EMBO/soda-nel huggingface dataset:
LY6E impairs coronavirus fusion and confers immune control of viral disease (2020)
Figure 3.

Ly6eΔHSC mice are more susceptible to murine hepatitis virus.a-b, Female Ly6efl/fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival (a) and weight loss (b). c-n, Mice were assessed at 3 (c-h) or 5 (i-n) days post-infection for serum ALT (c,i), viral titers in liver (d,j) and spleen (e,k), and liver necrosis and inflammation (f-h, l-n). For a-b, n=13 for days 0-14 (Ly6efl/fl), n=11 for day 0-3, n= 10 for days 4-5, n=5 for days 5-6, n=3 for days 6-14 (Ly6eΔHSC) from three pooled experiments. For c-g, n=9 (Ly6efl/fl), n=10 (Ly6eΔHSC), n=6 (PBS) from two pooled experiments. For i-m, n=9 (Ly6efl/fl,), n=7 (Ly6eΔHSC), n=6 (PBS) from two pooled experiments. In h,n, scale bars are 500 µM (4x) and 100 µM (20x). Statistical significance was determined by Mantel-Cox test (a), two-tailed unpaired student's t-test with Welch's correction (c-e, i-k), two-tailed Mann-Whitney U test (f-g, l-m). Error bars: SEM (b), SD (c-g, i-m). P values: a, *** p=0.0002; c, ** p=0.0031; d, ns p=0.2136; e, **** p=5.759 × 10−7; f, ns p=0.3698; g, ** p=0.0054; i, * p=0.0430; j, ns p=0.1321; k, ** p=0.0094; l, ** p=0.0072; m, **** p=8.741 × 10−5.
LY6E impairs coronavirus fusion and confers immune control of viral disease (2020)
Figure 4.

Murine hepatitis virus infection alters hepatic and splenic immune responses in female Ly6eΔHSC mice.a-e, Transcriptomic analysis from Ly6efl/fl (WT) and Ly6eΔHSC (KO) mice. a-b, Heat maps displaying significant changes (mean RPKM > 0.5; fold change > 2; FDR ≤ 0.05) in liver (a) and spleen (b). Dendrograms are normalized read data (row z-score) clustered with complete linkage method employing Spearman Rank correlation distance measurement. c, Pathway analysis of KO versus WT. Up-(red) or down-regulated (blue) pathways indicated by activation z-score. Numbers show significantly dysregulated genes as percentage of total gene number included in pathway. d-e, Expression of select genes in liver (d) and spleen (e). f-g, Immune cell counts from liver (f) and spleen (g). h, Infection of cultured splenocytes. For a-e, n=3. For f-g, n=8 MHV-injected, n=4 PBS-injected from two pooled experiments. For h, n=3 (Ly6efl/fl and Ly6eΔHSC) or n=2 (Ifnar−/−) from two pooled experiments. Significance for f-h was determined by two-tailed unpaired student's t-test with Welch's correction. Error bars: SD (d-h). P values (left-right): f, **** p=1.3644 × 10−6, ns p=0.7952, ns p=0.0781, ns p=0.7732, ** p=0.0016, ns p=0.6261, ** p=0.0068, ns p=0.9494, * p=0.0141, ns p=0.7639, * p=0.0436, ns p=0.9600, * p=0.0174, ns p=0.3096; g, ** p=0.0052, ns p=0.9447, ns p=0.1579, ns p=0.7769, ns p=0.4104, ns p=0.6924, *** p=1.754 × 10−4, ns p=0.9842, ** p=0.0034, ns p=0.9848, * p=0.0169, ns p=0.8591, ** p=0.0076, ns p=0.8623; h, ** p=0.0051, ns p=0.4525, ns p=0.7379, ns p=0.0702, * p=0.0119, ns p=0.6787, ns p=0.4112.
A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. (2008)
Figure 1

Dietary Restriction Increases the Level of Autophagy

LGG-1::GFP-positive puncta labeling autophagic membranes [22] were counted in wild-type or in food-limited animals.

(A) Micrographs of eat-2(ad1116) L3 larvae expressing GFP-tagged lgg-1/LC3. Arrow indicates autophagic focus. Magnification is indicated.

(B) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) mutants and N2 wild-type animals (WT), p < 0.0001.

(C) Average number of LGG-1::GFP-containing puncta in N2 wild-type, food-restricted animals grown in liquid media (WT, food limited) and N2 wild-type animals grown in liquid with a higher concentration of bacteria (WT, fully fed), p < 0.0001; see Methods.

Between three and ten seam cells were counted in each of 20–40 animals using high-power microscopy and averaged. n, total number of seam cells observed. Error bars: ±SEM. p-Values were calculated as unpaired, two-tailed t-test. Animals were raised at 20 °C. Please see Table S1 for quantification of all data.
A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. (2008)
Figure 2

Inhibition of Genes Required for Autophagy Shortens the Long Lifespan of <italic>eat-2</italic> Mutants

(A) Survival curves of eat-2(ad1116) animals fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan was 23.7 d for control and 19.6 d for bec-1 RNAi, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed a total of six times, and bec-1 RNAi shortened the lifespan of eat-2 animals ∼15%–30%. Please see Table 1 for additional data.

(B) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. These assays were performed concurrently with the eat-2 mutant lifespan analysis in Figure 2A. Mean lifespan was 17.3 d for control and 18.9 d for bec-1 RNAi. p = 0.045, Log-rank (Mantel-Cox) test. Depletion of bec-1 did not significantly change the lifespan of N2 or sterile fer-15(b26); fem-1(hc17) animals in any of six experiments. Please see Table 1 for additional data.

(C) Survival curves of eat-2(ad1116) animals fed either control bacteria or bacteria expressing vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.6 d for control and 22.8 d for vps-34 RNAi, p = 0.0003, Log-rank (Mantel-Cox) test. This experiment was performed a total of four times. Please see Table 1 for additional data.

(D) Survival curves of sterile fer-15(b26); fem-1(hc17) animals (WT) fed either control bacteria or bacteria expressing vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the eat-2 lifespan analysis shown in Figure 2C. Mean lifespan was 21.5 d for control and 23.3 d for bec-1 RNAi. p = 0.14, Log-rank (Mantel-Cox) test. Depletion of vps-34 did not significantly change the lifespan of N2 or sterile fer-15(b26); fem-1(hc17) animals in each of six different experiments. Please see Table 1 for additional data.
A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. (2008)
Figure 3

Inhibition of the FOXA Transcription Factor <italic>pha-4</italic> Decreases Autophagy in <italic>eat-2</italic> and <italic>rab-10</italic> Mutants

(A) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34 or pha-4 dsRNA their entire lives. p ≤ 0.0001 for bec-1, vps-34 and pha-4 RNAi treatments compared to control, respectively, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details.

(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) progeny of animals fed either control bacteria or bacteria expressing pha-4 dsRNA their entire lives; p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details and Table S1 for quantification of data. Feeding mutants for several generations with pha-4 dsRNA, inhibited development and sharply decreased the number of eggs laid ([46], data not shown). However, the loss of many embryos was unlikely to bias our findings, since many progeny of daf-2 mutants treated with pha-4 RNAi also died but those that did not exhibited a robust autophagy phenotype (Figure 6).
A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. (2008)
Figure 4

Animals with Low TOR-Pathway Activity Exhibit Increased Autophagy and Require the Autophagy-associated Gene <italic>bec-1</italic> during Adulthood to Live Long

(A) Average number of LGG-1::GFP-containing puncta in let-363/TOR RNAi-arrested animals compared to N2 wild-type animals (WT) grown on control (vector-only) bacteria, p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. We were not able to detect increased LGG-1::GFP puncta in long-lived let-363(RNAi) adults; however, one generation of daf-2 RNAi, our positive control, did not significantly increase the number of foci in adults either (data not shown). Please see Table S1 for quantification of all data.

(B) Average number of LGG-1::GFP-containing puncta in daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+) compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data. daf-15 encodes the TOR-binding partner Raptor.

(C) Survival curves of daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+, strain DR412) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan: daf-15/+ animals grown on control RNAi-bacteria: 25.1 d, daf-15/+animals on bec-1 RNAi: 20.8 d, p = 0.0008, Log-rank (Mantel-Cox) test. The lifespan of daf-15/+ animals grown on bec-1 RNAi-bacteria during adulthood was measured again, yielding similar results; the lifespan of daf-15/+ animals was also measured three times following whole-life RNAi exposure. In these experiments, bec-1 RNAi generally shortened the mean lifespan of daf-15/+ animals to a greater extent than it shortened the lifespan of wild type. (We also attempted to perform double-RNAi experiments, in which animals were cultured on a 50:50 mixture of let-363/TOR and bec-1 [or control] RNAi bacteria. Although the trends we saw were in the expected direction, the effects produced by half-strength RNAi were small and not statistically significant [data not shown].) Please see Table 2 for additional data.

(D) Survival curves of wild-type animals derived from strain DR412 (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA throughout their whole life at 20 °C. These assays were performed concurrently with the daf-15/+ lifespan analysis shown in Figure 4C. WT grown on control RNAi-bacteria: 21.4 d, WT on bec-1 RNAi-bacteria: 21.1 d (p = 0.34), p between daf-15/+ and WT grown on control RNAi-bacteria, p < 0.0001, Log-rank (Mantel-Cox) test. Please see Table 2 for additional data.
A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. (2008)
Figure 5

Inhibition of the Small GTPase <italic>rab-10</italic> Increases Autophagy

(A) Average number of LGG-1::GFP-containing puncta in N2 wild-type animals (WT) fed either control bacteria or bacteria expressing daf-2 (as a control) or rab-10 dsRNA for two generations. p < 0.0001 for either daf-2 or rab-10 RNAi treatment compared to control RNAi treatment, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data.

(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) mutants compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details. Please see Table S1 for quantification of all data.

(C) Survival curves of rab-10(ok1494) animals fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.9 d for control, 19.9 d for bec-1 RNAi, and 22.1 d for vps-34 RNAi, all pair-wise comparisons to control, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed two times. Please see Table 3 for additional data.

(D) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the rab-10 lifespan analysis shown in Figure 5C. Mean lifespan was 22.9 d for control, 23.6 d for bec-1 RNAi, and 24.5 d for vps-34 RNAi, pair-wise comparison to control, p = 0.057 and p = 0.16, respectively, Log-rank (Mantel-Cox) test. Please see Table 1 for additional data.
A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. (2008)
Figure 6

Increased Autophagy in <italic>daf-2/</italic>insulin/IGF Receptor Mutants Is Independent of the <italic>daf-16</italic>/FOXO and <italic>pha-4</italic>/FOXA Transcription Factors

(A) Average number of LGG-1::GFP-containing puncta in daf-2(e1370) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34, or pha-4 dsRNA for their entire lives. p < 0.0001 for bec-1 and vps-34 RNAi-bacteria compared to control RNAi-bacteria, respectively, p = 0.17 for pha-4 RNAi-bacteria compared to control RNAi-bacteria, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Feeding daf-2 mutants for several generations with pha-4 dsRNA sharply decreased the number of eggs laid (data not shown). The mean lifespan of daf-2(e1370) animals was shortened 12.5% by pha-4 RNAi ([28] and data not shown), and we measured an 11% decrease in puncta in daf-2(e1370) animals fed pha-4 RNAi. Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan.

(B) Average number of LGG-1::GFP-containing puncta in daf-16(mu86); daf-2(e1370) double mutants compared to daf-2(e1370) animals; p = 0.50, unpaired, two-tailed t-test. N2 wild-type animals (WT) are shown for comparison. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. The double mutant expressing the LGG-1 reporter had a mean lifespan similar to non-transgenic daf-16; daf-2 double mutants (data not shown, [65]). Please see Table S1 for quantification of all data.
An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk‐dependent repression of macrophage cholesterol efflux (2015)
Figure 1

Promoter enrichment‐quantitative mass spectrometry (PE‐QMS) identifies transcriptional regulators of Abca1 expression

RT–qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR+/+ and LXR−/− primary BMMs. Fold changes are shown relative to LXR+/+ 0 h. Error bars represent ± SEM for n = 4–8 (**P = 0.001 at 8 h and **P = 0.00004 at 16 h versus LXR−/−; **P = 0.0003 at 0 h versus LXR+/+); bottom, proposed model explaining the ligand‐dependent induction of Abca1. Following ligand stimulation, LXR–corepressor complexes are exchanged for LXR–coactivator complexes, which promote gene expression. The LXR–cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown.
Overview of the PE‐QMS experimental strategy to identify LXR ligand‐stimulated and LXR binding‐dependent interactions with the Abca1 promoter. Nuclear extracts were prepared from RAW 264.7 macrophages treated with control or 1 μM T0901317 (depicted as a black circle) for 18 h. The Abca1 promoter region was used to isolate gene regulatory complexes under the indicated conditions. Immobilized templates were washed, and bound proteins eluted and digested. Peptides were purified, then identified, and quantified by mass spectrometry.
Heat map of the PE‐QMS experiment. Ligand‐dependent subsets were quantified using label‐free and isotope labeling approaches and are indicated on the left. Colors represent Log2 relative abundance ratios (T0901317/vehicle) of identified proteins. LXR binding‐dependent subsets were quantified using an isotope labeling approach and are indicated on the right. Changes in binding > 1.5‐fold were employed as cutoffs for depicted subsets. ND = not determined. Full dataset is available as Supplementary Table S1.
An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk‐dependent repression of macrophage cholesterol efflux (2015)
Figure 2

Identification of NCOA5 as an LXR repressor

Protein–protein interaction network showing connectivity between the putative ligand‐stimulated and LXR binding‐dependent transcriptional regulators of Abca1 (shown in orange). Edges represent protein–protein interactions. NCOA5 node and edge are outlined red to indicate that this physical association is found in mouse interaction networks.
Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length SND1, SART1, or HMBOX1. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. A diagram of the reporter construct is shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 9 (**P = 0.001, *P = 0.011 for SND1, *P = 0.046 for SART1).
Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length NCOA5 or an NCOA5 mutant lacking the NH2‐terminus. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. Diagrams of the reporter constructs are shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 6–12 (**P 0.001, *P = 0.035).
RT–qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus. LXR ligand stimulations were performed with 1 μM T0901317 for 18 h. Fold changes are shown relative to vehicle‐stimulated control. Error bars represent ± SEM for n = 4 (*P = 0.029).
An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk‐dependent repression of macrophage cholesterol efflux (2015)
Figure 3

NCOA5 functions as an LXR corepressor

In vitro pulldown assays using recombinant GST‐LXRα to isolate the indicated in vitro translated NCOA5 constructs. Assays were performed for 2 h in the presence or absence of 2 μM T0901317. Samples were immunoblotted as indicated. Note the requirement of the NCOA5 NH2‐terminus for interacting with LXRα.
Immunoprecipitation of Protein C‐tagged LXRα from stable RAW 264.7 macrophage nuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h, followed by immunoblotting for endogenous NCOA5 or over‐expressed LXRα. Protein immunoblots of nuclear extracts are shown below.
NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the ligand‐stimulated association of NCOA5. Error bars represent ± SEM for n = 4–9 (*P = 0.02).
NCOA5 ChIP assays from LXR−/− BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5–6 (**P = 0.0003 for vehicle and **P = 0.002 for T0901317).
An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk‐dependent repression of macrophage cholesterol efflux (2015)
Figure 4

Attenuation of Abca1 transcription by NCOA5

RT–qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non‐silencing or Ncoa5‐specific shRNA. Fold changes are shown relative to shControl 0 h. Note the elevated Abca1 expression at 24–30 h following loss of NCOA5. Error bars represent ± SEM for n = 4–6 (**P = 0.0007 at 24 h and **P = 0.004 at 30 h, *P = 0.02).
Quantification of ABCA1 immunoblots from primary BMMs infected as in (A). Representative immunoblot is shown in Supplementary Fig S7E. LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h. Error bars represent ± SEM for n = 2 (**P = 0.007).
An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk‐dependent repression of macrophage cholesterol efflux (2015)
Figure 5

NCOA5 mediates TLR3–LXR signal crosstalk by antagonizing Abca1 expression and function

A, B RT–qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317 alone or together with 6 μg/ml PolyIC (A) or 10 ng/ml LPS (B). Fold changes are shown relative to vehicle‐stimulated shControl. Note only the loss of TLR3‐mediated repression following Ncoa5 silencing. Error bars represent ± SEM for n = 4–10 (**P = 0.0003 for A, **P = 0.0004 for shControl in B, **P = 0.0002 for shNcoa5 in B versus T0901317).
C, D RT–qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h unstimulated, or with 6 μg/ml PolyIC (C) or 10 ng/ml LPS (D). Fold changes are shown relative to unstimulated shControl. Error bars represent ± SEM for n = 4–6 (**P = 0.0006 for C, **P = 0.00000001 for shControl in D, **P = 0.000001 for shNcoa5 in D, *P = 0.013 versus T0901317).
E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the increased occupancy only in the presence of both lipid and inflammatory ligands. Error bars represent ± SEM for n = 8–12 (**P = 0.0007 versus T0901317).
F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg/ml PolyIC or left unstimulated. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5–6.
G Cholesterol efflux assays to APOA1 from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Agonist stimulations were performed for 6 h. Error bars represent ± SEM for n = 3 (*P = 0.02 versus T0901317).
An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk‐dependent repression of macrophage cholesterol efflux (2015)
Figure 6

NCOA5 represses Abca1 expression following TLR3–LXR signal crosstalk by inhibiting RNAPII function

A, B Unmodified/pSer5 RNAPII (A) or RNAPII pSer2 (B) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 TSS. Error bars represent ± SEM for n = 6–9 (**P = 0.0001 in A, **P = 0.002 in B versus T0901317).
C, D RNAPII ChIP assays as in (A, B) but performed from LXR−/− BMMs. Error bars represent ± SEM for n = 5–9 (**P = 0.000000001, *P = 0.02 versus T0901317).
E RT–qPCR of Abca1 expression from LXR+/+ versus LXR−/− BMMs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 together with 6 μg/ml PolyIC. Fold changes are shown relative to vehicle‐stimulated LXR+/+. Error bars represent ± SEM for n = 4 (**P = 0.004, *P = 0.012 versus T0901317).
F RNAPII pSer2 ChIP assays from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 + 6 μg/ml PolyIC. qPCR was performed for the Abca1 TSS. Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs (**P = 0.004 versus baseline). Error bars represent ± SEM for n = 3 from 11 mice.