EMBO/soda-nel huggingface dataset:
KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction (2014)
Figure 6

The disruptive activity of KSHV‐TK is dependent on FAK and paxillin

Immunoblot analysis reveals that GFP‐KSHV‐TK WT, Y2F and Y3F but not GFP‐KSHV‐TK‐DEAD associate with FAK.
GFP‐KSHV‐TK does not displace paxillin or zyxin from focal adhesions (white arrowheads) or induce contraction in the absence of FAK. Scale bar, 20 μm.
GFP‐KSHV‐TK does not induce loss of zyxin and p‐Tyr from focal adhesions or contraction of HeLa cells depleted of paxillin. Error bars represent SEM from three independent experiments, n = 196 for mock siRNA and n = 229 for paxillin siRNA; ***P 0.001. Scale bar, 20 μm.

Source data are available online for this figure.
KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction (2014)
Figure 7

Phosphorylated tyrosine 65/85 interact with Crk family members

A schematic representation of the Crk SH2 consensus binding motifs in KSHV‐TK.
Immunoblot analysis of GFP antibody immunoprecipitates reveals that GFP‐KSHV‐TK but not Y2F or GFP associates with CrkI, II and L.
Immunofluorescence analysis reveals CrkII associates with GFP‐KSHV‐TK but not the Y2F mutant. Scale bars, 20 μm.
Immunoblot analysis of RFP antibody immunoprecipitates reveals that RFP‐KSHV‐TK specifically associates with the SH2 domain of CrkII and CrkL.
Immunofluorescence analysis of phosphotyrosine epitopes in cells expressing GFP‐CrkII SH2, GFP‐CrkL SH2, RFP‐KSHV‐TK or a combination of CrkII/LSH2 and RFP‐KSHV‐TK. Arrowheads represent contracted KSHV‐TK‐expressing cells. The SH2 domains of CrkII or L block KSHV‐TK‐induced cell rounding. Scale bars, 20 μm.

Source data are available online for this figure.
KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction (2014)
Figure 8

KSHV‐TK associates with the p85 regulatory subunit of PI3‐Kinase

A schematic representation of the p85 regulatory subunit SH2 consensus binding motif of PI3‐Kinase in KSHV‐TK.
Immunoblot analysis reveals that GFP‐KSHV‐TK WT and Y2F but not Y120F associate with p85 of PI3‐Kinase.
Immunoblot analysis of p85 PI3‐Kinase antibody immunoprecipitates reveals that GFP‐KSHV‐TK and its Y2F mutant but not Y120F associate with p85 of PI3‐Kinase.
Immunoblot analysis of CrkII and CrkL antibody immunoprecipitates reveals that GFP‐KSHV‐TK and the p85 PI3‐Kinase associate with CrkII and CrkL. No association is observed with the Y2F, Y3F or kinase‐dead TK.
Tyrosine phosphorylation of CrkII and CrkL is only upregulated in cells expressing GFP‐KSHV‐TK.
Immunoblot analysis reveals that recombinant His‐CrkII binds to and is phosphorylated by KSHV‐TK but not the Y2F or Y3F mutants.

Source data are available online for this figure.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 1

Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP-TgAtg8 cell lines were separated by SDS-PAGE and analysed by Western blot using anti-TgAtg8 or anti-GFP antibodies. Overexpressed GFP-TgAtg8 and native TgAtg8 are indicated by arrows.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 2

T. gondii tachyzoites, assessed by the GFP-TgAtg8 marker.

A. Autophagic vesicles marker GFP-TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites. B. Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP-TgAtg8 signals were assessed. Data are mean from n = 4 independent experiments ±SEM.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 3

A. Extracellular tachyzoites were starved for 8 hours in HBSS medium and observed (2, 3). Unstarved control is shown for comparison (1). Several vacuoles (V) can be seen in starved parasites, with also cytosol- and organelle-sequestrating membranous structures resembling autophagosomes (Ap), as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). 2′, 3′ and 3" pictures are a magnification of squared regions. Scale bar = 1 µm in initial micrographs (1, 2, 3) and 0.2 µm in magnifications. B. Partial co-localisation of GFP-TgAtg8-labeled autophagosomes and Toxoplasma mitochondrion. Extracellular tachyzoites expressing GFP-TgAtg8 were incubated in complete medium with Mitotracker Red to label the mitochondrion; partial co-localisation could be observed in some parasites displaying autophagic vesicles. C. Identification of autophagic vesicles in extracellular tachyzoites by immuno-electron microscopy. Extracellular tachyzoites expressing GFP-TgAtg8 were starved for 8 hours in HBSS medium, fixed, cryosectioned and stained with an anti-GFP antibody, then with gold-conjugated secondary antibody and observed. Gold particles can be seen around cytosol-sequestrating membranous structures resembling autophagosomes (Ap), as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). Scale bar = 200 nm.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 4

A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control. B. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFP. C. The HSP fraction described in B. was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS-PAGE. Only a treatment with a detergent (DOC) resulted in GFP-TgAtg8 solubilisation from the HSP fraction. D. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn).
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 5

A. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM. C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 6

A. Intracellular tachyzoites expressing GFP-TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development. Data are mean from n = 5 independent experiments ±SEM. B. Fluorescence microscopy pictures of GFP-TgAtg8-expressing tachyzoites during their intracellular development and division. The arrowhead indicates the residual body made of maternal material left after endodyogeny. C. Detection of GFP-TgAtg8-labelled autophagosomes in dividing parasites. Progression of daughter cells division was followed by expression of an IMC1-RFP construct.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 7

TgAtg3 conditional knock out mutant.

A. Schematic representation of the strategy. Recombineered cosmid bearing chloramphenicol acetyl transferase (CAT) gene for selection of resistant parasites, flanked by homologous regions from TgAtg3, was used to replace endogenous genomic locus in the cell line expressing a regulatable extra copy of TgAtg3 (imyc-sTgAtg3). Mutants obtained after double homologous recombination events were selected with chloramphenicol. PCR amplifications and Southern blot strategy used for subsequent characterisation of the mutant are indicated in red and green, respectively. B. PCR verification of the removal of TgAtg3 in the mutant (imyc-sTgAtg3-ΔAtg3, A) compared to the parental cell line (imyc-sTgAtg3, B). C. Southern blot verification of the TgAtg3 gene replacement. ∼4 µg of genomic DNA from mutant (A) and parental (B) tachyzoites were digested by HindIII/AvrII, transferred on a nylon membrane and probed with 32P-labeled 5′ and 3′ DNA probes located as schematised in A.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 8

A. The vector for the expression of the ATc-regulatable sTgAtg3 extra copy allows myc-epitope tagging of this protein. Anti-myc detection of myc-tagged TgAtg3 protein in intracellular parasites either without treatment with ATc, or with a continuous 2 days treatment with 1.5 µg/ml ATc, or after reinvasion following a continuous 4 days treatment. DIC: differential interference contrast. B. Western blot detection of the myc-tagged sTgAtg3 protein (arrowed) in parasite lysates corresponding to the induction conditions described in A. Anti-SAG1 antibody was used as a loading control. C. Western blot analysis following urea SDS-PAGE of TgAtg3-depleted parasites extracts, showing an absence of upregulation of the autophasome-bound TgAtg8-PE form. Anti-ROP5 was used as a loading control.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 9

A. Confluent monolayers of fibroblasts were infected either with extra copy-expressing imyc-sTgAtg3 parental cell line in the presence of ATc (a), imyc-sTgAtg3-ΔAtg3 mutant cell line without ATc (b), mutant cell line in the presence of ATc (c), or mutant cell line pre-incubated with ATc before start of the experiment and maintained in the presence of ATc. Plaques (arrowheads) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed. B. Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3-depleted parasites (a, b, c: legend as in A). Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines (* p<0.005, Student"s T test). Data are mean from n = 3 independent experiments ±SEM. C. Numbers of parasites per vacuole are significantly lower in TgAtg3-depleted cell line (pre-incubated for 4 days with ATc) compared with controls 24 or 48 hours post invasion. Data are mean from n = 3 independent experiments ±SEM.
Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. (2011)
Figure 10

Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively). Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body. TgAtg3 depletion was verified by detecting myc-tagged regulatable extra-copy with specific antibody. TgAtg3-expressing cell line imyc-sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology (bottom). DNA was labelled with DAPI.
Genome-wide phenotypic analysis of growth, cell morphogenesis and cell cycle events in Escherichia coli (2018)
Figure 6

Figure 6. Nucleoid separation and cell constriction dynamics.

A. Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation. The gray scale indicates the density of dots in a given area of the chart. The dotted contours represent the 0.5, 0.75 and 0.95 probability contours of the 240 WT replicates. The Pearson correlation is ρ = 0.65, with a 95% CI = [0.63, 0.66]. The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time. Red dots highlight strains shown in panels C, D and E.

B. Close-up view of the cell cycle island 8 with each dot representing a Keio strain colored according to the two features driving the clustering of the 48 strains. The relative timing of nucleoid separation is the dominant feature of island 8, while the relative timing of cell constriction drives the layout of the strains within the island.

C. Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain. The cumulative distributions of the fraction of cells with two nucleoids (blue) and of the fraction of cells with a constriction degree above 0.15 (red) were plotted against cell age. Cell age was calculated according to the rank of each cell based on their cell length with the formula $\text{age}_{i}\left( F \right) = \ - \frac{ln(1 - \frac{F}{2})}{ln(2)}$, where F represents the fraction of cells with a cell length equal or below the length of cell i (Wold et al, 1994).

D. Same plots as in C for three strains clustering in island 8 with the ΔmatP strain. The WT curves shown in C were plotted in gray for comparison.

E. Same plots as in C for two island-8 strains, ΔycjV and ΔhslU, and the ΔhslV strain, which does not partition in island 8.
Genome-wide phenotypic analysis of growth, cell morphogenesis and cell cycle events in Escherichia coli (2018)
Figure 7

Figure 7. Cell length regulation mutants.

A. Scatter plot of the mean cell length versus the CV of the length for all the strains. The gray color levels indicate the density of points in the vicinity of each strain. The orange dots and error bars represent the mean and standard error of the mean per bin.

B. The cumulative distribution of the proportion of constricting cells for the ftsA* mutant and its parent were plotted against cell age. We measured the degree of constriction of all cells using Oufti. For each strain, we had two independently acquired image sets (n = 496 and 1,168 cells for the WT replicates and n = 692 and 1,506 cells for the ftsA* replicates). The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p-value of 0.01 using a Kruskal-Wallis multi-comparison test. Moreover, Bonferroni-corrected post-hoc pairwise tests did not allow a distinction between WT and ftsA* samples.

C. Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only. The contour lines represent the 0.5, 075 and 0.95 probability envelopes of the 240 independent WT replicates. The gray color levels indicate the density of points in the vicinity of each strain. The ∆mraZ strain discussed in the text is highlighted in red.
YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth (2022)
Figure 1

Figure 1. Hippo pathway inhibition or YAP activation impedes AR+ PCa growth in vitro

(A-D) Relative growth of (top) or western blot analysis of YAP in (bottom) the indicated prostate cancer cell lines expressing control, YAP-WT, or YAP-5SA constructs through lentiviral infection. Of note, LNCaP and C4-2 express full-length AR while 22RV1 and R1-D567 express AR splicing variants V7 and V567ES, respectively.

(E-H) Relative growth of the indicated prostate cancer cell lines treated with XMU-MP-1 (1 μM for C4-2; 2 μM for LNCaP and 22RV1; 5 μM for R1-D567) or 5 μM Enzalutamide.

(I) C4-2 cells treated with vehicle or 2 μM XMU-MP-1 were immunostained with DAPI anti-YAP antibody. Insets are enlarged views of cells indicated by the arrows. Scale bars are 100 μm.

(J) Western blot analysis of YAP in cytoplasmic and nuclear fractions of C4-2 cells treated with XMU-MP-1 at the indicated concentrations. Tubulin and Histone 3(H3) mark the cytoplasmic and nuclear fractions, respectively.

(K) Western blot analysis of YAP in (top) or relative growth of (bottom) C4-2 cells treated with two independent YAP siRNAs (siYAP_1 and siYAP_2) or control siRNA (siNC),

(L) Relative growth of C4-2 cells treated with YAP or control siRNA and with vehicle or 1 μM XMU-MP-1.

(M) Western blot analysis of LATS1, LATS2, YAP, phosphorylated YAP at S109 (pS109) and S127 (pS127) in C4-2 cells treated with two independent LATS1/2 siRNAs.

(N) Relative growth of C4-2 cells with control or the indicated LATS1/2 siRNA.

(O, P) Western blot analysis of YAP, phosphorylated YAP at S109 (pS109) and S127 (pS127) in (O) or relative growth of (P) C4-2 cells treated with 5 μM TRULI.

(Q, R) Western blot analysis of LATS1, LATS2, and YAP in (Q) or relative growth of (R) C4-2 cells treated with the indicated siRNA.

(S, T) Relative growth of LNCaP cells grown on hormone depletion medium without (S) or with (T) DHT adding back and without (Lenti-NC) or with YAP-5SA expression.

Data information: Results in A-H, K, L, N, P, R-T are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).
YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth (2022)
Figure 2

Figure 2. Hippo pathway blockage or YAP activation inhibits AR transcriptional program

A-D (A, C) Top 10 signaling pathways downregulated in C4-2 cells treated with vehicle or 3 μM XMU-MP-1 for 6 hours (A) or expressing Dox-induced YAP-5SA for 24 hours. Threshold P<0.001 and fold change>2. n=3. (B, D) Volcano plot shows the opposite effects of XMU-MP-1 treatment or YAP-5SA on androgen responsive signature genes (blue) and the Hippo pathway signature genes (red) in C4-2 cells treated with 3 μM XMU-MP-1 for 6 hours (B) or expressing Dox-induced YAP-5SA for 24 hours (D).Threshold P<0.05 and fold change>1.5.

(E) Relative mRNA levels of AR and the indicated AR target genes (left) and western blot analysis of AR (right) in C4-2 cells treated with increasing concentrations of XMU-MP-1.

(F) Relative mRNA levels of the indicated AR target genes in LNCaP cells grown in the absence or presence 10 nM DHT and treated with vehicle, 2 μM XMU-MP-1, or 20μM Enzalutamide.

(G) Relative mRNA levels of the indicated AR or YAP target genes in C4-2 cells treated with control (siNC) or YAP siRNA (siYAP_1 or siYAP_2) for 24 hours and with or without 2 μM XMU-MP-1 for 12 hours.

(H) Relative mRNA levels the indicated AR target genes in C4-2 cells treated with control (siNC) or LATS1/2 siRNA (siLATS1/2_1 or siLATS1/2_2).

(I) Relative mRNA levels the indicated AR or YAP target genes in C4-2 cells treated with the indicated siRNA.

(J-M) Relative mRNA levels of the indicated AR or YAP target genes (J, L) or western blot analysis of AR and YAP (K, M) in C4-2 (J, K) or 22RV1 (L, M) cells expressing control (NC), YAP-WT or YAP-5SA constructs through lentiviral infection.

Data information: Results in E-J, and L are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).
YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth (2022)
Figure 3

Figure 3. MST1/2 inhibition or YAP overexpression inhibits AR binding to its target promoters/enhancers

(A) Western blot analysis of AR (left) and quantification of AR (right) in C4-2 cells treated CHX for the indicated time.

(B) Western blot analysis of AR in C4-2 cells treated with DMSO or 2 μM XMU-MP-1 in the absence of presence of 20 nM MG-132 for 8 hours.

(C) Relative mRNA levels of the indicated AR target genes in C4-2 cells treated with DMSO or 2 μM XMU-MP-1 in the absence or presence of 20 nM MG-132 for 8 hours.

(D) ChIP-qPCR analysis of AR binding to the promoter/enhancer regions of the indicated AR target genes in C4-2 cells treated with vehicle or 2 μM XMU-MP-1 for 4 hours.

(E) ChIP-qPCR analysis of AR binding to the promoter/enhancer regions of the indicated AR target genes in C4-2 cells treated with vehicle or 2 μM XMU-MP-1 in the absence of presence of 20 nM MG-132 or 8 hours.

(F-H) Western blot analysis of AR ( top; F) and quantification (bottom; F), relative mRNA levels of the indicated AR target genes (G) and ChIP-qPCR analysis of AR binding to the promoter/enhancer regions of the indicated AR target genes (H) in C4-2 cells treated with 2 μM XMU-MP-1 for the indicated periods of time.

(I-N) Western blot analysis of AR and YAP (I, L), relative mRNA levels of the indicated AR target genes (J, M) and ChIP-qPCR analysis of AR binding to the promoter/enhancer regions of the indicated AR target genes (K, N) in C4-2 cells stably expressing Tet-O-YAP-5SA (I-K) or Tet-O-YAP-5SAS94A and treated with 0.2 μg/ml doxycycline (Dox) for the indicated periods of time.

Data information: Results in C-E, G-H, J-K and M-N are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).