EMBO/soda-nel huggingface dataset:
Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2- dependent antibacterial autophagy (2016)
Figure 5

Figure 5. Structure of the NDP52 zinc finger domain

A) Superposition of the 20 lowest energy solution structures of the NDP52 domain

B) Cartoon representation of the zinc finger domains of NDP52 and Optineurin (pdb id: 5AAZ)

C) Cartoon representation of the NDP52 zinc finger and ubiquitin. In red are those residues whose signals alter upon binding.

D) Phylogenetic alignment of the NDP52 zinc finger. Zinc-coordinating residues in bold, conserved residues in the helix in red. The asterisk indicates D439, the residue mutated in subsequent experiments. Hs – Homo sapiens, Bt – Bos taurus, Gg – Gallus gallus and Xe – Xenopus laevis.

E) Lumier binding assay. Binding of the indicated luciferase-tagged NDP52 variants to beads coated with GST, GST:galectin-8, or GST:ubiquitin. Coomassie stain of purified GST proteins. Western blot for luciferase-tagged NDP52 alleles in total cell lysates.

F) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-NDP52 fusion proteins. Infected cells were lysed at the indicated times post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical comparison with Tbk1-/- MEFs expressing TBK1ΔC is indicated. ***p<0.001, one-way ANOVA with Dunnett’s multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.
Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2- dependent antibacterial autophagy (2016)
Figure 6

Figure 6. Recruitment of TBK1 to S. Typhimurium via galectin-8 restricts bacterial proliferation and recruits WIPI1

A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. S. Typhimurium replication kinetics (A). Infected cells were lysed at the indicated times post inoculation (p.i.). and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett’s multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of three independent experiments. >200 bacteria counted per coverslip. *p<0.05, one-way ANOVA with Dunnett’s multiple comparisons test.
Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2- dependent antibacterial autophagy (2016)
Figure 7

Figure 7. Recruitment of TBK1 to S. Typhimurium via K48- or K63-ubiquitin chains restricts bacterial proliferation

A) Binding of NDP52 to ubiquitin tetramers of different linkage types. Beads coated with the indicated GST-tagged proteins were incubated with pooled M1-, K63-, and K48-linked tetra-ubiquitin chains. Anti-ubiquitin Western blot of input and proteins bound to beads.

B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett’s multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.
Placenta Derived Interferon Stimulated Gene 20 Controls ZIKA Virus Infection (2021)
Figure 1

Figure 1. ZIKV infection induces type I IFNβ expression in human first-trimester trophoblast cell line Sw.71 and human primary cultures.

Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring viral titers and gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture.

(A) ZIKV titers in Sw.71 and HPC trophoblasts. *p < 0.05 by One-way ANOVA.

(B) IFNα is not highly induced in response to ZIKV infection in trophoblast cells. IFNα mRNA expression was inhibited in the first 24 h.p.i. and maintained at low level during ZIKV infection. Difference not significant (n.s.) by One-way ANOVA.

(C) ZIKV infection induced a time-dependent increase in IFNβ mRNA expression in Sw.71 and HPC. *p < 0.05, **p < 0.01 by One-way ANOVA.

(D) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay. Supernatants were collected from ZIKV-infected and control trophoblast cultures at different times, and IFNβ protein secretion was quantified by ELLA assay. Note the increase of secreted IFNβ in the ZIKV-infected supernatant in a time-dependent manner. ****p < 0.0001 by Student's t-test against individual time point NT control.
Placenta Derived Interferon Stimulated Gene 20 Controls ZIKA Virus Infection (2021)
Figure 2

Figure 2. IFNβ promotes ISG20 mRNA and protein expression in a dose and time-dependent manner in trophoblast cells.

Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 mRNA and protein expression.

Data represent as mean ± SEM; n=3 biological replicates. NT, no treatment group; HPC, human primary culture.

(A) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for

8 h and RNA were collected for determining ISG20 mRNA expressions by qRT-PCR.

Note the increase of ISG20 mRNA expression in a dose-dependent manner. *p < 0.05, **p < 0.01 by One-way ANOVA.

(B) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a time-dependent manner. *p < 0.05 by One-way ANOVA.

(C) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for

24 h and protein was collected for determining ISG20 protein expressions by western blot. Note the increase of ISG20 protein expression in a dose-dependent manner. β-actin served as a loading control.

(D) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot. Note that there was no ISG20 protein expression in the no treatment group in both Sw.71 and HPC, only after IFNβ treatment, ISG20 protein expression exhibited a time-dependent manner. β-actin served as a loading control.
Placenta Derived Interferon Stimulated Gene 20 Controls ZIKA Virus Infection (2021)
Figure 3

Figure 3. Lack of ISG20 in trophoblast cells leads to enhanced ZIKV replication and viral shedding.

(A) ISG20 protein expression was increased in ZIKV infection. Sw.71 cells were infected with ZIKV (MOI=2) over time, and proteins were collected for western blot analysis. β-actin served as a loading control.

(B) No ISG20 protein expression in ISG20-/- Sw.71 in ZIKV infection. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and proteins were collected for western blot analysis. β-actin served as a loading control.

(C) Higher ZIKA titer was shown in ISG20-/- Sw.71 cells. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and RNA was collected for determining the viral titer and gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.

(D) More viral shedding in ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Supernatant from ZIKV-infected Sw.71 and ISG20-/- Sw.71 cells were collected and plaque assay was performed using Vero cells. A representative plaque assay picture is presented. Note that more plaques were formed in Vero cells by incubating with ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Scale bar=130μm.

(E) Plaques were counted and plaque forming-unit (pfu) ratio was calculated for the comparison of the viral titers. Data represent as mean ± SEM; n=3 biological replicates. *p < 0.05 by Student's t-test.
Placenta Derived Interferon Stimulated Gene 20 Controls ZIKA Virus Infection (2021)
Figure 4

Figure 4. Lack of ISG20 in trophoblast cells weakens the protection provided by IFNβ against ZIKV infection.

(A) Induction of IFNβ mRNA in response to ZIKV infection in Sw.71 and ISG20-/- Sw.71 trophoblast cells. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and RNA was collected for qRT-PCR. Note that ZIKV infection induces IFNβ mRNA expression in Sw.71 as well as in ISG20-/- Sw.71 cells. Data represent as mean ± SEM; n=3~4 biological replicates; *p < 0.05, **p < 0.01 by Student's t-test.

(B) ISGs mRNA expression stimulated by IFNβ in Sw.71 and ISG20-/- Sw.71 trophoblast cells. Sw.71 and ISG20-/- Sw.71 cells were treated with 30 ng/ml IFNβ for 24 h, and RNA was collected for determining the ISGs gene expression by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; *p < 0.05, **p < 0.01 by Student's t-test against control.

(C) No ISG20 protein expression in ISG20-/- Sw.71 cells after IFNβ treatment. Sw.71 and ISG20-/- Sw.71 cells were treated with 30 ng/ml IFNβ for 24 h, and proteins were collected for western blot analysis. β-actin served as a loading control for western blot.

(D) IFNβ pre-treatment significantly prevented trophoblast cells from ZIKV infection, however, this protection was evidently attenuated due to lack of ISG20. Sw.71 and ISG20-/- Sw.71 cells were pre-treated with or without 30 ng/ml IFNβ for 24 h, followed by ZIKV infection (MOI=2) for 1 h and refreshed with regular media for 24 h, and RNA was collected to determine the viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.
Placenta Derived Interferon Stimulated Gene 20 Controls ZIKA Virus Infection (2021)
Figure 5

Figure 5. Design and characterization of the anti-viral effect of recombinant ISG20 protein.

(A) Structure of the recombinant ISG20-Fc protein. The components are: 1) ISG20 sequence (Pink); 2) Linker (Orange); 3) Fc domain (White); 4) Tev sequence, cleavage between Q and G (Cyan); 5) Signal sequence (ENPP7) (Red); 6) His Tag (Green).

(B) Workflow of clone production and selection.

(C) Selection and verification of positive clones. In the positive clone, ISG20 protein expressed both in the cytosol (endogenous) and supernatant (secreted) compared to the non-transfected CHO cells and transfected negative clones. CHO, Chinese hamster ovary cell without transfection; β-actin served as a loading control.

(D) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells. Supernatants from negative and positive clones were collected and added to ISG20-/- Sw.71 trophoblast cells together with ZIKA virus (MOI=2) for 1 h, followed by refreshing with new growth media for 48 h. RNA was then collected to determine viral titers by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01 by Student's t-test.

(E) Recombinant ISG20-Fc degrades ZIKA viral RNA and HSV2 viral DNA. 50ng purified viral RNA (ZIKV) or DNA (HSV-2) were incubated with increasing concentrations of rISG20-Fc (5, 50, 500 ng) in the presence of RNase inhibitor for 90min at 37°C followed by quantification of viral titers by qRT-PCR, and agarose gel was used to evaluate the RNA degradation by electrophoresis. The representative picture of agarose gel is presented. rISG20-Fc was able to degrade both ZIKV RNA and HSV-2 DNA in a dose dependent manner; however, rISG20-Fc was more efficient in degrading viral RNA than DNA. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01. ***p < 0.001 and ****p < 0.0001 by One-way ANOVA.
Placenta Derived Interferon Stimulated Gene 20 Controls ZIKA Virus Infection (2021)
Figure 6

Figure 6. In vivo efficacy of rISG20-Fc improving pregnancy outcome in IFNAR1-/- pregnant mice during ZIKV infection

(A) Mouse experiment protocol. Adult (8-12 weeks of age) IFNAR1-/- pregnant mice were infected i.p. with 1*105 pfu ZIKV or 1% FBS DMEM/F12 media (vehicle) on embryo day 8.5 (E8.5). 1 h after ZIKV/vehicle injection, treatment with rISG20-Fc (1mg/kg) or PBS (control) was administrated i.p. to the pregnant mice. On E9.5 and E10.5, same protein/PBS injection was performed on the pregnant mice. On E14.5, the mice were sacrificed and organs were collected for viral titer quantification. N=3 for control PBS group, and n=4 for treatment groups.

(B) Macroscopic evaluation of the pregnant uterus and fetus. Compare to the fetal death and reabsorptions in ZIKV-infected mice, rISG20-Fc treatment rescued this phenotype by decreasing pregnancy loss (resorption).

(C) Effect of rISG20-Fc treatment on pregnancy loss. rISG20-Fc treatment significantly decreased the pregnancy loss caused by ZIKA infection. The pregnancy loss rate (number of dead fetus + resorptions/ total number of implantations x100) was calculated. Data represent as mean ± SEM; n=3-4 mice per group; *p < 0.05 and **p < 0.01 by One-way ANOVA.

(D) rISG20-Fc treatment improves fetal development. Measurement of crown-rump length (CRL) and occipitofrontal diameter (OFD) were determined on E14.5. n=3-4 mice per group and 6-8 fetuses from each pregnant mouse were evaluated; *p < 0.05, **p < 0.01 by One-way ANOVA.
Placenta Derived Interferon Stimulated Gene 20 Controls ZIKA Virus Infection (2021)
Figure 7

Figure 7. In vivo efficacy of rISG20-Fc inhibiting ZIKA viral replication and promoting placental integrity in IFNAR1-/- pregnant mice

(A) rISG20-Fc treatment alleviates maternal viral burden. Note that rISG20-Fc treatment significantly decreased the maternal serum viral titers. Data represent as mean ± SEM; n=3 mice for control PBS group, and n=4 mice for treatment groups; *p < 0.05 by Student's t-test.

(B) rISG20-Fc treatment reduces ZIKV titers in the fetal brain. Although there was no difference of ZIKV titer in the placenta, at the fetal side, there was a significant decrease of viral titer in the fetal brain, suggesting rISG20-Fc can block viral transmission from placenta to fetus. Data represent as mean ± SEM; n=4 for each group, and 3-4 placentas/fetal brains from every mouse were analyzed; *p < 0.05 by Student's t-test.

(C) rISG20-Fc treatment decreases Eotaxin significantly in maternal circulation. Data represent as mean ± SEM; n=2 for PBS control group, and n=3 for every treatment group; *p < 0.05 by One-way ANOVA; difference not significant for IL-9 and IFNγ by One-way ANOVA.

(D) Representative hematoxylin and eosin staining of the mouse placenta on E14.5. Labyrinth layers were marked with a solid line. Note the major alteration on placenta structure characterized by multifocal loss of tissue architecture (necrosis) and the defective blood vessel formation of labyrinth in ZIKV+PBS group, and rISG20-Fc treatment improves vascularity and decreases decidual edema and cellular fragmentation at the labyrinth. Scale bar=580μm (upper images); Scale bar=230μm (bottom images).
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment (2022)
Figure 1

Figure 1 Stress granule-deficient cells display aggravated impairment of the UPS

A) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 in control (siControl, left panel) and G3BP1/2-depleted (siGRBP1/2, right panel) MelJuSo cells. Cells were left untreated (- HS), exposed to 43°C for 30 minutes (+ HS) and followed for 4 hours after heat shock (HS + 4h Rec.). Arrows indicate the stress granules in thermally stressed control cells. Scale bar, 10 μm.

B) Analysis of puromycin incorporation in MelJuSo cells that were left untreated (- HS), exposed to 43°C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.). The blots were probed with antibodies against puromycin, and GAPDH as loading control.

C) Representative images of MelJuSo cells expressing Ub-YFP, which were either left untreated (- HS), exposed to 43 °C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.). Scale bar, 20 μm.

D) Quantification of mean cellular YFP fluorescence intensities from experiment shown in C), normalized to untreated control cells (siControl - HS). The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01, ns: not significant).

E) Kinetics of Ub-YFP image intensities in control (siControl) and stress granule-deficient MelJuSo cells (siG3BP1/2) in response to a 30 min 43°C heat shock, followed by 4 hours at 37°C (Recovery). Error bars indicate the standard error of the mean (n=3 independent experiments, 12 images analyzed per condition, Student's unpaired t-test, ** P<0.01).

F) Quantification of MelJuSo cells expressing YFP-CL1. Normalized to untreated control cells (siControl - HS). The frequency and distribution of the relative fluorescent intensity per cell are shown as violin plots. The solid lines in each distribution represent the median, and dashed lines represent the upper and lower interquartile range limits. (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01, ns: not significant).

G) Quantification of MelJuSo Ub-YFP cells transfected with G3BP1/2 siRNA AND siRNA resistant G3BP1 mutants YFP intensities. Normalized to mCherry untreated control cells (- HS). The frequency and distribution of the relative fluorescent intensity per cell are shown as violin plots. The solid lines in each distribution represent the median, and dashed lines represent the upper and lower interquartile range limits. (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01, ns: not significant).
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment (2022)
Figure 2

Figure 2 Compromised nuclear UPS in stress granule-deficient cells.

A) and B) Representative images of stress granule proficient (siControl) and deficient (siG3BP1/2) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1 (A) and the cytoplasmic UPS reporter NES-GFP-CL1 (B). The cells were left untreated (- HS), exposed to 43°C for 30 minutes (HS), or exposed to 43°C for 30 minutes and followed by 4 hours recovery (HS + 4h Rec.). Nucleolar accumulation of NLS-GFP-CL1 during the recovery is indicated by arrows in A). Scale bar, 20 μm.

C) and D) Quantifications of the mean cellular GFP fluorescence intensities of A) and B) normalized to untreated control cells (siControl - HS). The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01, ns: not significant).

E) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and the nuclear UPS reporter NLS-GFP-CL1 in control (siControl, left panel) and G3BP1/2-depleted (siGRBP1/2, right panel) MelJuSo cells exposed to a 30 min 43°C heat shock, followed by 4 hours at 37°C (Recovery). Nucleolar accumulation of NLS-GFP-CL1 is indicated by arrows. Scale bar, 10 μm.

F) Quantification of the nucleolus/nucleoplasm ratios of NLS-GFP-CL1 intensities in images of experiment shown in E). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, 100 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment (2022)
Figure 3

Figure 3 Stress granule-deficient cells accumulate defective ribosome products (DRiPs) in nucleoli

A) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. Scale bar, 10 μm.

B) Quantification of the nucleus/cytoplasm ratios of puromycin intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).

C) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin-labelled proteins in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. Scale bar, 10 μm.

D) Quantification of the nucleolus/nucleoplasm ratio of puromycin intensities in images from B). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).

E) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) U2OS cells. Cells were subjected to 43°C heat shock Scale bar, 10 μm.

F) Quantification of the numbers of puromycin positive TIA dots per cell in images from E). The numbers of positive foci per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).

G) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. Scale bar,10 μm.

H) Quantification of the nucleolus/nucleoplasm ratio of puromycin intensities in images from G). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).