or 50nM MMV048 for 20-24h. Subsequently, IFA was performed ~24h post-invasion to detect RAP1 and MSP1 (left panel) as described for figure 4C. Right panel, Quantitation of parasites exhibiting abnormal staining for RAP1 outside the PV was observed (mean± SEM, n=3; biological replicates, **p<0.01, t-test; Scale bar-1 μm).
PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in 
P. falciparum (2021)
Figure 6

Figure 6. PfPI4K inhibitors impair PL biosynthesis

A-B. 3D7 parasites were synchronized and treated with 10nM UCT943 or 50nM MMV048 for 10-12h. Subsequently, parasites were incubated with 14C-Cho (A) or 14C-Eth (B) and were harvested after 12h. Total lipids were extracted and separated by TLC.

The radiolabeled lipids were detected by phosphorimaging and quantitated by densitometry (upper panel). Bottom Panel (A and B), fold change in PC and PE formation in inhibitor treated parasites with respect to DMSO treatment was determined (mean±SEM, n=4; biological replicates, **p<0.01, ***p<0.001, ANOVA; ns, not significant).

C-D. UCT943 and MMV048 treated 3D7 (C) or EK-GFP overexpressing parasites (D) were subjected to Western blotting (as described in Figure 5B) using anti-PMT (C) and anti-GFP (D) antibodies (left panels). For experiments in Fig. 6C, the lysate used was the same as the one used for Fig. 5B for PfCDPK7 Western blotting. Therefore, the corresponding actin blot for loading control is shown in Fig. 5B. Right Panels: densitometry was performed for bands corresponding to PMT (C) or EK-GFP (D), which were normalized with respect to Actin. Fold change in PMT or EK was determined with respect to DMSO treated parasites (Mean± SEM, n=4; biological replicates, *p<0.05, ***p<0.001, ANOVA).
Aregs exhibit robust non-and anti-adipogenic properties that arise with age and involve RA signaling (2022)
Figure 2

Figure 2. Age-dependent molecular and phenotypic emergence of bona fide CD142+ ASPCs

Boxplot showing the distribution of the "CD142+ score" (Appendix Table S1) in adult (darker colours) and P12 (lighter colours) ASPCs across the three main ASPC subpopulations (adipose stem cells (ASCs) in green, pre-adipocytes (PreAs) in red and F3(CD142)+ ASPCs in blue) based on the integration of three single-cell datasets of mouse ASPCs (see Methods and Appendix Fig. S11);

FACS-based gating strategy of pre-weaning (new-borns (P0))- and post-weaning (11-week-old (wo))-derived Lin- (defined as CD31- CD45- TER119-), SCA-1+ (total ASPC), CD142- and CD142+ ASPC cellular fractions within the subcutaneous adipose SVF; at least 8 biological replicates were performed, shown here is one representative biological replicate;

Bar plots showing the normalised parental percentage of pre- and post-weaning indicated cellular fractions; the graph on the right represents the fractions of CD142+ ASPCs plotted separately; marker shapes correspond to different biological replicates, n=8-10;

Representative fluorescence microscopy images of P0-, P12-17-, 4wo-, 7wo- and 11wo-derived total, CD142- and CD142+ APSCs after in vitro adipogenic differentiation;

Fraction of differentiated cells per ASPC type shown in D, as quantified by the "adiposcore"; bar colour shading corresponds to pre- and post-weaning-derived cells as indicated; marker shapes correspond to different biological replicates, n=14-65, 12-13 biological replicates, 2-6 independent wells for each;

PCA based on the bulk RNA-seq data of freshly isolated P0-, P16-, 4wo-, 7wo- and 11wo-mice-derived total, CD142- and CD142+ ASPCs;

Boxplot showing the distribution of the "CD142+ score" (Appendix Table S1) across ages and tested cellular fractions (freshly isolated total, CD142- and CD142+ ASPCs) showing the age-dependent emergence of the CD142+ signa- ture; see colour legend in F, n=2-5, 2-5 biological replicates, 1 independent well for each;

Boxplot showing the distribution of the "CD142+ score" (Appendix Table S1) across ages and tested cellular fractions (freshly isolated total, CD142- and CD142+ ASPCs) using the log normalised expression corrected for age-driven source of variation; see colour legend in F, n=2-5, 2-5 biological replicates, 1 independent well for each.

Data information: In all images, nuclei are stained with Hoechst (blue) and lipids are stained with Bodipy (yellow); scale bars, 50 µm; bar colours: total ASPCs - brown, CD142- ASPCs - yellow, CD142+ ASPCs - blue; pre-weaning data are represented in lighter shades;

*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, pairwise two-sided t-test (A, C, E, H) or one-way ANOVA and Tukey HSD post hoc test (G, null hypothesis: no difference in means across age), for statistical details see Methods.
Aregs exhibit robust non-and anti-adipogenic properties that arise with age and involve RA signaling (2022)
Figure 4

Figure 4. Molecular (auto-)regulation of CD142+ ASPC (Areg)'s inhibitory activity

Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) carrying control (scr) or siRNA-mediated knockdowns of selected CD142+ ASPC (Areg)-specific candidate genes: F3, Mgp, and Gdf10 after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods);

Fraction of differentiated CD142- ASPCs, as quantified by the "adiposcore", co-cultured with CD142+ ASPCs (Aregs) subjected to specific knockdowns (or scrambled (scr) siRNA control), as indicated in A; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each;

PCA based on bulk RNA-seq data of CD142- ASPCs co-cultured with CD142+ ASPCs (Aregs) subjected to specific knockdowns as indicated in A; see colour legend at the bottom of the figure;

Expression heatmap of top CD142+ markers (i.e., Areg markers, Appendix Table S1) with a positive log2FC when performing differential expression analysis between CD142- ASPCs that were exposed to "active Aregs" (WT, scr, siGdf10) versus CD142- ASPCs that were exposed to "dysfunctional Aregs" (siF3, siMgp) across the bulk RNA-seq of the same samples and of CD142- and CD142+ ASPCs after exposure to an adipogenic cocktail; genes are ordered from the highest (top) to the lowest (bottom) log2FC; significantly differentially expressed genes (FDR < 0.05) are coloured in blue; Areg candidates are highlighted in bold; log normalised expression scaled by row; see colour legend at the bottom of the figure;

Heatmap showing the Euclidian distance of the transcriptomic data of CD142- ASPCs co-cultured (via transwell) with distinct CD142+ ASPC knockdown types or controls as well as CD142+ or CD142- ASPCs post-differentiation, calculated on the five first principal components of the PCA shown in Appendix Fig. S23D;

Correlation of "white fat cell differentiation score" versus "CD142+ score"; see colour legend at the bottom of the figure;

Volcano plot showing the coefficient of the linear regression performed between "white fat cell differentiation score" (WFCDS) and log normalised gene expression (WFCDS ~ log(normalised expression)) (x-axis) versus the

-log10(p-value) (y-axis); top CD142+ markers (i.e., Areg markers, Appendix Table S1) are highlighted in blue and genes linked to "cellular response to RA" (GO:0071300) and "RA receptor signalling pathway" (GO:0048384) terms in green.

Data information: In all images, nuclei are stained with Hoechst (blue) and lipids are stained with Bodipy (yellow); scale bars, 100 µm;

*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, pairwise two-sided t-test (B), for statistical details see Methods
SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology (2020)
Fig. 4.

Cross-reactivity of antisera raised against SARS-CoV structural and non-structural proteins.Selected antisera previously raised against SARS-CoV nsps and structural proteins cross-react with corresponding SARS-CoV-2 proteins. SARS-CoV-2-infected Vero E6 cells (m.o.i. of 0.3) were fixed at 12 or 24 h p.i. For immunofluorescence microscopy, cells were (double)labelled with (a) a rabbit antiserum recognising nsp4 and a mouse mAb recognising dsRNA; (b) anti-nsp4 rabbit serum and a mouse mAb directed against the N protein; (c-e) rabbit antisera recognising against nsp3, nsp13 and the M protein, respectively. Nuclear DNA was stained with Hoechst 33258. Bar, 20 µm.
Endothelial Primary Cilia Inhibit Atherosclerosis (2015)
Figure 1

Figure 1. Endothelial primary cilia are non-uniformly distributed in the adult mouse aorta and are dispensable for development.

(A-B) Primary cilia are enriched at branch points and the greater curvature of the aorta of wild type mice, schematized in (A) and quantified in (B). The color in (A) indicates which side of the aorta is quantified in (B). Numbers indicate the region of the aorta (A) whose proportion of ciliated cells is quantified in (B). Error bars are +/- 1 S.E.M and n=7 mice. (C) Aortic endothelial cilia are ablated in Tek-Cre Ift88C/- mice. Endothelial cells and cilia are labeled by staining for Pecam1 and Arl13b, respectively. Scale bars are 10μm.
Endothelial Primary Cilia Inhibit Atherosclerosis (2015)
Figure 2

Figure 2. Retinal angiogenesis and aortic endothelial polarity are unaffected in mice lacking EC cilia.

(A) The vasculature in P5 retinas from (A) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence.

(B) The fractional distance migrated to the edge of the retina was quantified. There was no statistically significant difference (p=0.305, Student’s 2-tailed t-test, n=6 mice for each group). The average value of 8 measurements (4 retina leaflets measured per eye on both eyes) was used for each mouse.

(C) The number of vascular branchpoints was counted. There was no statistically significant difference (p=0.25, Student’s 2-tailed t-test, n=6 for control and n=5 for mutant).

(D) Control and mutant adult mouse aortas were stained for GM130 (green) to mark the Golgi, CD144 (red) to mark ECs and DAPI (blue) to label DNA.

(E) The angle between the Golgi, nucleus and tissue axis was measured and binned as ‘left’, ‘right’, ‘upstream’ or ‘downstream’.

(F) Six areas in the aorta were analyzed.

(G) There was no difference in Golgi distribution at any single region or overall, nor was there a difference in the variance of the individual un-binned angles. Error bars are +1 S.E.M. Scale bars are 500μm. n=3 control and mutant mice for each region except region 2 where n=3 control and n=2 mutant mice.
Endothelial Primary Cilia Inhibit Atherosclerosis (2015)
Figure 3

Figure 3. Loss of EC cilia accelerates atherosclerosis.

(A) Scheme for induction of atherosclerosis Apoe-/- mice lacking Ift88 in the Tek-Cre and Mx1-Cre lineages (and control littermates). Following weaning, mice we’re placed on a high-fat, high-cholesterol diet for 8 weeks prior to analysis.

(B) Loss of EC cilia by Tek-Cre increases atherosclerosis in the aortas of Apoe-/- mice on a high-fat high-cholesterol diet as visualized by Oil Red O (ORO) staining (left panels) whereas loss of cilia genes in blood by Mx1-Cre has no effect (right panels).

(C) Cropped views of the boxed areas in (B) showing increased atherosclerosis in mice with Tek-Cre but not Mx1-Cre mediated loss of Ift88 in the aortic arch.

(D-G) Scatter plots of the percent ORO-positive area of the thoracic aorta in (D-E) Tek-Cre and (F-G) Mx1-Cre mice, separated by (D, F) females and (E, G) males. Tek-Cre Apoe-/- Ift88C/- mice had a 58.9% (p=0.0135, Student’s 2-tailed t-test, n=13 control and n=10 mutant mice) and 67% (p=0.0106, Student’s 2-tailed t-test, n=17 control and n=14 mutant) increase in ORO-positive area versus control littermates in females and males, respectively. Mx1-Cre Apoe-/- Ift88C/- mice had no significant difference in ORO-positive area in females (p=0.6643, Student’s 2-tailed t-test, n=7 control and mutant mice) or males (p=0.9381, Student’s 2-tailed t-test, n=9 control and n=7 mutant mice). Black bars represent the mean and error bars are +/- 1 S.E.M. Aortas depicted in (B-C) are red in the scatter plots.
Endothelial Primary Cilia Inhibit Atherosclerosis (2015)
Figure 4

Figure 4. Loss of EC cilia promotes pro-atherosclerotic pathways in Apoe-/- mice.

(A) Transcripts for the inflammatory cytokines Il1b, Il6, Tnf1 and Tgfb1, the adhesion molecules Vcam1 and Sele, the lymphocyte marker Nos2 and macrophage marker Cd68, and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe-/- mice lacking EC cilia by RT-qPCR, while transcripts of the Hh target genes Gli1, Ptc1, and Ptc2 were not. Error bars are +1 S.E.M. n=8 control and n=10 mutant mice of mixed sex. P-values calculated by Student’s 2-tailed t-test.

(B) Pkd2 localizes to EC cilia in vivo. Adult mouse aortas were stained for Pkd2, acetylated α-Tubulin, and Hoechst to label DNA. Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells. Arrowheads point out cilia. Example cilium highlighted in inset.

(C) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia. Aortas from control and mutant mice were stained for eNOS, p-eNOS-S1176, acetylated α-Tubulin, and Hoechst to label DNA.

(D) The ratio of the total fluorescence p-eNOS-S1176 to total eNOS in the upper thoracic aorta (near the first intercostal artery) was calculated for control and mutant mice. The relative p-eNOS-S1176 to eNOS ratio was reduced in the mutant to 72.7% the level of control siblings (p=0.009, paired 2-tailed ratio t-test, n=7 sets of mice). Error bar is +1 S.E.M. Four sets consisted of one control mouse and one mutant mouse. Three sets consisted of two control or mutant mice and the average value of the two was used. Scale bars are 10μm.
PGE2-EP3 axis promotes brown adipose tissue formation through 
stabilization of WTAP RNA methyltransferase (2022)
Figure 2

Figure 2. BA-specific deletion of EP3 impairs iBAT development and aggravates HFD-induced obesity in mice.

(A) Oil Red O staining of mouse BAs differentiated from normal or EP3-deficient brown pre-adipocytes. Scale bar: 50 μm.

(B) Mean lipid droplet area of differentiated BAs from normal or EP3-deficient brown pre-adipocytes. *P < 0.05 vs WT (n = 6).

(C) Relative mRNA levels of brown marker genes in differentiated mouse BAs from normal or EP3-deficient brown pre-adipocytes detected by RT-qPCR (n = 7-8).

(D) Representative image of hematoxylin and eosin (H&E) staining for iBAT from EP3F/F and EP3F/FMyf5Cre mice. Scale bar: 50 μm.

(E) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice (n = 8).

(F) Relative mRNA levels of brown marker genes in iBAT from EP3F/F and EP3F/FMyf5Cre mice detected by RT-qPCR (n = 8).

(G) Body weight analysis of EP3F/F and EP3F/FMyf5Cre mice fed HFD (n = 12).

(H) Food intake of EP3F/F and EP3F/FMyf5Cre mice during HFD feeding (n = 7).

(I-J) Intraperitoneal glucose tolerance test (GTT) (I) and insulin tolerance test (ITT) (J) in EP3F/F and EP3F/FMyf5Cre mice fed HFD (n = 7-8).

(K) Representative image of H&E staining of iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD. Scale bar: 50 μm.

(L) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD (n = 8).

(M) Relative mRNA levels of brown marker genes in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD detected by RT-qPCR (n = 6-8).

Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates (A-C) or pooled from two independent experiments with biological replicates (E-J, L, M). Statistical significance was evaluated by Mann-Whitney U tests (B, C, E, F, H, L, M), two-way ANOVA followed by Sidak's test for multiple comparisons (G, I, J). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).