EMBO/soda-nel huggingface dataset:
SPATA2 promotes CYLD activity and regulates TNFinduced NF-κB signaling and cell death (2016)
Figure 3

Figure 3. SPATA2 dependent recruitment of CYLD to the TNF-RSC

(A) A549 cells were infected to express a CRISPR/Cas9 system targeting luciferase (as control, indicated wt) or the spata2 gene (ko). Mixed cell cultures were treated with FLAG-huTNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, HOIP, SHARPIN, TRADD, TNF and tubulin as indicated. (B) MEF were infected to express a CRISPR/Cas9 system targeting luciferase (wt) or the spata2 gene (ko). Single clones were generated, which were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing M1-linked ubiquitin, CYLD, RIPK1, and tubulin as indicated.
SPATA2 promotes CYLD activity and regulates TNFinduced NF-κB signaling and cell death (2016)
Figure 4

Figure 4. SPATA2 promotes CYLD DUB activity

FLAG-tagged CYLD or catalytically inactive FLAG-CYLD (C598A), respectively, were stably expressed in CYLD-/- MEF, followed by infection with control retrovirus, or retrovirus encoding SPATA2, and purified by FLAG-IP. Purified CYLD or the inactive mutant were added to recombinant di-ubiquitin for 0, 20 or 40 min at 37°C as indicated. After electrophoresis, the gel was cut and the lower part was silver stained to visualize mono-and di-ubiquitin, while the upper part was transferred to a membrane and probed for FLAG and V5. In (A), the experiment was done with M1-linked di-ubiquitin substrate, in (B) K63-linked di-ubiquitin substrate was subjected to the assay, and in (C) K48-linked di-ubiquitin substrate was analyzed.
SPATA2 promotes CYLD activity and regulates TNFinduced NF-κB signaling and cell death (2016)
Figure 5

Figure 5. SPATA2 attenuates NFkB and MAPK activation, and promotes TNF-induced apoptosis

(A) MEF expressing CRISPR/Cas9 targeting luciferase (wt) or single clones generated from MEF expressing CRISPR/Cas9 targeting the spata2 gene (ko) were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBα IkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin. (B) 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting luciferase (as control) or single clones generated from 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting the spata2 gene were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBα IkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin. (C) TAK1-/- MEF were infected to express a CRISPR/Cas9 system targeting luciferase or the spata2 gene, from which single clones were generated. The cells were treated with mTNF (10 ng/ml) or mTNF along with Necrostatin-1 (Nec-1) for 1-3 h as indicated. The blot was sequentially probed with antibodies recognizing PARP, cleaved caspase-3 and tubulin. (D). The same cells as used in (C) were treated as before with TNF (10 ng/ml) or TNF along with Necrostatin-1 (Nec-1) for one and 2 h as indicated. Caspase activity was determined using the fluorogenic substrate DEVD-AMC. (E) Cells from the experiment as shown in (D) were analyzed for apoptosis by Annexin V staining after 2 h. In (D) and (E), a representative experiment is shown with error bars referring to technical replicates. (F) Cells as described before were treated with mTNF (10 ng/ml) for the indicated time and lysates were subjected to immunoprecipitation with an anti-FADD antibody. The immunoprecipitates were subjected to Western blotting and probed for RIPK1, FADD and actin.
Autophagy is essential for effector CD8+ T cell survival and memory formation (2014)
figf1

(a) Kinetics of the appearance of P14 cells in spleens of C57BL/6 mice given adoptive transfer of 1 × 104 P14 cells, monitored after infection of recipients with LCMV Armstrong strain. (b) Flow cytometry of cell size at various stages of P14 differentiation, assessed by forward scatter (FSC). (c) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation. Data are representative of two independent experiments with samples pooled from 5–20 mice for each time point.
Autophagy is essential for effector CD8+ T cell survival and memory formation (2014)
figf2

(a) MSCV retroviral constructs introduced in P14 cells (Supplementary Fig. 2a) to probe for autophagy activity, showing the transgenes encoding GFP-tagged wild-type LC3b (top; WT GFP-LC3b) and the GFP-tagged G120A mutant of LC3b (bottom; G120A GFP-LC3b). LTR, long terminal repeat; IRES, internal ribosomal entry site. (b) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5, 8 and 30 after infection of recipients with LCMV. Numbers in blue outlined areas indicate percent GFP− cells among transduced (Thy-1.1+) P14 cells. (c) Summary of results in b, showing autophagy activity as percent GFP− cells. *P ≤ 0.0005 (unpaired t-test). (d) Multispectral-imaging flow cytometry of P14 cells from mice treated as in b, assessed on day 8 after infection, showing GFP signals from GFP+ transduced (CD45.1+ Thy-1.1+) P14 cells. Original magnification, ×60. (e) Summary of results in d, presented as the frequency of cells with more than one GFP+ punctum among vector transduced Thy-1.1+ P14 cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). Data are representative of three independent experiments (b,d) or three independent experiments with three or more mice per group (e; error bars, s.e.m.) or six mice per group (c; error bars, s.e.m.).
Autophagy is essential for effector CD8+ T cell survival and memory formation (2014)
figf3

(a) mCherry-GFP-LC3b MSCV retroviral constructs (presented as in Fig. 2a) used for transduction of P14 cells (Supplementary Fig. 2a). (b) Flow cytometry of splenocytes from recipient mice (n = 6) given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5 and 8 after infection of recipients with LCMV (left), and quantification of P14 cells per spleen (right). Numbers adjacent to outlined areas (left) indicate percent P14 (CD45.1+) cells. *P ≤ 0.0005 (unpaired t-test). (c) Flow cytometry of the size of P14 cells transduced with wild-type mCherry-GFP-LC3b retrovirus (assessed as in Fig. 1b) in mice treated as in b, assessed at days 5 and 8 after infection. (d) Efficiency of retroviral transduction (left) and expression of GFP and mCherry (right) in retrovirus-transduced P14 cells, assessed by flow cytometry gated on CD45.1+ P14 cells (left) or CD45.1+Thy-1.1+ P14 cells (right). Numbers in quadrants indicate percent CD45.1+Thy-1.1− (untransduced) P14 cells (top left) or CD45.1+Thy-1.1+ (transduced) P14 cells (top right; magenta) (left) or percent cells in each (right). (e) Ratio of the geometric mean fluorescence intensity (gMFI) of GFP to that of mCherry (GFP/mCherry) for the cells in d; each symbol represents an individual mouse; numbers above lines connecting groups indicate difference in ratio. Data are representative of two independent experiments with three or more mice per group (error bars (b), s.e.m.).
Autophagy is essential for effector CD8+ T cell survival and memory formation (2014)
figf4

(a) Genomic deletion of Atg7 in H-2Db–gp33+ T cells and in total activated (CD44hiCD62Llo) CD8+ T cells from Atg7fl/fl mice (Cre −) and Atg7fl/flGzmb-Cre mice (Cre +) at day 8 after infection with LCMV Armstrong strain; results are presented relative to those of Atg7fl/fl mice, set as 100%. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). (b) Immunoblot analysis of Atg7 and the products of its enzymatic activity (left margin) in CD44hiCD62Llo CD8+ T cells isolated at day 8 after infection as in a; β-actin serves as a loading control. (c) Flow cytometry of T cells in the peripheral blood at day 8 after infection as in a, gated on CD8+ T cells (left), and of the H-2Db–gp33+ T cells identified at left, identifying terminal effector (KLRG1hiCD127lo) and memory precursor (KLRG1loCD127hi) populations (right). Numbers adjacent to outlined areas (left) indicate percent H-2Db–gp33+ T cells; numbers in quadrants (right) indicate percent cells in each. (d) Total antigen-specific CD8+ T cells in the spleen, liver and lungs at day 8 after infection as in a. (e) Production of IFN-γ and TNF in cells obtained 8 d after infection and stimulated ex vivo with various peptides (above plots), gated on total CD8+ T cells. Numbers in quadrants indicate percent cells in each. (f) Flow cytometry of CD8+ T cells in the peripheral blood of mice at day 15 after infection as in a (numbers in plots as in c, left). (g) Quantification of tetramer-positive cells in spleens at day 15 after infection as in a. (h) Kinetics of the appearance H-2Db–gp33+ CD8+ T cells among peripheral blood mononuclear cells (PBMCs) after infection as in a (left); each line represents one mouse. Right, frequency of H-2Db–gp33+ CD8+ T cells remaining on day 15 relative to that on day 8, set as 100%. (i) Quantification of tetramer-positive cells at day 30 after infection as in a. Horizontal dashed lines (g,h) indicate the limit of detection. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data were pooled from two independent experiments (a) or at least two independent experiments (c–i) with three to seven mice per group (error bars, s.e.m.) or are from one experiment with samples obtained from two independent experiments (b).
Autophagy is essential for effector CD8+ T cell survival and memory formation (2014)
figf5

(a) Flow cytometry of circulating LCMV-specific CD8+ T cells among peripheral blood mononuclear cells from Atg5fl/fl and Atg5fl/flGzmb-Cre mice on days 8, 15 and 50 after infection with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent H-2Db–gp33+ T cells, gated on CD8+ T cells. (b) Longitudinal kinetics of the appearance of H-2Db–gp33+ T cells in the peripheral blood of mice as in a (left), and percent H-2Db–gp33+ CD8+ T cells remaining on day 15 (right) (all presented as in Fig. 4h). (c) Flow cytometry of T cells in tissues on day 279 after infection as in a (numbers in plots as in a, among total gated CD8+ T cells). (d) Total T cells specific for H-2Db–gp33 (left) or H-2Db–NP396 (right) in the spleen, liver and lungs of mice as in a. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to seven mice per group (error bars (d), s.e.m.).
Autophagy is essential for effector CD8+ T cell survival and memory formation (2014)
figf6

(a,b) Flow cytometry of CD8+ T cell obtained from chimeras generated (as in Supplementary Fig. 5a) by reconstitution of wild-type mice with a mixture of bone marrow cells from Atg7fl/fl mice and wild-type C57BL/6 mice (Atg7fl/fl vs C57BL/6) or from Atg7fl/flGzmb-Cre mice and wild-type C57BL/6 mice and (Atg7fl/flGzmb-Cre vs C57BL/6), gated (far left) as populations specific for H-2Db–gp33 (a) or H-2Db–NP396 (b) and then assessed, on days 8, 15 and 30 after infection of recipients with LCMV Armstrong infection, as CD45.2+ (Atg7fl/fl or Atg7fl/flGzmb-Cre) donor cells or CD45.1+ (C57BL/6) donor cells (middle). Far right, appearance of tetramer-positive T cells (key, donor source) in the peripheral blood of chimeras from day 8 to day 30 after infection, presented relative to that at day 8 after infection, set as 100%. (c) Frequency of CD45.2+ antigen-specific CD8+ T cells in the spleen, liver and lungs on day 30 after infection as in a,b. Data are representative of two independent experiments with three to four mice per group (error bars, s.e.m.).
Autophagy is essential for effector CD8+ T cell survival and memory formation (2014)
figf8

(a) Kinetics of the appearance of H-2Db–gp276+ T cells in the peripheral blood of Atg7fl/fl and Atg7fl/flGzmb-Cre mice at various times (horizontal axis) after infection with LCMV clone 13. (b) Total IFN-γ-secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a, assessed after 5 h of stimulation ex vivo with various peptides (horizontal axes). (c) Viral titers in serum of mice at various times (horizontal axis) after infection as in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to five mice per group (error bars, s.e.m.).
Zika virus depletes neural stem cells and evades selective autophagy by suppressing FANCC (2020)
Figure 1

Fig 1. Activation of autophagy by ZIKV infection of NSCs

(A) Western blot of autophagy proteins LC3 and p62 at various time points after infection of human NSCs with strains MR766 or Paraiba.

(B-C) Quantitative representation of LC3-II and p62 protein levels during ZIKV infection at indicated time points. Mean ± SEM, n=3 biological replicates.

(D) GFP-LC3 puncta of mock- and ZIKV-infected hNSCs. Scale bar = 100 µm.

(E) Tandem EGFP-LC3 and mCherry punctae co-localization in control and ZIKV infected hNSC. Scale bar = 100 µm, Boxed magnified images with scale bar = 50 µm

(F) Number of GFP-LC3 puncta in MR766 and Paraiba infected hNSCs, n =>50 cells. Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.

***p < 0.001 by Student's t test.

(G) RT-qPCR of ZIKV RNA in hNSCs incubated with vehicle or 100 nM rapamycin, 20 μM chloroquine, 10 nM bafilomycin A1, or 5 mM 3-methyladenine for 24 h. Mean ± SEM of 3 biological replicates. *p < 0.05, ***p < 0.001 by Student's t test.

(H) GFP-LC3 puncta in ZIKV with or without rapamycin and 3-MA treated hNSC cells. Cells were treated with or without rapamycin (100 nM) or 3-methyladenine (5 μM). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test.

(I) RT-qPCR of ZIKV RNA in infected hNSCs transfected with siRNAs targeting LC3, BECN or ATG9A 48 h post infection. Mean ± SEM of 3 biological replicates. *p < 0.05 by Student's t test.

(J) Venn diagram of genes significantly downregulated in HeLa cells treated for 4 h with perifosine (50 μM), rapamycin (50 nM), and resveratrol (100 μM), compared with ZIKV-infected hNSCs (ZIKV data from (Tang et al., 2016)).

(K) Sankey diagram of pathways associated with genes commonly enriched in ZIKV-infected hNSCs and perifosine-, resveratrol-, and rapamycin-treated HeLa cells. The width of each band is proportional to the number of genes in the group/pathway.

(L) Transmission electron microscopy of hNSCs infected by ZIKV MR766 and Paraiba strains. Mitochondria (MT), endoplasmic reticulum (ER), Golgi bodies (GB), ribosomes (RS), phagophores (PH), lipid droplet (LD). Scale bars, first row, 1 μm (left), 500 nm (control right); 200 nm (Right).
Zika virus depletes neural stem cells and evades selective autophagy by suppressing FANCC (2020)
Figure 2

Fig 2. Autophagy enhances viral replication in vivo

(A-D) Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the SVZ (A-B) and hippocampus (C-D) of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone. (E) qRT-PCR for ZIKV RNA expression in brain, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test. (F-G) Western blot for autophagy protein LC3-II, p62 and quantification normalized with GAPDH, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.
Zika virus depletes neural stem cells and evades selective autophagy by suppressing FANCC (2020)
Figure 3

Fig 3. ZIKV dysregulates the Fanconi anemia pathway to suppress virophagy

(A) Venn diagram showing overlap between virophagy-related genes in HeLa cells (Orvedahl et al., 2011) and ZIKV-infected hNSCs (Tang et al., 2016).

(B) RT-qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs. Mean ± SEM of biological triplicates. *p < 0.05, **p < 0.01 by Student's t test.

(C-D) RT-qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC (C) or FANCC-specific siRNA (D). Mean ± SEM of biological triplicates. **p < 0.01 by Student's t test.

(E) Flow cytometry analysis of ZIKVE expression in mock-infected (red), ZIKV-infected (blue), ZIKV-infected siFANCC-transfected (orange), and ZIKV-infected siE2F4-transfected (green) hNSCs at 48 h post-infection.

(F) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs. Scale bar = 100 µm.

(G) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA (NTC) or siFANCC-targeting siRNA. Scale bar = 100 µm.

(H-I) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control (NTC) or siE2F4-targeting siRNA. Mean ± SEM of six imaging fields of biological triplicates, **p < 0.01 by Student's t test, Scale bar = 100 µm,

(J) Immunoblot of macroautophagy (p62, LC3I/LC3II) and selective autophagy (FANCC, TOMM20) proteins 24 h after overexpression of FLAG-tagged ZIKV NS1, NS2B, NS3, NS4A, NS4B and NS5 in 293FT cells.

(K) Analysis of E2F4 ChIP-seq datasets (Encode) showing direct chromatin-binding interaction between E2F4 and FA pathway genes in HeLa (GEO: GSM935365, Farnham lab), K562 (GEO: GSM935600, Farnham lab), and MCF10A (GEO: GSM: 935400, Struhl lab) cell lines. H3K9ac and H3K4me3 datasets identify promoter regions. The genomic region is displayed in log scale with the scale range identified in each panel (0-30). The summed interaction (Sum) across all three cell types is represented as a heatmap.

(L-M) Heatmaps of RT-qPCR analysis of FA pathway genes (K) or essential selective autophagy genes (L) in hNSCs 48 h after transfection with control (NTC) or E2F4-targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains. Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes.
Zika virus depletes neural stem cells and evades selective autophagy by suppressing FANCC (2020)
Figure 4

Fig 4. Downregulation of FANCC increases ZIKV replication and inhibits neurosphere growth

(A) Representative images of mock- or ZIKV-infected human neurospheres expressing control (NTC) or FANCC-targeting siRNAs at 1-4 days post-infection. Scale bar = 250 µm.

(B-C) Violin plot (B) and box plot (C) of relative size of neurospheres treated as described in (A) and measured on day 4 (B) or days 1-4 (C). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.

(D) Pie chart displaying the percentage of early apoptotic, late apoptotic, or necrotic hNSCs at 48 h after mock infection (blue), ZIKV infection (green), or siFANCC transfection (red).

(E) Model of the effects of ZIKV infection on ER stress, autophagy induction, and FA gene expression leading to impaired virophagy and mitophagy.
Zika virus depletes neural stem cells and evades selective autophagy by suppressing FANCC (2020)
Figure 5

Fig 5. ZIKV induces autophagy and downregulates Fancc in vivo

(A-B) Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the hippocampus (A) and SVZ (B) of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.

(C) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Ifnar-/- mice. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.

(D) RT-qPCR analysis of relative Fancc mRNA levels from whole brains or testes in mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. Mean ± SEM of n=3 biological replicates, *p < 0.05 and ns by Student's t test.

(E) RT-qPCR analysis of relative Fancc or Fancl mRNA levels from whole brains from mock- and ZIKV Paraiba infected Ifnar-/- newborn mice. Mean ± SEM of n=3 biological replicates, ***p < 0.001 by Student's t test.

(F) Western blot analysis of Fancc protein in brain regions from mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb.

(G) Bar graph showing the quantification of Fancc proteins, mean ± SEM of n=3 biological replicates, *p<0.05 and ns by Student's t test.

(H) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Fancc KO neonatal mice six days post-infection. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.

(I) qRT-PCR for Ifnab gene expression in wild type and Fancc KO mice, Mean ± SEM of n=3 biological replicates, ns by Student's t test.
Zika virus depletes neural stem cells and evades selective autophagy by suppressing FANCC (2020)
Figure 6

Fig 6. ZIKV infection of Ifnar-/- and Fancc KO mice induces autophagy and NPCs cell death

(A) Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the hippocampus and olfactory bulb of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Bottom row shows enlargements of the hippocampus region. GCL = granular cell layer, SGZ = subgranular zone, DG = dentate gyrus, InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.

(C-E) Western blot of autophagy protein LC3 and p62 in different brain region of Fancc KO neonatal mice six days post-infection of ZIKV (MR766). Mean ± SEM of n=3 biological replicates, *p<0.05, **p<0.005, by Student's t test. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb.

(F) Representative immunofluorescent images showing immunoreactivity of neural progenitor marker (Sox2) with apoptosis marker (Cleaved-caspase-3, CC3) in hippocampus region of Fancc KO mice. Scale bar=50µm.

(G) Quantitative analysis of number of Sox2+ cells and Sox2/CC3+ co-labelled cells in Fancc KO mice hippocampal brain region. Mean ± SEM, n=3 biological replicates, *p < 0.05 by Student's t test.

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