EMBO/soda-nel huggingface dataset:
The eukaryotic replisome tolerates leading-strand base damage by replicase switching (2021)
Figure 6

Figure 6. The replisome bypasses 8oxoG by a replicase-switch mechanism

(A) Standard replication assay on undamaged (UD), 8-oxoguanine (8oxoG), and thymine glycol (Tg) templates in the absence of TLS components.

(B) Pulse chase reaction on UD and 8oxoG templates. Where present, Pol δcat (10 nM) was added directly after addition of the chase.

(C) Pulse chase reactions on the 8oxoG template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel.
NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 1

Figure 1. Induced expression of NB-3 in the lesion area after SCI.

A, B X-gal staining showed NB-3/LacZ expression in the sham-operated (A) and injured (B) spinal cords from adult NB-3+/− mice.

C, D Western blot analysis (C) and quantification (D) of NB-3 expression in the lesion area post-injury. NB-3 expression levels were normalized by -tubulin. Data are represented as mean ± SEM. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni’s post-test. Data were analyzed from 10 independent experiments each including 2 mice per group.

E, F Single-labeling immunostaining showed NB-3 expression in the sham-operated (E) and injured (F) spinal cords from adult NB-3+/+ mice.

G–K Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+ spinal cord 14 days post-injury. (G1–K1) High-magnification images of boxed areas in (G–K), respectively.

L Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (L1) High-magnification image of the boxed area in (L). The arrows indicate the lesion sites.

M–Q Co-immunostaining of NB-3 (green), Fibronectin (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+ spinal cord 14 days post-injury. (M1–Q1) High-magnification images of boxed areas in (M–Q), respectively.

R Co-immunostaining of NB-3 (green), Fibronectin (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (R1) High-magnification image of the boxed area in (R). The arrows in (G–R) indicate the lesion sites.

Scale bars, 400 µm (A and B), 400 µm (E and F), 400 µm (G–L), 100 µm (G1–L1), 400 µm (M–R), and 100 µm (M1–R1).
NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 2

Figure 2. Induced expression of NB-3 in corticospinal neurons and axons after SCI.

A Diagram of the NB-3 induction (green) after SCI.

B NB-3 expression in corticospinal neurons 14 and 28 days post-injury (dpi). FG (blue) was applied to retrogradely label corticospinal neurons.

C Quantification of the fluorescence intensity of NB-3 in (B). **p < 0.01; one-way ANOVA followed by Bonferroni’s post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.

D, E Western blot analysis (D) and quantification (E) of NB-3 expression in the sensorimotor cortex after SCI. NB-3 expression levels were normalized by -tubulin. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni’s post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.

F–K Induced NB-3 expression at the distal terminations of post-traumatic corticospinal axons and in reactive astrocytes at the lesion border 14 (H and I) and 28 days post-injury (dpi) (J and K). BDA (red) was applied to anterogradely label corticospinal axons. GFAP (blue) was used to label reactive astrocytes. (G) High-magnification image of the boxed area in (F). (I and K) High-magnification and x-y-z reconstruction images of boxed areas in (H and J), respectively, show co-localization of NB-3 and GFAP in astrocytes at the lesion border and co-localization of NB-3 in BDA-labeled axons and GFAP-positive astrocytes at the contact surface of corticospinal axons and astrocytes (arrowheads). The arrows in (H and J) indicate the lesion sites. The asterisks in (H and J) indicate the terminations of the BDA-labeled projecting axons.

Data are represented as mean ± SEM (C, E). Scale bars, 30 μm (B), 400 µm (F, H, and J), 25 µm (G), and 25µm (I and K).
NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 3

Figure 3. Deficiency in NB-3 leads to enhanced regrowth of post-traumatic corticospinal axons.

A Diagram showing BDA-labeled corticospinal axons at certain distances from the spinal lesion border after SCI. BDA (red) was applied into sensorimotor cortex to anterogradely label corticospinal axons. GFAP (blue) was used to label reactive astrocytes and outline the lesion border in the injured spinal cord. The red dashed lines indicate the rostral and caudal borders of the lesion; the green dashed lines indicate various distances from the rostral lesion border (0 mm); the blue dashed lines indicate various distances from the caudal lesion border (0 mm).

B–K Representative images of serial sagittal sections showing the main BDA-labeled corticospinal axons in NB-3+/+ (B–D) and NB-3−/− (E–G) mice 14 weeks after complete spinal transection. (H–J) High-magnification images of the areas in (E–G) (single asterisks), respectively. (K) High-magnification image of the area in (E) (double asterisk). The arrows indicate the lesion sites; the white dashed lines indicate the regenerative corticospinal axons extending into the distal spinal cord; the single asterisks in (E–G) indicate the lesion epicenters; the double asterisk in (E) indicates the axons extending from the caudal lesion border. Scale bars, 400 µm (B–G) and 100 µm (H–K).

L Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (B–G). Data are represented as mean ± SEM. *p < 0.05, and **p < 0.01; two-way ANOVA followed by Fisher’s LSD. n = 25 mice per group.
NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 4

Figure 4. Increase in BMS Scores, electrophysiological improvement and synapse reformation of NB-3–deficient mice after SCI.

A Time course of the change in BMS scores in NB-3+/+ and NB-3−/− mice as measured with the BMS rating scale after spinal cord complete transection. *p < 0.05; two-way ANOVA followed by Bonferroni’s post-test. n = 25 mice per group.

B Diagram showing electrophysiological analysis of transmission across the spinal lesion site.

C Electrophysiological transmission across the spinal lesion site. In each group of NB-3+/+ and NB-3−/− mice, the electrophysiological responses in the caudal spinal cord were recorded in the condition of sham-operated or 12 weeks post-injury. The latencies and amplitudes of responses were analyzed and quantified in each group. n.s., not significant, *p < 0.05, and **p < 0.01; two-way ANOVA followed by Bonferroni’s post-test. n = 10 mice per group.

D Diagram showing analysis of synapse reformation between regenerative axons and neurons in the injured spinal cord 0.5 mm rostral to the lesion site or 0.5 mm caudal to the lesion site.

E Representative images of coronal sections showing synapse formation between the glutamatergic corticospinal terminals and spinal neurons in NB-3+/+ and NB-3−/− mice. Corticospinal axons, neurons, and glutamatergic terminals of corticospinal axons were labeled with BDA (red), Tuj1 (blue) and vGlut1/2 (green), respectively. Each group showed the data from the rostral and caudal spinal cord in the condition of sham-operated or 12 weeks post-injury. Scale bar, 20 μm.

F Quantification of the number of glutamatergic corticospinal synapses per neuron in each group. n.s., not significant, **p < 0.01; two-way ANOVA followed by Fisher’s LSD. n = 15 mice per group.

Data are represented as mean ± SEM (A, C, and F).
NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 5

Figure 5. Interruption of NB-3 homophilic binding leads to enhanced regrowth of post-traumatic corticospinal axons.

A, B Western blot analysis (A) and quantification (B) of NB-3 expression in cultured pyramidal neurons infected with lentivirus encoding negative control (LV-nc-GFP) and that encoding NB-3 shRNA (LV-NB-3 shRNA-GFP). NB-3 expression levels were normalized by -tubulin. ***p < 0.001; one sample t-test. Data were analyzed from 5 independent experiments.

C–E Sagittal sections showing the BDA-labeled corticospinal axons in injured spinal cords following cortical injection of LV-nc-GFP (C) and LV-NB-3 shRNA-GFP (D). High-magnification image (E) of the area in (D) (asterisk).

G–I Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (G) and LV-NB-3 shRNA-GFP (H) into the lesion sites. High-magnification image (I) of the area in (H) (asterisk). The arrows in (C–E and G–H) indicate the lesion sites; the asterisks in (D and H) indicate the lesion epicenters; the white dashed lines in (D and H) indicate the regenerative corticospinal axons extending into the distal spinal cord.

F, J Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (C, D, G, and H). *p < 0.01; two-way ANOVA followed by Fisher’s LSD. n = 19 mice per group in (F), and n = 17 mice per group in (J).

Data are represented as mean ± SEM (B, F, and J). Scale bars, 400 µm (C and D), 40 µm (E and I), and 400 µm (G and H).
NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 6

Figure 6. Self-interactions of NB-3 and its inhibition of neurite extension.

A, B NB-3–Fc–coated bead aggregation assay (A). BSA–coated beads as a negative control (B).

C Co-immunoprecipitation of NB-3–Myc with NB-3–HA from co-transfected COS1 cells.

D, E Cis interactions of NB-3–Myc with NB-3–HA in COS1 cells. (E) High-magnification image of the boxed area in (D). Cells were co-stained for Myc (red), HA (green), and DAPI (blue). The arrowheads indicate cell surface co-localization of NB-3–Myc and NB-3–HA.

F, G Trans interactions between NB-3 expressed in COS1 cells and treated NB-3–Fc. Cells were co-stained for Myc (red), Fc or NB-3–Fc (green), and DAPI (blue). The arrows indicate co-localization of NB-3–Myc and NB-3–Fc at the cell membrane (G).

H Cell membrane co-localization of NB-3–Myc and NB-3–HA separately expressed in 293T and COS1 cells. Cells were co-stained for Myc (red), HA (green), and DAPI (blue).

I, J NB-3 expression in cultured pyramidal neurons and astrocytes. Pyramidal neurons were co-stained for NB-3, Ctip2 and Tuj1 (I). Astrocytes were co-stained for NB-3, GFAP, and DAPI (J).

K Western blot analysis and quantification of NB-3 in cultured pyramidal neurons and astrocytes. NB-3 expression levels were normalized by -tubulin and expressed relative to that of neurons. ***p < 0.001; one sample t-test. Data were analyzed from 3 independent experiments.

L–Q NB-3+/+ (L and M) and NB-3−/− (O and P) pyramidal neurons were co-cultured with astrocytes that did not (L and O) or did (M and P) express NB-3, and immunostained for Tuj1, NB-3, and GFAP (L, M, O and P). NB-3+/+ pyramidal neurons (N) and NB-3−/− pyramidal neurons that overexpressed NB-3ΔECD–Myc (Q) were co-cultured with astrocytes that overexpressed NB-3ΔECD–Myc, and immunostained for Tuj1, Myc, and GFAP (N and Q).

R The length of neurites on astrocytes relative to total neurite length. n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The neurite lengths of more than 150 pyramidal neurons from 4 independent experiments in each group were quantified.

Data are represented as mean ± SEM (K and R). Scale bars, 15 µm (A and B), 20 µm (D), 6 µm (E), 15 µm (F and G), 10 µm (H–J), and 10 µm (L–Q).
NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 7

Figure 7. NB-3 trans-homophilic interactions inhibit neurite outgrowth and lead to growth cone collapse.

A Layer V cortical explants from E18.5 NB-3+/+ and NB-3−/− mice were cultured in medium supplemented with either Fc or NB-3–Fc (50 μg/ml).

B Quantification of neurite outgrowth in (A). n.s., not significant, **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 50 layer V cortical explants from 5 independent experiments in each group.

C Pyramidal neurons of NB-3+/+ and NB-3−/− mice were cultured in medium supplemented with either Fc or NB-3–Fc (50 μg/ml). The neurons were co-stained for Ctip2, NB-3, and Rhodamine phalloidin. (C1–C4) High-magnification images of the growth cones (white arrowheads) of the neurons in (C), respectively.

D Quantification of neurite length in (C), and the percentage of collapsed growth cones in (C). n.s., not significant, *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 300 corticospinal neurons from 6 independent experiments in each group.

Data are represented as mean ± SEM (B and D). Scale bars, 200µm (A), 10 µm (C), and 20 µm (C1–C4).
NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 8

Figure 8. Trans-homophilic interactions of NB-3 downregulate mTOR signaling activity in corticospinal neurons.

A, C Images of coronal sections of layer V sensorimotor cortex from NB-3+/+ and NB-3−/− mice after SCI. Corticospinal neurons were retrogradely labeled with Fluorogold (FG), and coronal sections were co-stained with p-mTOR (A) and p-S6 (S240/244) (C) (arrowheads).

B, D Quantification of fluorescence intensity of p-mTOR and p-S6 (S240/244) in FG-labeled corticospinal neurons in (A and C). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.

E–H Western blot analysis and quantification of the expression of p-Akt/Akt (E and F) and p-S6 (S240/244) (G and H) in sensorimotor cortex from NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.

I, K, M, O Expression of Akt (I), p-Akt (K), p-mTOR (M), and p-S6 (S240/244) (O) in NB-3+/+ pyramidal neuron–astrocyte co-cultures. The astrocytes were transfected either with NB-3–Myc or a control vector. The dashed lines outline the astrocytes. The asterisks indicate the NB-3–expressing astrocytes.

J, L, N, P Quantification of fluorescence intensity of Akt (J), p-Akt (L), p-mTOR (N), and p-S6 (S240/244) (P), respectively, in pyramidal neurons of the co-cultures in (I, K, M, and O). n.s., not significant, and *p < 0.05; one sample t-test. The intensities of more than 300 pyramidal neurons from 6 independent experiments in each group were quantified.

Data are represented as mean ± SEM (B, D, F, H, J, L, N, and P). Scale bars, 20 µm (A and C) and 10 µm (I, K, M, and O).
Cyprinid-specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors (2022)
Figure 1

Figure 1. CiTLR5a and CiTLR5b can form homodimers and heterodimer.

CFM on FHM cells transfected with CiTLR5a-GFP and CiTLR5b-RFP (A), CiTLR5a-GFP and CiTLR5a-RFP (B), or CiTLR5b-GFP and CiTLR5b-RFP (C). Green, red, blue, and yellow indicate CiTLR5a, CiTLR5b, nucleus, and co-localization, respectively (scale bars: 10 μm).

mRNA and protein expression of CiTLR5a-HA (D), CiTLR5b-HA (D), CiTLR5a-GFP (E), and CiTLR5b-GFP (E) plasmids in FHM cells were examined by qRT-PCR (upper panel) with EF1α gene as an internal control and by immunoblotting (IB) (lower panel) with β-tubulin as a reference (n = 4).

FHM cells in 10 cm2 dishes were transfected with the indicated plasmids for 24 h. Co-IP was performed with anti-HA monoclonal antibody. Mouse IgG was used as control. Immunoblotting was performed with anti-HA and anti-GFP antibodies, respectively.

Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (D) and (E). n represents biological replicates. ***P < 0.001.
Cyprinid-specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors (2022)
Figure 2

Figure 2. CiTLR5a and CiTLR5b sense flagellin as heterodimer and sense poly(I:C) as homodimers.

CIK cells were seeded in 12-well plates for 24 h, stimulated with PBS (control), LPS, PGN, dsDNA, K. pneumonia lysate, poly(I:C), FlaA, FlaB, FlaC, A. hydrophila lysate, or GCRV for different times (0, 3, 6, 12, 24, 48 h), then mRNA expression of CiTLR5a and CiTLR5b were measured by qRT-PCR (n = 4).

Effects of MAMPs and bacteria on NF-κB2 (B) and IFN1 (C) promoter activities were measured by DLR assays in FHM cells. PBS serves as a control (n = 3).

mRNA expression in 12 tissues, based on qRT-PCR analysis (n = 4). 18S rRNA gene was employed as an internal control.

E mRNA expression of CiTLR5a and CiTLR5b were examined by qRT-PCR in grass carp spleen and head kidney tissues at different time points post FlaC, A. hydrophila, poly(I:C), or GCRV challenge (n = 6).

F-H MAMPs (FlaA, FlaB, FlaC, dsDNA, poly(I:C), PGN, LPS, and a negative control) were coated to 96-well plates and then blocked with bovine serum albumin. The binding abilities between CiTLR5a-ectodomain (E), CiTLR5b-ectodomain (F), GST (G) and different MAMPs were assessed by ELISA (n = 4).

I-L Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b were respectively incubated with FlaA, FlaB, FlaC, biotin-poly(I:C), biotin-ssRNA (1 μg/mL) at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads or streptavidin agarose beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA or anti-His antibodies.

M Amino acid sequence alignment of the EJM and putative transmembrane regions of CiTLR5a, CiTLR5b, DrTLR5a, DrTLR5b, and HsTLR5. The acidic amino acid residues in the EJM were highlighted in red. The transmembrane sequences were marked by shadow.

N, O CiUNC93B1-Myc and CiTLR5a-HA (M) or CiTLR5b-HA (N) were transfected into FHM cells. Co-IP and immunoblotting were conducted to test the interactions between CiUNC93B1-Myc and CiTLR5a-HA or CiTLR5b-HA.

P, Q Effects of CiUNC93B1 on CiTLR5a- and CiTLR5b-mediated IFN1 (O) and NF-κB2 (P) promoter activities were examined in FHM cells by DLR assays (n = 3). Both poly(I:C) and FlaC are 1 μg/mL.

R, S FHM cells were transfected with CiTLR5a-GFP (Q) or CiTLR5b-GFP (R) and RAB5-RFP, an early endosomes protein marker, RAB7-RFP, a late endosome protein marker, or LAMP2-RFP, a lysosome protein marker, seeded on microscope cover glasses in 12-well plates for 24 h, then fixed and examined by CFM. Green represents CiTLR5a-GFP or CiTLR5b-GFP, red represents RAB5-RFP, RAB7-RFP, or LAMP2-RFP, and blue indicates nucleus. Yellow represents co-localization between CiTLR5a or CiTLR5b and early endosomes or lysosomes (scale bars: 10 μm).

T, U Effects of chloroquine (in 5 μg/mL) on CiTLR5a- and CiTLR5b-dependent signaling were examined by DLR assays in FHM cells (n = 3). HsTLR5 and CiMDA5 (C. idella MDA5) were employed as positive controls.

Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (B), (C), (P), (Q), (T), and (U). n represents biological replicates. **P < 0.01.
Cyprinid-specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors (2022)
Figure 3

Figure 3. CiTLR5a homodimer slightly promotes while CiTLR5b homodimer potently restricts GCRV replication in CIK cells.

Effects of the indicated constructs on IFN1 and IFN3 promoter activities after GCRV challenge were measured by DLR assays in CIK cells (n = 3).

Standard plaque assays in CIK cells with indicated overexpressing constructs or siRNAs and different dilution of GCRV.

Relative plaque intensity ratios of (B) were analyzed by ImageJ (n = 3).

E, F CIK cells overexpressing (E) or interfering (F) were infected with GCRV at an MOI = 0.1, and supernatants were collected at 48 h for viral titer assays by TCID50 (n = 3).

G, H CIK cells were treated with the supernatants for titer assays for 24 h, and mRNA expression of GCRV VP4, VP5, VP6, VP7, and NS38 genes were quantified by qRT-PCR (n = 4).

I, J Antiviral activities (cell viability) of the indicated constructs or siRNAs were measured by MTT assays in CIK cells at deferent time points post GCRV challenge (n = 4).

K, L mRNA expression of antiviral regulator and effector molecules IFN1, Mx2, Gig1, and IL-22 were quantified by qRT-PCR in overexpressing (K) or interfering (L) CIK cells post GCRV challenge (n = 4).

M, N mRNA expression of IFN1, IFN3, NF-κB1 and NF-κB2 in spleen tissue were investigated by qRT-PCR after RNAi in vivo for 24 h, and A. hydrophila or GCRV challenge for 24 h (n = 6).

O, P Protein expression of IFN1 (O) and NF-κB1 (P) in spleen tissue were investigated by immunoblotting (IB) after RNAi in vivo for 24 h, and A. hydrophila or GCRV challenge for 24 h (n = 3).

Q Viral titers in spleen homogenates were examined with gene knockdown in vivo for 24 h and GCRV challenge for 24 h in CIK cells (n = 6).

Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (A) and (C-Q). n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.
Cyprinid-specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors (2022)
Figure 5

Figure 5. CiTLR5a and CiTLR5b share conserved flagellin binding regions and sites.

Comparison of the structures and relevant sequence regions in CiTLR5a and CiTLR5b with counterparts in DrTLR5a and DrTLR5b. The two representative conserved amino acids in flagellin binding regions for following studies were denoted with triangles. Identical amino acid residues were shown by asterisks (*), and high and low similarities were indicated with colons (:) and points (.), respectively.

Effects of the two selected flagellin binding sites on NF-κB2 promoter activity were investigated by site-directed mutagenesis and DLR assays in FHM cells (n = 3).

Lysates of FHM cells overexpressing mutants were incubated with FlaC for pull-down assays and analyzed by immunoblotting (IB). Vector and wild type (WT) were used as controls. The images of (D) and (E) were one of the three independent biological experiments in Fig EV4A and B. Relative band intensity ratios were analyzed by ImageJ (n = 3).

Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (B-E). n represents biological replicates. *P < 0.05, **P < 0.01.