EMBO/soda-nel huggingface dataset:
Effector loading onto the VgrG carrier activates type VI secretion system assembly (2019)
Figure 3

Fig. 3. The presence of Tde effectors is required for efficient TssBC sheath assembly.

A. Representative images of cells of A. tumefaciens C58 strains ΔtssB, ΔtssLΔtssB, and ΔtdeiΔtssB cells each expressing TssB-GFP from pTrc200. Upper panel: phase contrast images; Lower panel: green fluorescence images. Examples of GFP streaks were indicated by white arrowheads. Scale bar: 2 μm.

B. Quantification of TssB-GFP foci in different genetic background. The number of the fluorescent foci were counted using the "analysis particle" function in Fiji with a manually set threshold. The number of cells were counted manually. For each strain, a total of three images were obtained from one independent experiment and each image contained more than 300 cells for quantification and statistical analysis. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Data are mean ± SEM and two groups with significant differences (P=0.000272) are indicated with different letters (a and b). Similar results are reproduced in two independent experiments.

C. Western blots of the isolated sheaths, which were prepared via ultracentrifugation of cell lysates from A. tumefaciens C58 wild type, ΔtssL, and Δtdei. T: total proteins from the cell lysate. P: pellet samples containing TssBC sheaths after ultracentrifugation. S: supernatant after ultracentrifugation. Proteins were analyzed in western blots probed with indicated antibodies. Fha serves as a cytoplasmic control. Molecular weight markers (in kDa) are indicated on the left. Similar results were obtained from at least two independent experiments. Data are from one independent experiment and reproduced in at least two independent experiments.

D. Pellet samples of wild type and Δtdei were visualized with transmission electron microscopy (TEM). Sheaths are indicated by red arrowheads and flagella, which are distinguishable on the basis of smoother, more solid, and thinner structures (~13 nm in width), are indicated by green arrowheads [33]. Data are from one independent experiment and reproduced in at least two independent experiments. Scale bar: 0.5 μm (upper panel), 100 nm (upper panel).
Effector loading onto the VgrG carrier activates type VI secretion system assembly (2019)
Figure 4

Figure 4. Impacts of vgrG-associated genes on T6SS secretion and antibacterial activity of A. tumefaciens strains other than C58.

A. The vgrG genetic modules of the tested A. tumefaciens strains. Genes are color coded according to the predicted function or results of functional assays [24]. The vgrG-associated genes are v1-v6 for 12D1, v1-v9 for 1D1108, v1-v7 for 15955.

B. Secretion assay of wild type and various mutants of 12D1, 1D1108 and 15955.

C. Antibacterial activity assay of wild type and mutants of 12D1, 1D1108 and 15955 was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in three independent experiments.

D. Secretion assay of wild type 12D1, ΔtssL, Δv1, Δv3-4 containing pRL662 empty vector (V) or its derivatives with v1 or v3-4 were analyzed for secretion.

E. Secretion assay of wild type 1D1108, ΔtssL, Δv4, Δv6 containing pRL662 empty vector (V) or its derivatives with v4 or v6.

Data information: In (B, D, E), cellular and extracellular fractions were collected from A. tumefaciens strains grown in liquid 523 medium. Proteins were collected and analyzed in western blots probed with indicated antibodies. RpoA is RNA polymerase subunit alpha and located in the cytosol of A. tumefaciens. Molecular weight markers (in kDa) are indicated on the left. Data are from one independent experiment and reproduced in at least two independent experiments.
Effector loading onto the VgrG carrier activates type VI secretion system assembly (2019)
Figure 5

Figure 5. Effect of tae on T6SS secretion and antibacterial activity in the C58 Δtdei mutant grown in minimal medium.

A. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, and various single, double, and triple toxin-immunity deletion strains.

B. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, Δtdei, and Δ3TIs harboring the indicated plasmids.

Data information: In (A, B), A. tumefaciens cells were grown in I-medium (pH 5.5) and cellular and extracellular fractions were collected for western blot analysis probed with antibodies for indicated proteins. Plasmids: pRL662 vector (V), pTae (tae expressed pRL662), pTai (tai expressed on pTrc200). Molecular weight markers (in kDa) are indicated on the left. Data are from one independent experiment and reproduced in at least two independent experiments.
Effector loading onto the VgrG carrier activates type VI secretion system assembly (2019)
Figure 6

Figure 6. Impact of tae-tai on T6SS secretion and antibacterial activity of A. tumefaciens strains 12D1 and C58 grown in minimal medium.

A. T6SS secretion assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains.

B. T6SS secretion assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids.

C. Antibacterial activity assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in two independent experiments.

D. T6SS secretion assay of A. tumefaciens C58 wild type, ΔtssL, and various mutant strains.

Data information: In (A, B, D), A. tumefaciens cells were grown in I-medium (pH 5.5) and cellular and extracellular fractions were collected for western blot analysis probed with antibodies for indicated proteins. Plasmids: pRL662 vector (V) and derivative expressing v3-v4. Molecular weight markers (in kDa) are indicated on the left. Data are from one independent experiment and reproduced in at least two independent experiments.
Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons (2021)
Figure 1

Figure 1

Centrosomes display MTOC function and Trim46 appearance during axon specification

A. Schematic illustration of centrosomal MTOC function in neurodevelopmental stages 1, 2 and 3 in human iPSC-derived NSCs/neurons.

B. Typical examples of stage 1 (day 0), stage 2 (day 7) and stage 3 (day 12) human iPSC-derived NSCs/neurons immunostained for γ-Tubulin and Centrin, marked by arrows. Cells were co-immunostained with Ki67 and DAPI (day 0) or Trim46 and MAP2 (day 7 and 12) to define their neuronal stages. Arrowheads mark the AIS. Scale bar = 10µm in overview, 2µm in zooms.

C. Quantifications of normalized γ-Tubulin fluorescent intensities at centrosomes in stage 1, 2 and 3 human iPSC-derived neurons. n = 46-51 cells in 2 independent experiments.

D. 3D STED imaging of αTubulin in stage 1 (day 0), stage 2 (day 7) and stage 3 (day 13) hiPSC-derived neurons. Scale bar = 5µm in overview, 1µm in zoom.

E. Human iPSC-derived NSCs (day 0) immunostained for Trim46, γ-Tubulin and Centrin. Zoom represents centrosome structure. Scale bar = 5µm in overview, 2µm in zooms.

F. Typical examples of centrosomes of human iPSC-derived NSCs (day 0) with STED imaging of Trim46 and Centrin immunostaining, and confocal imaging of γ-Tubulin immunostaining. Scale bar = 1µm.

G. Typical examples of stage 2 and stage 3 human iPSC-derived neurons immunostained for Trim46 and MAP2. Inserts represent centrosomes. Zooms on the right represent a non-polarized neurite or a developing axon in stage 2 or stage 3 neurons, respectively. Scale bar = 20µm in overview, 1µm in insert, 10µm in zoom.

Data information: Data represent mean ± SEM. One-way ANOVA including post-hoc analysis with Bonferroni correction (C). *** p<0.001, ns p>0.05
Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence (2014)
f1

(a) Δ133p53α expression is induced by cycloheximide (CHX). MRC-5 fibroblasts (at population doubling levels (PDLs) 51) were treated with CHX (25 μg ml−1) for indicated time periods and examined in immunoblot for Δ133p53α and full-length p53. β-actin was a loading control for normalization. The relative expression levels of Δ133p53α and full-length p53 are shown below the images. (b) Δ133p53α expression is induced by phosphoinositide 3-kinase inhibitors. MRC-5 fibroblasts (PDLs 51) were treated for 4 h with LY303511 (10 μM), LY294002 (10 μM), Wortmannin (50 nM) or dimethylsulphoxide alone (as control) and examined in immunoblot for Δ133p53α and β-actin expression. (c) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1. The immunoblot analyses were performed in early-passage (Y) and replicatively senescent (S) human fibroblast strains MRC-5 and WI-38. The examined PDLs were 30 (Y) and 60 (S) for MRC-5; and 30 (Y) and 54 (S) for WI-38. Bafilomycin A 1 treatment was at 100 nM for 4 h. The relative expression levels of Δ133p53α, full-length p53 and p62/SQSTM1 (normalized with β-actin) are shown. Anti-LC3B antibody detected LC3-I and LC3-II, which was used in quantitative analysis shown in d. (d) Replicative senescence is not associated with enhanced autophagy in general. LC3-II expression levels were normalized with β-actin (data in c) and are shown in the bar graphs as relative values to replicatively senescent cells without bafilomycin A1 (S/−; defined as 1). The LC3-II levels in replicatively senescent cells were higher than those in early-passage cells whether in the presence or absence of bafilomycin A1, which may suggest increased synthesis of autophagosomes at replicative senescence. However, the differences in LC3-II levels between the presence and absence of bafilomycin A1 were similar in early-passage and replicatively senescent cells: in MRC-5, 1.9 for Y (2.1−0.2) and 2.0 for S (3.0−1); in WI-38, 1.6 for Y (2.2−0.6) and 1.6 for S (2.6−1). These data indicate that early-passage and replicatively senescent cells have similar rates of autophagic flux, that is, the number of autophagosomes that are delivered to lysosomes for degradation.
Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence (2014)
f2

(a–c) Knockdown of pro-autophagic proteins restores Δ133p53α expression. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with three independent siRNA oligonucleotides (lanes 1–3) against ATG5 (a), ATG7 (b) and Beclin-1 (c), along with their control siRNA (lane C), for 4 days. Knockdown of ATG5 was confirmed by the loss of ATG5–ATG12 conjugate (detected by anti-ATG5 antibody). Knockdown of the other two proteins was confirmed using the corresponding antibodies. Δ133p53α, full-length p53 and LC3B (LC3-I and LC3-II) were examined in all samples. The relative expression levels of Δ133p53α and full-length p53 (normalized with β-actin) are shown below the images. (d) Knockdown of pro-autophagic proteins and treatment with bafilomycin A1 show similar effects on Δ133p53α and p62/SQSTM1. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with control siRNA (c), ATG5 siRNA (A5, no. 1 in a), ATG7 siRNA (A7, no. 1 in b) and Beclin-1 siRNA (B1, no. 2 in c) as in a–c, untreated (−) or treated with bafilomycin A1 (+, 100 nM for 4 h), and examined in immunoblots for indicated proteins. The relative expression levels of Δ133p53α and p62/SQSTM1 (normalized with β-actin) are shown below the images.
Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence (2014)
f3

(a) Endogenous STUB1 protein is downregulated at replicative senescence. Immunoblot was performed using early-passage (Y) and replicatively senescent (S) fibroblasts (the same set of cells without treatment as in Fig. 1c). (b) STUB1 mRNA is downregulated at replicative senescence. The same set of cells as in a were examined by qRT–PCR. Data were from triplicate experiments (mean±s.d.). *P0.01; **P0.001 (Student’s t-test). (c) Knockdown of STUB1 represses Δ133p53α. MRC-5 fibroblasts (30 PDLs) were transfected with two siRNAs against STUB1 (no. 1 and no. 2) and control siRNA. At 4 days after transfection, immunoblots were performed using anti-STUB1, anti-Δ133p53α, DO-1 (detecting full-length p53), CM1 (detecting both Δ133p53α and full-length p53), anti-p62/SQSTM1 and anti-β-actin antibodies. (d) STUB1 knockdown inhibits cell proliferation. MRC-5 fibroblasts (30 PDLs) were seeded on six-well plates on day 0 (4 × 104 cells per well in 6-well plate; nine wells for each siRNA). Cells were counted on days 1, 4 and 7 (three wells each; mean±s.d.), while siRNA transfection was performed on days 1 and 4. *P0.01 (Student’s t-test). (e) STUB1 knockdown induces cellular senescence. The siRNA transfection was performed as in c and repeated 4 days later, followed by incubation for 3 days and SA-β-Gal staining. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P0.001 (Student’s t-test). (f,g) STUB1 knockdown upregulates p21WAF1 and IL-8. The same set of cells as in e were examined in qRT–PCR for p21WAF1 mRNA (f) and IL-8 mRNA (g). Data were from triplicate experiments (mean±s.d.). **P0.001 (Student’s t-test). (h) Δ133p53α rescues STUB1 knockdown-induced senescence. MRC-5 fibroblasts were transfected with STUB1 siRNA (no. 1) as in e. At the next day after the second transfection (on day 5), the cells were transduced with a lentiviral vector driving Δ133p53α or its vector control. At 5 days after the lentiviral transduction, the cells were stained for SA-β-Gal activity. Representative pictures (left) and quantitative data (right) are shown. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P0.001 (Student’s t-test).
Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence (2014)
f4

(a) STUB1 knockdown-induced repression of Δ133p53α is abrogated by bafilomycin A1 treatment. The siRNA transfection in MRC-5 fibroblasts was performed as in Fig. 3e. At 7 days after the initial transfection, the cells were untreated, treated with bafilomycin A1 (100 nM for 4 h) or treated with MG-132 (15 μM for 4 h), and examined in immunoblots as indicated. The results of Δ133p53α, full-length p53 and p62/SQSTM1 in untreated cells replicate those shown in Fig. 3c. The inhibition of autophagy by bafilomycin A1 was confirmed by increased levels of p62/SQSTM1 and LC3B. The inhibition of proteasomal degradation by MG-132 was confirmed by increased full-length p53. (b) STUB1 knockdown-induced repression of Δ133p53α is abrogated by knockdown of pro-autophagic proteins. MRC-5 fibroblasts (PDLs 30) were transfected for 4 days with control siRNA (−) or STUB1 siRNA (no. 1, +), in combination with control siRNA (c), p62/SQSTM1 siRNA (62), ATG5 siRNA (A5, no. 1 in Fig. 2a), ATG7 siRNA (A7, no. 1 in Fig. 2b) or Beclin-1 siRNA (B1, no. 2 in Fig. 2c), and examined in immunoblots for indicated proteins. (c) Δ133p53α is recruited to autophagosomes upon STUB1 downregulation. MRC-5 fibroblasts at early passage (PDLs 30; Y), those at or close to replicative senescence (PDLs 58; S (Rep)) and those induced to senesce by STUB1 knockdown (S (KD)) were used in immunoprecipitation (IP) with anti-STUB1, anti-Hsp70 and anti-LC3B antibodies, followed by immunoblots (IB) as indicated. Whole-protein lysates without IP (input) were also examined for Δ133p53α and β-actin levels. (d) Bafilomycin A1 stabilizes LC3B-interacting Δ133p53α in senescent cells. The same set of cells as in c were treated with bafilomycin A1 (+, 100 nM for 4 h) or untreated (−). These cells were used in IP with anti-LC3B antibody, followed by IB with anti-Δ133p53 antibody. Whole-protein lysates without IP were also examined in IB with anti-LC3B antibody. While untreated samples replicate the result shown in c, the differences between early passage and senescent cells become more evident with bafilomycin A1 treatment. (e) Schematic model of the regulation of Δ133p53α degradation by STUB1.
Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence (2014)
f5

(a) Δ133p53α protein is polyubiquitinated. FLAG-tagged versions of wild-type Δ133p53α (wt) and mutant Δ133p53α (mut; from c below), along with vector control (−), were retrovirally transduced into MRC-5 fibroblasts. The wild-type Δ133p53α-expressing cells had a pair of STUB1 siRNA-transfected (+, siRNA no. 1 for 4 days) and untransfected (−) counterparts. These cells were maintained for 8 h under amino acid- and serum-starved conditions with bafilomycin A1 (100 nM). Protein lysates were used either in IP with anti-ubiquitin (Ub) antibody, followed by IB with anti-FLAG antibody (top), or directly in IB for FLAG-Δ133p53α, STUB1 and β-actin (lower three panels). Smear signals indicate polyubiquitinated FLAG-Δ133p53α protein (Poly-Ub). Asterisk indicates a nonspecific band in negative control. These experimental conditions resulted in similar amounts of FLAG-Δ133p53α in the presence and absence of STUB1 knockdown, allowing quantitative analysis of ubiquitination. The relative densitometric values of wild-type FLAG-Δ133p53α polyubiquitination in the presence and absence of STUB1 knockdown (normalized with total FLAG-Δ133p53α) are shown. (b) Δ133p53α is ubiquitinated at the C-terminal lysine residues. MRC-5 fibroblasts expressing wild-type FLAG-Δ133p53α in the absence of STUB1 knockdown were used in IP with anti-FLAG antibody, and the resulting immunoprecipitates were analysed using mass spectrometry. Representative MS/MS spectrum of an [M+3H]3+ peptide from FLAG-Δ133p53α shows sites of ubiquitin modification. The peptide sequence for the spectrum is shown, with the sites of modification marked as asterisks: amino-acid positions 248 and 249, which correspond to positions 381 and 382 in full-length p53. The same peptide was also observed in the unmodified state. (c) Lysine (K)-to-arginine (R) substitutions at amino-acid residues 248 and 249. (d) Mutation of the ubiquitination sites renders Δ133p53α resistant to degradation. The same three cells as in a were cultured under amino-acid- and serum-starved conditions for 8 h (without bafilomycin A1), transfected with STUB1 siRNA (no. 1 for 4 days) or untreated, and examined in IBs for FLAG-Δ133p53α, STUB1, p62/SQSTM1 and β-actin. The effect of starvation was confirmed by decreased p62/SQSTM1 in each cell. The upregulation of p62/SQSTM1 by wild-type and mutant FLAG-Δ133p53α is likely associated with enhanced cell proliferation by Δ133p53α overexpression.
Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence (2014)
f6

(a) Δ133p53α is colocalized with p62/SQSTM1 upon autophagy induction. MRC-5 fibroblasts expressing FLAG-Δ133p53α (wild-type) were cultured under amino-acid- and serum-starved conditions for 4 h, and co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-p62/SQSTM1 antibody (red). Two representative sets of images, including nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and merged images, are shown. Scale bars, 10 μm. (b) Knockdown of p62/SQSTM1 stabilizes endogenous Δ133p53α. MRC-5 fibroblasts at late passage (PDLs 51) were transfected with p62/SQSTM1 siRNA (p62) or control siRNA (C) for 3 days, and examined in immunoblots for Δ133p53α and p62/SQSTM1 expression. The relative expression levels of Δ133p53α (normalized with β-actin) are shown. (c) Δ133p53α is colocalized with LC3B upon autophagy induction. MRC-5 fibroblasts with FLAG-Δ133p53α under starved conditions (as in a) were co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-LC3B antibody (red). Two representative sets of images, including DAPI and merged images, are shown. Scale bars, 10 μm. (d) Quantitative analysis of Δ133p53α-LC3B colocalization. The number of colocalized signals out of total number of Δ133p53α signals or out of total number of LC3B signals was counted in 26 cells. Data (percent colocalization) are mean±s.d. Almost all Δ133p53α signals were colocalized with LC3B, while ~80% of LC3B signals were colocalized with Δ133p53α.
A secreted factor NimrodB4 promotes the elimination of apoptotic corpses
by phagocytes in Drosophila (2021)
Figure 1

Figure 1. NimB4 is enriched in phagocytes.

(A) RT-qPCR analysis of NimB1, NimB2, NimB3, NimB4 and NimB5 transcripts, normalized to RpL32, from carcass, macrophages, fat body and gut of wild type (w1118) L3 wandering larvae. Data are represented as mean ± SD from three independent experiments.

(B) Representative confocal imaging of NimB4-sGFP macrophages of embryo at stage 16; lateral (left) and ventral (right) views; Tissues were stained with anti-GFP (green, corresponding to NimB4-sGFP) and anti-SIMU antibodies (red, corresponding to macrophages). The arrows show the presence of NimB4-sGFP inside the embryonic macrophages. Scale bars = 20 μm

(C) Representative live confocal imaging of NimB4-sGFP macrophages of L3 wandering larvae at 30 and 60 min after dissection. Scale bars = 10 μm.

(D) Representative confocal imaging of NimB4-sGFP macrophages of L3 wandering larvae after fixation. Cells were stained with phalloidin (gray), DAPI (blue) and anti-GFP (green, corresponding to NimB4-sGFP). Scale bars = 10 μm.
A secreted factor NimrodB4 promotes the elimination of apoptotic corpses
by phagocytes in Drosophila (2021)
Figure 2

Figure 2. NimB4 binds to apoptotic cells ex vivo

(A-C) Representative confocal imaging of CFSE-stained apoptotic bodies (green) incubated with secreted NimB4-RFP (B, C) or SPvkg-RFP (A). Apoptotic bodies were pre-incubated in absence (A, B) or presence (C) of annexin V (25 μg/mL). Scale bar = 10 μm.

(D) Quantification of the colocalization of NimB4-RFP or SPvkg-RFP with the apoptotic bodies in presence or absence of Annexin V, as measured by Pearson's correlation coefficient. Values from at least four independent experiments are represented as mean ± SD (** P<0.01 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).
A secreted factor NimrodB4 promotes the elimination of apoptotic corpses
by phagocytes in Drosophila (2021)
Figure 3

Figure 3. The NimB4sk2 mutant is defective in cell corpse clearance.

(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and apoptosis-null NimB4 sk2; Df(3L) H99 embryonic macrophages at stage 16 of development; ventral and lateral view; Tissues were co-stained with Dcp-1 (red, corresponding to apoptotic corpses) and anti-SIMU (green, corresponding to macrophages) antibodies. The arrows show the presence of apoptotic cells inside embryonic macrophages. Scale bars = 20 μm

(B) Quantification of caspase-positive particles (anti Dcp-1, red) within macrophages (anti-SIMU, green). Values from at least five independent experiments are represented as mean ± SD total volume of caspase-positive particles (** P<0.01, by Mann-Whitney test).

(C) Representative projections from confocal stacks of entire third instar wild type (w1118) and NimB4 sk2 larval brains stained with anti-Draper (green) and anti-Dcp1 (red). The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain. Scale bars = 100 μm

(D) Quantification of caspase-positive particles in the central brain area of larval brain. Values from at least five independent experiments are represented as mean ± SD total volume of caspase-positive particles (** P<0.01, by Mann-Whitney test).