EMBO/soda-nel huggingface dataset:
Suppressing PARylation by 2'-5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death (2020)
Figure 6

Figure 6.

Accumulation of PAR in H2O2-treated cells depends on OAS1 abundance

Representative images and analysis of Western blots for PAR, performed with lysates of HME control (vec) OAS single KO cells (KO1-3), treated with H2O2 for 15 or 40 min. β-actin is shown as a loading control. Signal intensity was measured using ImageJ software. Average lane densities, normalized to vector from three independent experiments, are plotted below as mean + SD, * p < 0.05 as assessed by Student's t-test.

Same as (A), with lysates of HME control (vec) cells and cells expressing ectopic OAS1, OAS2 or OAS3 (OAS1-3hi). Experiments were repeated at least three times. Average lane densities normalized to vector are plotted below below as mean + SD, * p < 0.05 as assessed by Student's t-test.

HME cells, pre-incubated with IFNβ for 2 days to increase OAS abundance, were treated with H2O2 for 0-60 min at 37oC. Western blots of PAR accumulation (top), shift in the position of PARylated PARP1 (middle), OAS2 induction by IFNβ, and β-actin as a loading control are shown. Experiments were repeated twice.

HME control (vec) and KO1 cells were pretreated with 0-500 IU/ml IFNβ for 2 days, and then treated with 500 μM H2O2 for 10 or 30 min. Western blots of PAR accumulation (top, high and medium exposure; middle, low exposure), OAS1-3 induction by IFNβ, and β-actin as a loading control are shown. The experiment was repeated four times; ns, non-specific band.

Source data are available online for this figure.
Suppressing PARylation by 2'-5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death (2020)
Figure 7

Figure 7.

Suppression of PARylation by catalytically active OAS1 inhibits DNA damage-induced cell death

HME cells were treated with 750 μM of H2O2, with and without pre-treatment with olaparib. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.01 in two-way ANOVA. The data represent the results of three experiments.

HME control (vec) and KO1-3 cells were incubated with olaparib for 30 min followed by treatment with 500 μM H2O2 for 15 min. PAR accumulation was assessed by the Western method, with anti-PAR. β-actin is shown as a loading control.

HME vec, KO1 and OAS1hi cells were incubated with olaparib for 30 min, then treated with 500 or 1000 μM of H2O2 and allowed to grow for 72 h. Bars represent means of triplicates ± SD from three experiments. * p<0.05; ** p<0.01 (relative to H2O2-treated vec cells) in Student's t-test.

KO1 cells transfected with vector (KO1-vec), wild-type OAS1 (KO1-OAS1 wt), or mutant OAS1 (D75A/D77A, KO1-OAS1 mut) were treated with 500 μM of H2O2 for the indicated times and analyzed by the Western method. Loading controls: β-actin and overall protein levels were visualized by stain-free technology. The experiment was repeated three times; a representative image is shown.

Quantification of the relative abundance of PAR in (D). Images obtained in three independent experiments were analyzed; the data are plotted as mean + SD *, p<0.05 by Student's t-test.

KO1 cells transfected with vector, wild-type OAS1 (OAS1 wt), or mutant OAS1 (D75A/D77A, OAS1 mut) were treated with 500 μM of H2O2. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ****, p<0.001 in two-way ANOVA. The data represent the results of three experiments.

Source data are available online for this figure.
Suppressing PARylation by 2'-5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death (2020)
Figure 8

Figure 8.

OAS1 suppresses the nuclear translocation of Apoptosis-Inducing Factor (AIF)

A, B. Representative images and analysis of AIF accumulation in the nuclei of HME control (vec) and OAS single-knockout cells (KO), and HME control (vec) and OAS double- and triple-knockout cells (DKOs and TKO), 24 h after treatment with 1000 μM H2O2.

C. Representative images and analysis of AIF accumulation in the nuclei of PC3 DKO2/3 and PC3 DKO2/3+1 cells, 24 h after treatment with 100, 200 or 400 μM H2O2.

Data information: Nuclei were visualized by DAPI staining. Arrowheads: AIF translocated to nuclei. Untreated control cells are shown on the left. Right panels, AIF-positive nuclei were counted from pictures taken from three independent fields in two independent experiments. Means ±SD are plotted. HME: * p<0.005; ** p<0.001 compared to H2O2-treated control (vec) cells; PC3: *** p<0.0001 compared to untreated cells of each genotype as assessed by Student's t-test. Scale bars, 10μm.

Source data are available online for this figure.
A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI-NET sequencing (2022)
Figure 2

Figure 2. Homozygosity mapping followed by RNA-seq uncovers a deep intronic recessive mutation in TAPT1.

(A) Schematic representation of the shared IBD region between both Jordanian families, located on Chromosome 4 (4p16.1 - p15.31) with a size of ~12 cM. Whereas WES analysis did not reveal any mutations in the coding sequences located in the IBD region, RNA-seq analysis helped us to identify the disease causative gene from this locus.

(B) Volcano plot showing differentially expressed genes between WT (WT1 and WT2) and patient (V.1 (F1), V.5 (F1)) primary dermal fibroblasts. The vertical axis (y-axis) shows the -log10 P-value, whereas the horizontal axis (x-axis) displays the log2 fold change value. The red dots represent the upregulated transcripts; the blue dots represent the downregulated transcripts. A total of 172 genes were found significantly dysregulated. TAPT1, a gene located in the IBD region, appeared among the most significantly downregulated genes in the patients.

(C) Plot showing the alternative splicing analysis results from WT (WT1 and WT2) and patient (V.1 (F1), V.5 (F1)) primary dermal fibroblasts. The vertical axis (y-axis) shows the -log10 FDR (False Discovery Rate), whereas the horizontal axis (x-axis) represents the exon inclusion level (value ranging from -1 to 1). The red dots represent transcripts with exon inclusion events; the blue dots represent transcripts affected by exon skipping. A total of 63 aberrantly spliced genes were found in the patient cells, being TAPT1 the most significant exon skipping event.

(D) (Left) Schematic representation showing the complete loss of exon 12 from TAPT1 transcript in patient cells, as defined by our splicing analysis data. (Right) Chromatogram showing the novel intronic mutation (c.1237-52 G>A) we found entirely segregating with the disease in all available family members. For display purposes, results from the targeted Sanger sequencing in WT, IV.3 (F1) and V.5 (F1) individuals are shown. The mutation is present in heterozygosis in IV.3 (F1) (unaffected mother) and in homozygosis in V.5 (F1) (affected patient).
A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI-NET sequencing (2022)
Figure 4

Figure 4. Exon-12 skipped transcripts undergo NMD to create a protein-null allele

(A) Schematic representation showing that the complete loss of exon 12 in TAPT1 results in a premature stop codon, which targets the transcript for nonsense-mediated mRNA decay.

(B) qPCR results using specific primers for TAPT1 and TAPT1-AS1 in 3 WT (WT1, WT2 , V.2 (F1)) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts. TAPT1 mRNA is significantly reduced in all patients compared to WTs, whereas TAPT1-AS1 transcript levels are unaffected. Fold change relative to V.2 (F1) is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).

(C) Western blot analysis of endogenous TAPT1 protein (~60 kDa) using whole protein extracts from primary dermal fibroblasts from WT (WT1 and WT2), heterozygous (IV.3 (F1)) and homozygous (V.1 (F1), V.5 (F1) and IV.1 (F2)) individuals and two different commercial antibodies (top: Sigma, HPA042567; bottom: Sigma, HPA048658). Results show a complete absence of TAPT1 protein in patient samples. GAPDH was used as a loading control.

(D) qPCR analysis of TAPT1 expression in 3 WT (WT1, WT2, WT3) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts treated with cycloheximide (CHX). CHX was used to block nonsense mediated decay (NMD). Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells. For each graph, fold change relative to non-treated condition is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).
A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI-NET sequencing (2022)
Figure 5

Figure 5. TAPT1 cellular localization and functional data.

(A) TAPT1 predicted topology: a membrane-spanning protein consisting of 5 transmembrane helices (Uniprot database).

(B) Western blot analyses for TAPT1 (~60 kDa) using cytosolic, Mito/ER/Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs (WT1 and WT2) and two patients (V.5 (F1) and IV.1 (F2)). TAPT1 protein is highly enriched in the Mito/ER/Golgi fraction, and to a lower extent in the nuclear fraction. GAPDH, TGN46 and BiP served as a cytosolic, Golgi network and ER markers, respectively. Adenylate Kinase (AK2) was used as a mitochondrial marker. Laminin A/C was used as a nuclear marker.

(C) Immunofluorescence staining of mitochondria using anti-TOM20 (green), ER using anti-CANX (red) and Golgi using anti-GLG1 (red) in primary dermal fibroblasts from WT1 and V.5 (F1). Similar staining patterns are observed with the three antibodies in both cell lines. Scale bar represents 10 µm.

(D) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients (-/-) (V.I (F1), V.5 (F1) and IV.1 (F2)) primary dermal fibroblasts compared to WTs (+/+) (WT1, WT2 and WT3) cells. Fold change relative to WT is plotted as mean ± SD. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).

(E) CMV cell infection assay on 2 patient (V.1 (F1) and V.5 (F1)) and 3 WT (WT1, WT4 and WT5) primary dermal fibroblast cell lines, using β-galactosidase activity as a readout. MRC5 cell line was used as a positive control. All of the cells were infected by the HCMV strain RC256 at a MOI=0.1. Data are shown as mean ± SD of three technical replicates. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).
A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI-NET sequencing (2022)
Figure 6

Figure 6. Integrated analysis of SI-NET-seq and RNA-seq data revealed enrichment of collagen and ECM-related pathways in TAPT1-null cells.

(A) Volcano plot showing differentially expressed genes as determined by RNA-seq in patient primary dermal fibroblasts (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) cells. The y-axis shows the -log10 P-value, whereas the x-axis displays the log2 fold change value. The red dots represent 75 significantly upregulated genes, and the blue dots represent 97 significantly downregulated genes.

(B) qPCR validation test for 3 top dysregulated genes (RARRES2, ZIC1, and ZIC4) detected by RNA-seq. The analysis was performed on RNA samples independent from those sent for RNA-seq for 2 WTs (WT1 and WT2) and 2 patients (V.5 (F1) and IV.1 (F2)). Fold change relative to WT1 is plotted as mean ± SD of three technical replicates. Asterisks indicate statistical significance (Student t-test; *** p-value < 0.001, **** p-value < 0.0001).

(C) Volcano plot showing genes with an altered occupancy of transcriptionally engaged Pol II in patients (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) primary fibroblast cells using SI-NET-seq. The y-axis shows the -Log10 P-value, whereas the x-axis indicates the log2 fold change value for the Pol II occupancy. The Pol II density is increased in 222 genes (red dots), and decreased in 95 genes (blue dots). The yellow dot represents TAPT1.

(D) Bubble plot showing enrichment of collagen and extracellular matrix (ECM) pathways from the integrated reactome pathway analysis (Jassal et al, 2020) of the SI-NET-seq (light blue circles) and RNA-seq (dark blue circles) data. Enriched pathways are indicated on the y-axis, and the corresponding p-values are shown on the x-axis. The size of the circles represents the number of altered genes from each pathway.
Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis. (2015)
Figure 1

Retinoic acid signaling is involved in HSC activation

Phenotypic changes of HSCs by citral. Phase-contrast images (left), oil red O-stained images (middle), and autofluorescence images (right) are shown for HSCs after passage 1 with or without the treatment of citral (100 μM) for 24 h. Scale bar = 10 μm.

Western blot analysis of cell lysates from the citral-treated HSCs in (A). α-tubulin was used as a loading control. The quantitative densitometric data are expressed as the percentage of untreated control and represent the means (SD) for three independent experiments. **P-value = 0.002, paired t-test (compared to control).

Protein expression of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-d3) and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting.

Real-time PCR analysis for the expression levels of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-d3. **P-value, paired t-test (n = 3) (compared to HSCs-d3), ALDH1A1/HSCs-P3: 0.003, ALDH1A2/HSCs-P1: 0.0018, ALDH1A2/HSCs-P3: 0.001.

Effect of ALDH1A suppression on HSC activation. HSCs-P1 were transfected with non-targeting siRNA or siRNA specific to ALDH1A1 and/or ALDH1A2, and after 48 h, whole-cell lysates were subjected to Western blotting. **P = 0.0023, paired t-test (n = 3) (siRNA-ALDH1A1/2 compared to control).

Reverse-phase HPLC analysis of whole-cell lysates from the siRNA-ALDH1A-treated HSCs in (E). Typical chromatograms of all-trans retinoic acid and 13-cis retinoic acid. The retention time is 14 min for 13-cis-RA and 23 min for all-trans RA.

Phenotypic changes of HSCs by RAR antagonist. HSCs-d3 were incubated with AGN193109 (1 μM) ± all-trans RA (10 μM) for 3 days and subjected to Oil red O staining. Scale bar = 10 μm.

Western blot analysis of cell lysates from AGN193109 ± RA-treated HSCs in (G). **P = 0.003, paired t-test (n = 3) (AGN193109 compared to control).
Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis. (2015)
Figure 2

Downregulation of RA signaling may contribute to the anti-fibrotic effect of albumin

Albumin and R-III downregulate RA signaling. HSCs-P1 were either transfected with RARE Cignal reporter and expression plasmid containing vehicle or albumin (left panel) or treated with R-III (25–250 nM) after transfection with RARE Cignal reporter (right panel). After 36 h, the transfected cells were harvested for luciferase assay. Renilla luciferase activity was normalized to firefly luciferase activity. Ratios were normalized against the cells transfected with vehicle plasmid. **P-value, two-sample t-test (n = 9) (compared to control), albumin: 0.0037, R-III 125 nM: 0.0032.

Phenotypic changes of HSCs by the expression of albumin and/or ALDH1A. HSCs-P1 were transfected with plasmids encoding albumin, ALDH1A1, and ALDH1A2, either individually or in combination, and incubated in the presence of all-trans retinoic acid (50 nM) for 18 h. Cells were analyzed by oil red O staining. Scale bar, 20 μm.

Western blot analysis of cell lysates from albumin- and/or ALDH1A-transfected HSCs as in (B). **P = 0.002, paired t-test (n = 3) (albumin compared to control).

Reverse-phase HPLC analysis of whole-cell lysates from albumin- and/or ALDH1A-transfected HSCs as in (B).

RAR agonist counteracts R-III action. HSCs-P1 were treated with His-tagged R-III (final concentration, 0.15 μM) in the presence or absence of RAR agonist (AGN191183, 1 μM) for 18 h and analyzed by Western blotting. **P = 0.0024, paired t-test (n = 3) (R-III compared to control). The separating lines demarcate bands that come from non-consecutive lanes of the same gel.

Source data are available online for this figure.
Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis. (2015)
Figure 3

STRA6 may be involved in the cellular uptake of R-III into HSCs

Protein expression of STRA6 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-3d), and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting. Lysates from NIH3T3 cells were used as a negative control. The separating lines demarcate bands that come from non-consecutive lanes of the same gel.

Real-time PCR analysis for the expression level of STRA6 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-3d. **P-value, paired t-test (n = 3) (compared to HSCs-d3), HSCs-P2: 0.002, HSCs-P3: 0.0022.

R-III uptake parallels STRA6 level. HSCs at different stages of activation were incubated with His-tagged R-III (final concentration, 0.15 μM) for 30 min, washed, and subjected to Western blotting for R-III uptake.

STRA6-mediated uptake of R-III. HSCs-P1 were transfected with non-targeting siRNA or one of the two different STRA6-specific siRNAs, and after 48 h, cells were incubated with R-III for 30 min and analyzed by Western blotting.

Source data are available online for this figure.
Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis. (2015)
Figure 4

Therapeutic effect of R-III on CCl4-induced liver fibrosis

Representative macroscopic pictures of livers from control and CCl4-, CCl4/albumin-, CCl4/RBP-, and CCl4/R-III-treated mice.

Liver sections were stained with H&E and Sirius red, and also subjected to immunohistochemistry for type I collagen. Scale bar, 200 μm.
Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis. (2015)
Figure 5

R-III reduces CCl4-induced liver fibrosis

Hydroxyproline content in the livers from control and CCl4-, CCl4/albumin-, and CCl4/R-III-treated mice (μg/g liver). *P = 0.037, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).

Western blot analysis on liver extracts from the treated mice with antibody against type I collagen.

Immunohistochemical analysis of α-SMA on representative liver sections from the treated mice. Scale bar, 200 μm.
Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis. (2015)
Figure 6

Immunohistochemical staining of liver sections for TGF-β and F4/80

Liver sections from control and CCl4- and CCl4/R-III-treated mice were immunohistochemically stained for TGF-β and F4/80. Scale bar, 50 μm.
Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis. (2015)
Figure 7

Targeted delivery of R-III to hepatic stellate cells

Liver sections from treated mice were immunohistochemically stained for desmin, His-tag, and α-SMA. His-positive signals largely overlapped with desmin/α-SMA staining. Scale bar, 50 μm.
Diverse roles of TssA-like proteins in the assembly of bacterial Type VI secretion systems. (2019)
Figure 1

Figure 1. TssAVC and TssA2PA influence T6SS sheath assembly dynamics

A. Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 µm.

B. H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 µm.
Diverse roles of TssA-like proteins in the assembly of bacterial Type VI secretion systems. (2019)
Figure 2

Figure 2. Localization and dynamics of TssAVC and TssA2PA

A. Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 µm.

B-E. Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow).

F. Fluorescence microscopy of T6SS dynamics in a double tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 µm.

G-J. Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I) or stays attached to distal end after contraction (J).
Diverse roles of TssA-like proteins in the assembly of bacterial Type VI secretion systems. (2019)
Figure 3

Figure 3. Cryo-EM of TssAVC

A. Top view and side cutaway view of the TssAVC cryo-EM reconstruction, shown low-pass filtered at a lower (white, transparent) and higher (royal blue, non-transparent) threshold. Possible (-60˚;60˚) range of motion of Nt2 domain relative to the CTD ring plane is shown.

B. Top view of the ribbon diagram of TssAVC CTD model. Two interfaces are highlighted with green circles. Enlarged side view of interface between helices α9-α11 of two neighboring subunits (top right), enlarged side view of interface between α11-α12 linker of one subunit and helix α10 from the neighboring subunit based on conserved WEP-motif (bottom right). Part of Nt2-CTD linkers are highlighted with red circles.

C. Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2-dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2-dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right).

D. Side and top views of putative model of TssAVC Nt2-CTD ring fitted into cryo-EM reconstruction (royal blue, non-transparent) of the distal end of TssAVC T6SS (EMD-3878). TssAVC Nt2-CTD symmetrized reconstruction shown (white, transparent), fitted Nt2-CTD model shown as a ribbon diagram.