EMBO/soda-nel huggingface dataset:
Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza (2019)
Figure 2

Figure 2. Ile68 and Leu69 in the C-terminal part of PB1-F2 determine its stability.

A Amino acid sequences of PR8 PB1-F2, 1918 PB1-F2, and the cloned PB1-F2 mutants.

B-D A549 cells were transfected with the indicated plasmids encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and chimeric PB1-F2 mutants. At 18 h after transfection, cells were treated with or without MG132 for 6 h. The expression of PB1-F2 and control genes was detected by semi-quantitative RT-PCR (upper panel) or Western blotting (lower panel).

E Representative immunofluorescence images of PB1-F2 expression in A549 cells. Cells transfected with the Flag-tagged PB1-F2 plasmid were stained with anti-Flag (red) antibody and analyzed by immunofluorescence assay. Magnification, ×400; scale bar, 50 μm.

Results shown are representative of three independent experiments.
Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza (2019)
Figure 3

Figure 3. Inhibition of IFNβ induction and its association with proteasomal degradation of 1918 PB1-F2.

A Relative activity of NF-kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1-F2. The mRNA levels of pro-inflammatory cytokines were determined by semi-quantitative RT-PCR.

B Cells were transfected with PB1-F2-expressing plasmids. Relative IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (bottom).

C U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay.

D Cells were infected for 24 h with each virus at 1 MOI. The mRNA level of IFNβ was determined by semi-quantitative RT-PCR, and PB1-F2 protein expression was assessed by Western blotting.

E Cells were transfected with each plasmid encoding PB1-F2. 18 h after transfection, cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi-quantitative RT-PCR (left) and qPCR (right). PB1-F2 protein expression was assessed by Western blotting.

F A549 cells were transfected with PB1-F2 clones mutated at positions 68 and 69; 24 h after transfection, the IFNβ mRNA level and PB1-F2 protein expression were determined by semi-quantitative RT-PCR and Western blotting, respectively (left). The relative expression level of IFNβ mRNA was also analyzed by qPCR (right).

All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).
Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza (2019)
Figure 4

Figure 4. IAV (1918) PB1-F2 increases viral pathogenicity in Balb/c mice and its amino acid residues 68 and 69 contribute to virulence.

A Mice (n = 5 or 6 per group) were infected with IAV with indicated pfu. Survival was monitored daily for 2 weeks.

B Mice (n = 6-9 per group) were infected with each IAV as indicated. Body weight was recorded daily after infection for 2 weeks.

C Mice (n = 5 or 6 per group) were infected with viruses (1000 pfu) carrying PR8 PB1-F2 with the I68T, L69P, or I68T+L69P mutation. Body weight changes and survival rate were monitored daily. Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level (80%) of mortality.

D Three cohorts of mice (5 or 6 per group) were infected with 500 pfu of one of the three IAVs. Viral protein expression in mouse lung was determined by Western blotting 2 days after infection.

E Mice (6 per group) were infected with 500 pfu of one of the three IAVs. Viral titration by plaque assay was performed at 2 days post-infection using lung homogenates.

F Mice (5-9 per group) were infected with 500 pfu of each virus. At 2 days post-infection, IFNβ mRNA levels were determined by qPCR.

G Mice (5-9 per group) were infected with 1 × 105 pfu of each virus. At 2 days post-infection, IFNβ protein levels in lung homogenates were measured by ELISA.
Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza (2019)
Figure 5

Figure 5. 1918 PB1-F2 inhibits IFNβ production via an interaction with DDX3.

A SDS-PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA-antibody. Bands were stained by Coomassie Blue.

B Venn diagrams classifying the proteins identified in immunoprecipitated samples. A total of 318 proteins were identified by LC-MS/MS analysis.

C The list of top ten networks obtained from Ingenuity Pathway Analysis of the 1918 PB1-F2 interactome.

D The "viral infection" network of interactome proteins. Proteins shaded in gray directly interact with 1918 PB1-F2. Interferon signaling pathway is shown in yellow. Solid lines, protein-protein interactions; solid arrows, phosphorylation; dotted arrows, induction.

E Analysis of interaction between DDX3 and 1918 PB1-F2. Cells were transfected with indicated plasmids; 18 h later, cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-HA antibody. Proteins were detected by immunoblotting with the indicated antibodies (left panel). IAV-infected cell lysates were immunoprecipitated with anti-PB1-F2 antibody and immunoblotted with the indicated antibodies (middle panel). Purified recombinant 1918 PB1-F2 protein was incubated with recombinant LysRS or LysRS-DDX3, and the interaction was analyzed by immunoblotting (right panel).

F Analysis of interaction between DDX3 and PB1-F2 mutants. A549 cells were transfected with the indicated clones; 18 h after transfection, cells were treated with MG132 for 6 h and analyzed by immunoprecipitation.

G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting (top). A549 cells were infected with indicated IAV. At 24 h post-infection, cell lysates were analyzed by Western blotting (bottom).

H Representative fluorescence images showing expression level of GFP-DDX3 (green). A549 cells were co-transfected with plasmids encoding the GEP-DDX3 and Flag-tagged PR8 or 1918 PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to fluorescence analysis.

I Mice were infected with 1×105 pfu of each virus, and lung homogenates were examined for endogenous Ddx3 expression.

J A549 cells were transfected with the indicated plasmids or treated with poly I:C. Cytoplasmic and nuclear fractions were separated and subjected to Western blotting.

K A549 cells were transfected with the indicated plasmids, and IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (middle). IFNβ promoter activity was determined by luciferase reporter assay (bottom).

Results were confirmed in three independent experiments, and representative data are shown.
Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza (2019)
Figure 6

Figure 6. DDX3 rescues mice from 1918 PB1-F2 virus infection.

A-D Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally.

A Scheme of the experiments.

B Survival was monitored daily for 2 weeks.

C Two days post-infection, virus titers in lung homogenates were determined by plaque assay.

D Two days post-infection, the expression level of IFNβ in the lungs was analyzed by semi-quantitative RT-PCR (left panel) and qPCR (right panel).

E Proposed model for innate immune evasion by the 1918 pandemic virus.

A p value of <0.05 was considered significant (** p < 0.01).

Expanded View Figure Legends
A distinct metabolic state arises during the emergence of 2-cell-like cells (2019)
Figure 2

Figure 2. Increased glucose uptake supports higher flux into the Pentose Phosphate pathway in Zscan4+ cells.

A) ATP content in ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 4 independent biological replicates.

B) Extracellular acidification rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser.

C) Glucose uptake rates in Zscan4+ (red) and 2-cell-like cells (green) were measured using a luciferase-based assay across 4 independent biological replicates and are represented relative to those of control ES cells (blue).

D) Schematic representation of measured fluxes (left). In order to ascertain whether the increased glucose uptake observed leads to higher flux into the hexosamine biosynthesis pathway (HBP) or the pentose phosphate pathway (PPP), one enzyme of each pathway was disrupted through siRNA-mediated knockdown (right).

E) Experimental design. ESC cultures were transfected with siRNAs targeting Gnpnat1 (HBP), G6pdx (PPP) or a negative control siRNA (Neg). After 48 hours of culture cells were FACS-sorted into a 96-well plate based on their fluorescent reporters and glucose uptake rates were measured using a luciferase-based assay.

F) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES, Zscan4+ and 2-cell-like cells. Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA. Shown are the mean ± s.d. of the indicated number of independent cell cultures, performed across 2 or more independent biological replicates each.

G) Glucose-6-phosphate dehydrogenase activity was measured in ES (blue), Zscan4+ (red) and 2-cell-like cell (green) lysates using a fluorometric assay. Measurements were obtained from 3 independent biological replicates, performed in 3 technical replicates each. n.s. - not significant; one-way ANOVA. Data information: In panels A-C and G, boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each individual technical replicate.
A distinct metabolic state arises during the emergence of 2-cell-like cells (2019)
Figure 3

Figure 3. Induction of Zscan4+ and 2-cell-like cells by defined metabolites.

A) Experimental design. ESC cultures were supplemented with increasing concentrations (see Table EV1) of 20 distinct metabolites for 48 hours. The abundance of the Zscan4+ and 2-cell-like cells was quantified by FACS according to the reporters shown in Figure 1B.

B) Heatmap showing the effect of the 20 metabolites tested on 2-cell-like cell levels.

C-F) Percentage of Zscan4+ and 2-cell-like cells in cultures treated with increasing concentrations (C and D) or varying combinations (E and F) of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 (C and D) or 2 (E and F) biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each of the 6 (C and D) or 4 (E and F) technical replicates.
A distinct metabolic state arises during the emergence of 2-cell-like cells (2019)
Figure 4

Figure 4. Sodium acetate induces Zscan4+ and 2-cell-like cells.

A) Immunofluorescence staining for OCT4, ZSCAN4 and 2C::tbGFP in control and acetate treated ESC cultures. Green arrows indicate 2-cell-like cells, and inlets highlight their DAPI structure. Scale bar, 20 μm.

B) RT-qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h. Shown are the mean ± s.d. of 3 independent cell cultures, performed in 2 technical replicates.

C) Percentage of 2-cell-like cells obtained upon transfection of control or Dux-targeting siRNAs in control conditions or in combination with sodium acetate treatment. Measurements were obtained from 2 independent cell cultures, performed across 3 independent biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate.

D) Percentage of 2-cell-like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment. Shown are the mean ± s.d. of 4 independent cell cultures, performed across 2 independent biological replicates.

E) Experimental design. ESC cultures were FACS-sorted to remove Zscan4+ and 2-cell-like cells and plated in a glass bottom 96-well-plate. Cells were then imaged for 96 hours in the presence of sodium acetate (32 mM), sodium L-lactate (32 mM) or in control conditions.

F) Proportion of Zscan4+ cells at various timepoints during the time-lapse experiment. Shown are the mean ± s.d. of 3 independent experiments.
Regulator of Calcineurin 1 (RCAN1) helps coordinate whole body metabolism and thermogenesis (2018)
Figure 1

Figure 1. Rcan1-KO mice are resistant to diet-induced obesity.

A, B Body weights of WT and Rcan1-KO mice fed normal chow diet (NC) or a high-fat diet (HFD) for 20 weeks starting at 8-10 weeks of age (male, n=15 each).

C Food consumption per gram of body weight gained (g/g ∆BW) over the first 15 weeks on NC or HFD, starting at 8 weeks of age (male, n=9 each).

D, E Cholesterol and triglyceride remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). Cholesterol and triglyceride content of NC and the HFD chow (input) were measured for comparison.

F-I MRI measures of total body weight, percentage lean mass, and percent fat mass prior to and after 6 weeks on the HFD (males, n=5). MRI image of WT and KO males following 8 weeks on a HFD. Yellow delineates visceral WAT. Pink delineates subcutaneous WAT. Quantification graphed on the right.

J Representative H&E staining of gWAT from WT and KO males after 25 weeks on NC or HFD. Scale bar = 100 μm.

K Quantification of cross-sectional area (CSA) of adipocytes in K (n=3 animals, 3 images/each, 75-100 adipocytes). Scale bar = 100 μm.

L Transcript levels for inflammatory markers Monocyte chemoattractant protein-1 (Mcp-1), macrophage antigen F4/80 (F480), tumor necrosis factor-alpha (Tnfα), and tumor necrosis factor-beta (Tnfβ) in gWAT of WT and KO after 25 weeks on NC or HFD (males, n=4-5). Transcript levels were normalized to 18S

Data information: values shown are mean ±SD except for graph in panel I where ±SEM is indicated. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).
Regulator of Calcineurin 1 (RCAN1) helps coordinate whole body metabolism and thermogenesis (2018)
Figure 2

Figure 2. Rcan1-KO mice maintain normal lipid handling and insulin sensitivity on a HFD.

A, B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD, with quantification of relative stain intensity (n=3, 3 images each). Scale bar = 100 μm.

C Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD. Stearoyl-Coenzyme A desaturase (Scd1), fatty acid transporter (CD36), glycerol-3-phosphate acyltransferase (Gpat), carnitine palmitoyltransferase I (Cpt1), peroxisome proliferator-activated gamma (PPARγ), pyruvate dehydrogenase acetyl-transferase (Pdk4), PPARγ coactivator 1 alpha (PGC1α), diacylglycerol O-acyltransferase (Dgat), and facilitated glucose transporter (Glut4). (males, n=4-5). Transcript levels were normalized to 18S.

D-F Serum levels of free fatty acids (FAA), insulin, and leptin in WT and KO after 25 weeks on NC or HFD (males, n=8-10).

G, H Glucose tolerance test (GTT) and Insulin tolerance test (ITT) after 25 weeks on NC or HFD. GTT was performed following an overnight fast, ITT following a three hour fast (males, n=6).

Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).
Regulator of Calcineurin 1 (RCAN1) helps coordinate whole body metabolism and thermogenesis (2018)
Figure 4

Figure 4. Depletion of Rcan1 reduces adipocyte lipid accumulation in vitro.

A Transcript levels for Rcan1.1 and Rcan1.4 during differentiation of 3T3-L1 adipocytes, measured at 0, 1, 2, 3, 4, 6 and 24 hours following the addition MDI as well as at 3, 7 and 10 days as indicated (n=3). * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, vs 0 time point.

B Western blot for RCAN1.1, RCAN1.4 and tubulin (TUB) in whole cell extracts of 3T3-L1 adipocytes transfected with a control si-RNA or ones targeting either Rcan1.4 or Rcan1.1 (20 μg per lane).

C Representative images of 3T3-L1 adipocytes transfected with the indicated si-RNAs prior to differentiation and stained with Oil Red O on day 10. Scale bar = 100 μm.

D Western blot for adiponectin (ADPOQ) released into the media during differentiation by 3T3-L1 adipocytes transfected with the indicated si-RNAs. Ponceau stained image provides loading control.

E Transcript levels for Pgc1-α, in 3T3-L1 adipocytes transfected with the indicated si-RNAs and assayed over 10-days of differentiation (n=3).

F Representative bright-field images showing lipid accumulation at 10-days differentiation of Ing-svf cells treated with the indicated siRNA. Scale bar = 50 μm.

G Transcript levels for Adrenoceptor beta 3 (Adrb3), uncoupling protein 1 (Ucp1), PPARγ coactivator 1 alpha (PGC1α), and deiodinase type II (Dio2) Ing-svf cultures transfected with control siRNA (C) or one targeting Rcan1 (KD) then maintained in growth media (G), or differentiated for ten-days (D) with and without the addition of 100 mM isoproterionol for 6 hours prior to harvesting (D+ISO) (n=2 in triplicate). * = p < 0.05; ** = p < 0.01, two-way ANOVA with multiple comparisons.

H, I Transcript levels for the indicated panel of genes relevant to adipocyte differentiation in Platelet-derived growth factor receptor alpha-positive (PDGFRα+) and lineage-negative (Lin-) stromal vascular cells isolated by FACS sorting from sWAT of WT (black bars) and KO (red bars). In H, isolated cells were maintained in growth media. In I, cultures were treated with MDI then differentiated for 10 days. * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, WT vs. KO.

Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD
Regulator of Calcineurin 1 (RCAN1) helps coordinate whole body metabolism and thermogenesis (2018)
Figure 5

Figure 5. Rcan1-KO mice show enhanced beiging of adipocytes in sWAT and elevated expression of SLN skeletal muscle.

A Representative H&E histological sections of sWAT from WT and KO mice probed with anti-UCP1 antibodies. Mice were either maintained at 24°C (RT) or housed at 6°C for 5 days. Scale bar = 200 μm.

B Transcript levels of Pgc1α and Ucp1 in sWAT of male and female WT and KO mice after 24 hours at RT or 4°C (n=5).

C Transcript levels of Ucp1, Pgc1α, Adrb3, retinoblastoma 1 (Rb1), zinc finger protein 423 (Zfp423), and PR domain containing 16 (Prdm16) were quantified in sWAT of WT and KO mice after 25 weeks on a HFD (n=4-5).

D Transcript levels of Pgc1α-1b and Pgc1α-1a isoforms in sWAT of WT and KO 8 week old females maintained at 24°C (RT) or housed at 4°C for 24 hours (n=5).

E. Western blot for RCAN1 in various skeletal muscle groups. Each lane was loaded with 20 μg of total protein from plantaris (PL), soleus (SOL), extensor digitorum longus (EDL), tibialis anterior (TA), red gastrocnemius (RG), white gastrocnemius (WG), white vastus lateralis (WV), heart ventricle (HT), atria, diaphragm (Dia), and Tong. The lane containing brain extract was loaded with 10 μg of protein. Antibody specificity was validated by running 20 μg of protein from the heart of an Rcan1-KO animal (HT-KO). The asterisks * indicate the location of putative proteolytic fragments of RCAN1.1.

F Transcript levels of Sln in skeletal muscle (tibialis anterior) of 8-10 week old WT and KO mice housed at RT. (n=4-5 each gender).

G Transcript levels of Sln in skeletal muscle (tibialis anterior) of 8-10 week old male WT and KO animals housed at RT. (n=5).

H Representative western blot showing SLN protein in soleus muscle of 8-10 week old WT and KO animals housed at RT. GAPDH was used as a loading control.

I Alignment of the genomic region 1.5 Kb upstream of the mammalian Sln gene using the ECR Browser (http://ecrbrowser.dcode.org/). Human was used as the reference genome. The location of conserved NFAT binding sites are indicated by blue diamonds and CREB sites by yellow. Sites were conserved across all mammalian species available in the database except opossum. Mouse, rat, and dog are provided as representative examples. The structure of the Sln-Luc reporter construct is aligned below.

J Activity of the Sln-Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin (CnA*) and Rcan1. Luciferase activity was normalized to beta-galactosidase activity from a co-transfected vector. (n=3, assayed in duplicate).

Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons in B, D, F, and J; unpaired student's t-test in C and G.
Regulator of Calcineurin 1 (RCAN1) helps coordinate whole body metabolism and thermogenesis (2018)
Figure 6

Figure 6. Rcan1 expression is positively correlated with indices of metabolic syndrome. A Correlation of Rcan1 expression in liver and gonadal adipose tissue with HOMA-IR, using male mice from over 100 strains from the hybrid mouse diversity panel. Regression line (red), r = biweight midcorrelation, p = p-value. B, C Correlation of Rcan1 expression in gonadal adipose and liver with body weight, plasma insulin and plasma triglycerides, in a data set comparing backgrounds susceptible (BTBR background) or resistant (C57BL/6 background) to diabetes when carrying the leptinob/ob (ob) mutation. Measures are reported as the ratio of the mean log10 intensity (ml ratio). Regression line (black), r = linear regression, p = p-value. D Model for RCAN1 suppressive control over two prominent mechanisms of mammalian nonshivering thermogenesis (NST): one in white adipose tissue, where uncoupling of the mitochondrial membrane potential by UCP1 increases heat generation at the expense of ATP production; the other in skeletal muscle, where SLN increases ATP consumption by SERCA. In adipose, RCAN1 suppresses the conversion of white adipocytes to UCP1-positive, beige adipocytes by acting as a brake on calcineurin (CN)-dependent activation of Pgc1α-1b expression, thereby blunting downstream activation of Ucp1 expression. In skeletal muscle, RCAN1 suppresses CN-dependent expression of Sln directly. Loss of these repressive functions underlies the elevated metabolic rate and lean phenotype of the Rcan1-KO animals. Table 1 Table 2: Sources for key resources Forward primer for cloning pSln-Luc ACGCGTGCTAGCTTCTAAGCCCCATTTAGC Reverse primer for cloning pSln-Luc AAGCTTAGATCTCCGGCTGTCTGAGCTCCTGG Expanded View Figure Legends