EMBO/soda-nel huggingface dataset:
Evolutionary remodelling of N-terminal domain loops fine-tunes SARS-CoV-2 spike. (2022)
Figure 3

Figure 3. Length polymorphism of the NTD loops across the sarbecoviruses.

A. Surface representation of spike colour-coded for conservation across the sarbecoviruses (as shown in the key), spike is shown from the side and from above. NTD, annotated, is a hotspot of diversity.

B. Maximum likelihood phylogeny of the Spike NTD nucleotide sequences (without loops) for 86 sarbecoviruses. Nodes with bootstrap support below 70 have been dropped. Clades containing nearly identical HKU-3-like and RaTG15-like viruses (see Dataset EV1) have been collapsed for clarity. Branches are coloured based on clade groupings.

C. NTD loop lengths were quantified across the sarbecoviruses; data points are colour-coded as in 3B, SARS-CoV-2 is represented as a star. In each column, the bar represents mean loop length.

D. Example protein alignments for each loop displaying SARS-CoV-2 (Wu-Hu-1), Pangolin CoV (Guangdong isolate, GD) and SARS-CoV. The length of each loop is provided with each label. Lentiviral PV encoding a luciferase reporter gene were used to evaluate spike-mediated entry by Pangolin CoV GD.

E. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting.

F. Representative raw luciferase activity upon PV infection of the HeLa ACE2 and HEK 293T cells by the stated PV, annotated values represent fold enhancement in entry compared to Wu-Hu-1, n=3 technical repeats.

G. Pangolin CoV entry into HeLa ACE2 and HEK 293T cells relative to Wu-Hu-1 control. Data points represent the mean of independent experiments, n=8 biological repeats.

H. Surface representation of spike protein illustrating the NTD swaps between Wu-Hu-1 and Pangolin CoV.

I. Entry of PV bearing the stated spike proteins into HeLa ACE2 and HEK 293T. Infection is expressed relative to Wu-Hu-1. Data points represent the mean of independent experiments, n=10 biological repeats.

J. Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting.

Data information: In all plots, error bars indicate standard error of the mean, statistical analysis (T-test and ANOVA, compared to control Wu-Hu-1) performed in GraphPad Prism. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = not significant.
Evolutionary remodelling of N-terminal domain loops fine-tunes SARS-CoV-2 spike. (2022)
Figure 4

Figure 4. Length polymorphism of the N2 loop alters SARS-CoV-2 spike function independent of immune evasion

A. Protein alignment illustrating the N2 loop variants designed for this study; loops of equivalent length to Wu-Hu1 and Alpha are annotated. The location of the N74 glycosylation site, and N74K mutation, is highlighted in bold.

B. Molecular models to illustrate the N2 loop lengths (generated using AlphaFold/ColabFold); shorter loops tend to adopt a β-hairpin. N74 is annotated.

C. N2 loop variant spike PV infection of the stated cell lines, expressed relative to Wu-Hu-1 (15 residue loop, coloured grey), n=3 biological repeats.

D. Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting.

E. Experiments performed with authentic Public Health England strain SARS-CoV-2 without the polybasic cleavage site (PHE ΔPBCS), compared to an N2 loop deleted mutant that emerged spontaneously in cell culture (Δ67-76). AlphaFold/ColabFold molecular models illustrate the N2 loop in either virus.

F. Three-day Vero cell growth curves. One representative experiment is shown, n=4 technical repeats.

G. and H. Single round titres and qPCR Ct values in cell culture supernatant from either virus, values represent the mean of n=4 biological repeats.

I. Representative inverted greyscale images of crystal violet-stained cells in a plaque assay, lighter areas represent areas of cell death. PHE ΔPBCS Δ67-76 exhibits larger plaques. Note, the white squares in the corners of each micrograph are a feature of the imaging system and cannot be removed.

Data information: In all plots, error bars indicate standard error of the mean, statistical analysis performed in GraphPad Prism. (ANOVA and T-test, compared to controls N2 loop=15 residues (C), PHE ΔPBCS (G and H)) * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = not significant.

Expanded View Figure Legends
Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart (2013)
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(a) Ten-month-old adult mice and 20-month-old aged mice were treated with an intraperitoneal injection of 5 mg kg−1 CCCP and their hearts were collected 12 h later. Representative electron micrographs of hearts from CCCP-treated adult and aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents an autophagic vacuole containing mitochondria; original magnification × 12,000; scale bar, 500 nm. Mitochondria incorporated into an autophagic vacuole in baseline and CCCP-treated conditions were quantified blindly from 8–10 images from different fields (original magnification × 5,000) (n=3 per group). (b,c) CCCP-induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria-rich fraction in adult and aged mice (b), and DOX-treated mice (c). Representative immunoblots are shown from four independent experiments. (d) Parkin was overexpressed in young and senescent MEFs using adenovirus-mediated transduction for the in vitro cell-based bioassay to assess the ability to clear damaged mitochondria. Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM. Results from four independent experiments performed in duplicate are shown. MEFs at passage 3 and passage 9 were used as young and senescent MEFs, respectively. (e) Mitochondrial DNA content was assessed by real-time PCR. Results are shown from six-independent experiments. (f) Mitochondrial content was assessed by immunoblotting for the mitochondrial chaperone, GRP75 (matrix protein) and complex I subunit, NDUFA9 (inner membrane protein). MEFs were treated with 100 nM bafilomycin-A1, (Baf-A1, an inhibitor of the vacuolar-type proton ATPase) to inhibit autophagy. Representative immunoblots are shown from three independent experiments. (g) Representative images of GFP-LC3 expressing MEFs treated with 20 μM CCCP for 10 h before the immunostaining of mitochondria with anti-TOM20 (red); original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm. Line scans below the images indicate colocalization between LC3 (green) and mitochondria (red) and correlate to the arrows drawn in the images. WHL indicates whole-heart lysate. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student’s t-test).
Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart (2013)
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(a) The senescence-associated molecules p53, p21, p16, H-Ras 12V and Rb were overexpressed in MEFs by retrovirus-mediated transduction. After puromycin selection, mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus-mediated Parkin expression. Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM. Results are shown from four independent experiments performed in duplicate. (b) Representative images of MEFs infected with the GFP-LC3 adenovirus and stained with anti-TOM20 (red) to visualize mitochondria 10 h after mitophagy induction; original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm. (c) Mitochondrial content of H-Ras 12 V expressing WT, p53−/− and p21−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. Results are shown from four independent experiments performed in duplicate. (d) Mitochondrial content of WT and p53−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. WT and p53−/− MEFs were transfected with control or p53-specific siRNAs or cultured in complete medium for 24 h in the presence or absence of 20 μM pifithrin-α before mitophagy induction. Results are shown from four independent experiments performed in duplicate. (e) Murine HL-1 cardiac myocytes were infected with retrovirus vectors encoding p53, p21 or p16 and after puromycin selection, underwent mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown. (f) HL-1 cells were transfected with control, p53, p21 or p16-specific siRNAs, followed by treatment with 0.02 μM DOX and mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate. (g) CCCP-induced recruitment of Parkin and expression of Mfn1 and Drp1 in DOX-treated WT, p53−/− and p21−/− mice were determined by immunoblotting of the heart mitochondria-rich fraction. Representative immunoblots are shown from four independent experiments. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student’s t-test).
Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart (2013)
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(a) Whole-cell lysates of HL-1 cells treated with 0.02 μM DOX and transfected with FLAG-tagged Parkin or the FLAG-empty vector were immunoprecipitated with the anti-Flag-M2 antibody and blotted with anti-p53 and Flag antibodies. (b) Whole-cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti-Parkin antibody. (c) Cytosolic heart lysates of adult and aged mice were immunoprecipitated with anti-Parkin, p53 and control IgG antibodies. (d) Schematic representation of N-terminally FLAG-tagged WT Parkin and various mutants of Parkin. (e) The association of endogenous p53 with various Parkin mutants in DOX-treated HeLa cells. The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53. (f) Schematic representation of the GST–p53 fusion proteins. (g) GST pull-down assays using GST–p53 fusion proteins and in vitro transcribed/translated FLAG-Parkin. Residues 81–160 of p53 were sufficient to bind Parkin.
Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart (2013)
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(a) HL-1 cells transfected with control or p53-specific siRNAs no.1 and cultured in complete medium for 24 h in the presence or absence of 0.02 μM DOX. After treatment with 20 μM CCCP for 6 h, the fractionation experiment was performed. Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting. Representative immunoblots are shown from three independent experiments. (b) These samples were subjected to immunoprecipitation with the anti-VDAC antibody to evaluate ubiquitination. Representative immunoblots are shown from three independent experiments. (c,d) Representative images of YFP-Parkin overexpressing p53−/− HCT116 cells re-transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8 (c) and 36 h (d) before the immunostaining of mitochondria with anti-TOM20 (red) and endogenous poly-ubiquitin with a specific antibody FK-2 (white); original magnification, × 630; scale bar, 20 μm. (e–g) Parkin mitochondrial translocation (e) and ubiquitination (f) 8 h after CCCP treatment and mitochondrial clearance (g) 36 h after CCCP treatment were quantified. A minimum of 300–400 YFP-positive cells were scored in three independent experiments. The single-letter amino-acid code is used. NES indicates nuclear export signal; NLS, nuclear localization signal; ER, endoplasmic reticulum; Ub, ubiquitin. Data are shown as the means±s.d. *P0.05, compared with the corresponding control (two-tailed unpaired Student’s t-test).
Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart (2013)
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(a) Representative electron micrographs of the heats of 20-month-old aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph shows an autophagic vacuole containing mitochondria; original magnification, × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8–10 images from different fields (original magnification × 5,000) (n=3 per group). (d,e) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser. With the initial presence of 5 μg mitochondria per well, 0.5 mM NADH and 4 μM FCCP, injections were performed as follows: port A, 50 μl of 20 μM rotenone (2 μM final); port B, 55 μl of 100 mM succinate (10 mM final); port C, 60 μl of 40 μM antimycin A (4 μM final); port D, 65 μl of 100 mM ascorbate plus 1 mM TMPD (10 μM and 100 μM final, respectively). Representative results of aged littermates are shown (d) and oxygen consumption by each complex was calculated from four independent experiments, five replicates per sample, and four samples per experiment (n=8 per group, e). (f) NADH-driven H2O2 production was determined using Amplex Red. Results are shown from five hearts analysed in duplicate. (g) Maximum dP/dt was examined using cardiac catheterization with graded dobutamine infusion in WT and Parkin Tg aged littermates (n=8 per group). (h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section. (i) Real-time PCR assessing the expression of cell cycle inhibitors and proinflammatory cytokines (n=8 per group). (j) Representative immunoblots assessing the expression of cell cycle inhibitors are shown; Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student’s t-test). (k) Proposed model for mitochondrial compromise in heart failure.
Antagonism of interferon signaling by fibroblast growth factors promotes viral replication (2020)
Figure 1

Fig 1: Inhibition of FGFR signaling in keratinocytes promotes IFN and ISG expression

(A,B) qRT-PCR for different ISGs relative to Rps29 using epidermal RNA from 8 week-old (A) or 5 day- or 9 day-old (B) K5-R1/R2 and control (CTRL) mice.

(C) Confocal microscopy images of epidermal sheets from back skin of K5-R1/R2 and CTRL mice stained for IRF7 and K14, counterstained with DAPI. Arrows denote nuclear IRF7 (red) in basal and suprabasal keratinocytes of K5-R1/R2 mice. The strong red staining of the stratum corneum (sc) is unspecific background and is more pronounced in K5-R1/R2 mice due to the increased thickness of this layer.

(D) qRT-PCR for different ISGs, Ifnb, and Ifnl3 relative to Rps29 using RNA from freshly isolated, non-cultured keratinocytes of K5-R1/R2 and control (CTRL) mice at P7.

(E) qRT-PCR for ISGs relative to Rps29 using RNA from primary keratinocytes of neonatal CTRL and K5-R1/R2 mice.

(F) qRT-PCR for ISGs and Cre relative to Rps29 using RNA from primary keratinocytes of neonatal K5-Cre and wild-type (WT) mice.

(G) Western blot analysis of total (L) and nuclear (N) lysates from immortalized (imm.) keratinocytes of CTRL and K5-R1/R2 mice for IRF1, IRF7, IRF9, Lamin A/C (nuclear marker, loading control) and GAPDH (cytosolic marker, loading control).

(H) qRT-PCR for RSAD2 and ISG15 relative to RPLP0 using RNA from HaCaT keratinocytes treated for 48 h with the FGFR inhibitor BGJ398 (3.5 μM) or vehicle in the presence of serum.

Data information: Scatter plots show mean +/- S.E.M. Mean expression levels in CTRL mice or cell cultures were set to 1, and mRNA levels relative to this value are shown. In (F) expression levels in K5-Cre mice were set to 1. A: N=10 mice per genotype, B: N=6-14 mice per genotype. C: Representative images of two experiments. Magnification bar: 20 μm. D: N=5 mice per genotype. E, F: N=4-7 cultures (each from a different mouse) per genotype. G: Representative of three experiments. H: N=6-9 per treatment group. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (Mann-Whitney U test). Exact P-values are provided in Dataset EV2.
Antagonism of interferon signaling by fibroblast growth factors promotes viral replication (2020)
Figure 2

Fig 2: FGF7 and FGF10 suppress the IFN response in keratinocytes

(A) Growth factor-starved primary keratinocytes from WT mice were treated for 3 or 6 h with FGF7 (10 ng/ml) or vehicle (CTRL). RNA samples were analyzed by qRT-PCR for the indicated ISGs relative to Rps29.

(B) Serum-starved HaCaT keratinocytes were treated for 6 h with FGF7 or FGF10 (each 10 ng/ml). RNA samples were analyzed by qRT-PCR for IRF7 and RSAD2 relative to RPLP0.

(C) Serum-starved HaCaT keratinocytes were treated for 16 h with FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for IRF1, IRF3, IRF9 and GAPDH.

(D) RNA samples from untreated, serum-starved primary keratinocytes from WT and Ifnar1-KO mice were analyzed by qRT-PCR for Irf7, Rsad2, Oasl2 and Ifnar1 relative to Rps29.

(E) Growth factor-starved primary keratinocytes from neonatal WT, Ifnar1-KO or Ifnar1/Ifnlr1-KO mice were treated for 24 h with FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for Irf7 relative to Rps29.

(F) HaCaT keratinocytes were transiently transfected with an ISRE-Luciferase reporter construct and starved 24 h after transfection for 9 h. They were then incubated in the presence or absence of FGF7 (10 ng/ml) for 20 h. Lysates were analyzed using a DualGlo Luciferase Assay.

(G, H) HaCaT keratinocytes were pre-treated for 2 h with the proteasome inhibitors MG132 (10 μm (G) or epoxomicin (100 nM) (H), followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.

Data information: Scatter plots show mean +/- S.E.M. Mean expression levels in CTRL cell cultures (not treated with FGF7 or any inhibitor) were set to 1. In (D) and in the left panel of (E) expression levels in wt cells were set to 1, while expression levels in untreated IFN receptor knockout cells were set to 1 in the middle and right panel of (E). A: N=8-11 cultures (each from a different mouse) from four experiments. B: N=3-5 from one experiment. C: Representative of 3 experiments. D, E: N=5-11 cultures (each from a different mouse) from two experiments. The results with Ifnar1-KO mice were reproduced in two additional experiments. F: N=16-18 from 4 experiments. G: Representative of four experiments, N=3 per treatment group for each experiment. H: N=6 per treatment group from two experiments. ns: non-significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P<0.0001 (Mann-Whitney U test (A, B, D, E, F, H) or t-test for assessment of FGF7 effect (G), with Welch correction for IRF1. Exact P-values are shown in Dataset EV2.
Antagonism of interferon signaling by fibroblast growth factors promotes viral replication (2020)
Figure 3

Fig 3: FGF7 suppresses IFN-induced ISG expression

(A-C) Serum-starved HaCaT keratinocytes were treated with FGF7 (10 ng/ml (A, C) or different concentrations as indicated (B)) and/or IFN-α (500-1000 U/ml) for 12-16 h. RNA was analyzed by qRT-PCR for ISGs relative to RPLP0 (A, B) and protein samples by Western blot for total and pSTAT1 (Y701 and S727), IRF1, IRF9 and RSAD2 and GAPDH (C). The vertical line in the RSAD2 blot indicates a place where the gel was broken. Densitometric quantification of the different blots (N=3) is shown on the right.

(D) Human primary keratinocytes were starved and treated for 12 h with FGF7 (10 ng/ml) and/or IFN-α (1000 U/ml). RNA samples were analyzed by qRT-PCR for ISGs relative to RPLP0.

Data information: Scatter plots show mean +/- S.E.M. Mean expression levels in CTRL cell cultures were set to 1. A: N=5-6 from two independent experiments. B: N=2-3 from one experiment. C: Representative blot from two experiments. The intensities of the bands were used for the quantification (N=3). D: N=6 replicates from two experiments with cells from two donors. ns: non-significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P<0.0001 (2-way ANOVA with Tukey's multiple comparisons test (A, D) or t test with Welch correction for assessment of FGF7 effect (C)). Exact P-values are shown in Dataset EV2.
Antagonism of interferon signaling by fibroblast growth factors promotes viral replication (2020)
Figure 4

Fig 4: FGF7 suppresses poly(I:C)-induced IFN and ISG expression

(A) Serum-starved HaCaT cells were treated for 24 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for IFNL1, IFNB, RSAD2, IRF1, IRF7, CGAS, TLR3 or DDX58, relative to RPLP0.

(B) Serum-starved HaCaT cells were treated for 18 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for total and pSTAT1 (Y701 and S727), total and pSTAT2 (Y690), IRF1, IRF3, pIRF3 (S396), IRF9, RIG-1, RSAD2 and GAPDH.

Data information: Scatter plots show mean +/- S.E.M. Mean expression levels in cell cultures treated with poly(I:C) only were set to 1, since IFN mRNA levels in non-treated cells are hardly detectable. A: N=6-9 from two experiments. B: Representative blot from two experiments. Quantification of the bands is shown in Figure EV3. **P≤0.01, ***P≤0.001 (2-way ANOVA with Tukey's multiple comparisons test). Exact P-values are shown in Dataset EV2.