EMBO/soda-nel huggingface dataset:
Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor (2019)
Figure 4

Figure 4. STXBP4 functions in the actin cytoskeleton tension-mediated Hippo pathway regulation by forming a complex with α-catenin and a group of Hippo PY motif-containing proteins. (This figure is related to Appendix Figures S6 and S7; Table EV7)

(A) A summary map of cytoscape-generated protein-protein interaction network for STXBP4, α-catenin and a group of Hippo pathway proteins.

(B) STXBP4 forms a protein complex with α-catenin and a group of Hippo pathway proteins.

(C) STXBP4 promotes the association of α-catenin with AMOT and LATS1. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.

(D) Loss of STXBP4 diminishes the association of α-catenin with AMOT, LATS1 and YAP. HEK293A and STXBP4 KO cells were transfected with the SFB-tagged α-catenin construct and subjected to the pulldown assay.

(E) STXBP4 induces the co-localization between α-catenin and AMOT as well as LATS1 and YAP. HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed. Scale bar, 20 μm.

(F-H) Identification of several STXBP4 missense mutations that disrupt its interaction with α-catenin and AMOT. The missense mutations within the STXBP4 α-catenin-binding region and the 9-amino acid sequence of the STXBP4 WW domain were indicated and annotated (F). The identified missense mutations respectively disrupted the STXBP4- α-catenin (G) and STXBP4-AMOT (H) complex formation.

(I) Inhibition of actin cytoskeleton promotes the STXBP4-associated protein complex formation. HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre-immune serum and anti-STXBP4 serum under the indicated treatments.

(J) The missense mutations of STXBP4 (F) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension. YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.
Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor (2019)
Figure 5

Figure 5. STXBP4 is a tumor suppressor in human kidney cancer (This figure is related to Appendix Figure S8).

(A and B) STXBP4 is downregulated in human kidney cancer. The mRNA level of STXBP4 is analyzed in the Firebrowse web database (http://firebrowse.org) (A), where 14,729 tumor sample data generated by TCGA were included. The first quartile, median and third quartile values were indicated as the boxplots. Outliers were plotted as individual points. Error bars indicated the standard deviation above and below the mean of the data. The expression of STXBP4 was also examined using kidney tissue microarray, where percentage of the indicated tissue samples with downregulated STXBP4 was shown (B). The p value was calculated by using the paired Student's t-test.

(C) Kaplan-Meier curves of overall survival of patients with ccRCC is stratified by STXBP4 expression level. Clinical data of STXBP4 were analyzed in TCGA-KIRC project containing total 611 patient samples. The p value was calculated by using the Log-rank (Mantel-Cox) test.

(D) Immunohistochemical staining of STXBP4 and YAP were performed in a kidney cancer tissue microarray, where the indicated regions in the box were shown three times enlarged. Brown staining indicates positive immunoreactivity. Scale bar, 100 μm.

(E) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables. Statistical significance was determined by chi-square test. R, correlation coefficient. N, nuclear localization. C, cytoplasmic localization.

(F) STXBP4 expression is examined in a panel of ccRCC cell lines by Western Blotting.

(G and H) Both the association with α-catenin and the functional WW domain are required for the STXBP4's tumor suppressive function in 786-O cells. Overexpression of STXBP4, but not the indicated STXBP4 missense mutants, significantly suppressed the 786-O cell xenograft tumor formation. Xenograft tumors are shown in (G), and the tumor weight is quantified in (H) (n = 5 mice, mean ± s.d.). ** p < 0.01 (Student's t-test). Scale bar, 1 cm.

Expanded View Figure Legends
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 1

<italic>T. gondii</italic> induces Akt activation via PI3K signaling in non-hematopoietic cells.

A, Primary HBMEC were challenged with type I (RH) or type II (ME49) strains of T. gondii for the indicated time points. Cell lysates were used to probe for total Akt and phospho-Ser473 Akt by immunoblot. B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation. D, HBMEC were challenged with either live or killed T. gondii. Cell lysates were subject to immunoblotting as indicated. E, HBMEC were transiently transfected with a plasmid encoding PH-Akt-GFP. Cells treated with or without LY294002 (20 µM) for 1 h prior to challenge with T. gondii-RFP. Cells were examined by immunofluorescence at 5 min post-challenge to examine accumulation of PH-Akt-GFP around the parasites. F, HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T. gondii. Densitometry data represent means ± SEM of 4 experiments. A vertical line was inserted between densitometry data from control and LY294002-treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. G, HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA. Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T. gondii 48 h after transfection. Cell lysates were subject to immunoblotting as indicated. Densitometry data are shown as above and represent means ± SEM of 3 experiments. Results shown are representative of 3–4 independent experiments.
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 2

Blockade of Akt induces accumulation of the autophagy protein LC3 around the parasite, vacuole-lysosome fusion and killing of <italic>T. gondii</italic> dependent on the autophagy proteins.

A, HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge. B, HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge. C, RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge. D, mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IV followed by challenge with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite. E, HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole. F, Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite G, H, mHEVc cells were transfected with Beclin 1 siRNA (G), Atg7 siRNA (H) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 24 h. I, mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii. 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge. J, Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii. Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 3

Role of G protein coupled receptors and MyD88 in <italic>T. gondii</italic>-induced Akt phosphorylation.

A, HBMEC were treated with or without pertussis toxin (PTx; 100 ng/mL) for 4 h prior to challenge with T. gondii. Cell lysates were obtained at 5, 15 or 30 min post-incubation with T. gondii and used to examine total Akt and phospho-Akt serine 473 by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and PTx-treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. HBMEC were also treated with or without LPA (10 µM) in the presence or absence of PTx. Cell lysates were obtained at 5 min and subjected to immunoblotting. B, HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h. Cell lysates were used to examine total Akt and phospho-serine 473 Akt by immunoblot. Results shown are representative of 3 independent experiments.
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 4

<italic>T. gondii</italic> infection induces EGFR activation in mammalian cells.

A, HBMEC cells were challenged with RH T. gondii and cell lysates were obtained to probe for total EGFR, phospho-tyrosine 1068 EGFR and phospho- tyrosine 1148 EGFR by immunoblot. B, HBMEC were challenged with live vs killed tachyzoites. Total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot. C, D, Human RPE cells (C) and mouse microglia (D) were challenged with RH T. gondii and total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot. Results shown are representative of 3 independent experiments.
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 5

EGFR mediates Akt activation induced by <italic>T. gondii</italic>.

A, mHEVc cells were transfected with control siRNA or EGFR siRNA. Expression of EGFR and actin were assessed by immunoblot 48 h post-transfection. mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. B, Mouse microglia were treated with or without AG1478 (1 µM) 1 h prior to challenge with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478-treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 6

Blockade of EGFR induces accumulation of the autophagy protein LC3 around the parasite, vacuole-lysosome fusion and killing of <italic>T. gondii</italic> dependent on the autophagy proteins.

A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h. B, Human RPE cells transfected with either EGFR or control siRNA were challenged with T. gondii followed by examination of monolayers by light microscopy at 24 h. C, Mouse bone marrow-derived macrophages were incubated with AG1478 and challenged with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h. D, Parental CHO and CHO-EGFR cells were challenged with RH T. gondii. Monolayers were examined microscopically 2 h or 24 h post-challenge. E, mHEVc-LC3-EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite. F, HBMEC cells treated with or without AG1478 were challenged with either T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. The percentages of endothelial cells with LAMP-1 accumulation of around the parasite were determined. G, H, mHEVc cells transfected with Beclin1 siRNA (G) or Atg7 siRNA (H) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T. gondii. Monolayers were examined by light microscopy 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 7

Role of metalloproteinases and G protein coupled receptors in <italic>T. gondii</italic>-induced EGFR phosphorylation.

HBMEC were treated with or without GM6001 (10 µM) for 1 h prior to challenge with T. gondii (A) or with Pertussis Toxin (PTx; 100 ng/ml) for 4 h prior to parasite challenge (B). Cell lysates were used to examine total EGFR and phospho-tyrosine 1068 EGFR by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and GM6001- or PTX-treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 8

<italic>T. gondii</italic> micronemal proteins appear to induce EGFR and Akt activation.

A, B, C HBMEC were challenged with ΔHx (WT), MIC1 ko, MIC3 ko, MIC1-3 ko T. gondii at MOIs that yielded similar percentages of infected cells (A). Cell lysates were obtained and used to examine total EGFR and phospho-tyrosine 1068 EGFR (B) or total Akt and phospho-Ser 473 Akt (C) by immunoblot. HBMEC were incubated with 10 nM of recombinant MIC4, MIC3, M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho-tyrosine 1068 EGFR (D) or total Akt and phospho-Ser473 (E) by immunoblot. Densitometry data represent means ± SEM of 4 experiments. Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells (Un), which were given an arbitrary number of 1. Results shown are representative of 4 independent experiments.
Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. (2013)
Figure 9

EGFR and <italic>T. gondii</italic> micronemal proteins modulate CD40-induced recruitment of LC3 around <italic>T. gondii</italic> and parasite killing.

A, hmCD40 mHEVc expressing LC3-EGFP were treated with or without CD154 followed by challenge with T. gondii-RFP in the presence or absence of EGF (50 ng/ml) or AG1478 (1 µM). LC3 accumulation around T. gondii was assessed by immunofluorescence. B, mHEVc-LC3-EGFP cells were infected with T. gondii-RFP. Cells were treated with vehicle or rapamycin (1 µM) 2 h after challenge with T. gondii in the presence or absent of EGF or AG1478. Recruitment of LC3 around the parasite was examined at 5 h after challenge with T. gondii. C, hmCD40 mHEVc-LC3-EGFP cells were treated with or without CD154 followed by challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko, MIC1-3 ko+MIC1-3 tachyzoites. LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post-challenge. D, hmCD40 mHEVc-LC3-EGFP treated with or without CD154 were challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko or MIC1-3 ko+MIC1-3 tachyzoites. Parasite load was examined 24 h post-challenge. E, HBMEC cells were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites followed by treatment with vehicle or rapamycin. Parasite load was examined 24 h post-challenge. F, HBMEC treated with or without CD154 or IFN-γ/TNF-α were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites. Monolayers were examined microscopically at 24 h post-challenge. G, hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1-3 ko tachyzoites in the presence of complete medium (CM) alone or medium plus MIC3, MIC4, M2AP or MIC6 (all 10 nM). Parasite load was determined microscopically 24 h post challenge. Results shown are representative of 3–4 independent experiments.
Protease protection assays show polypeptide movement into the SecY channel by power-strokes of the SecA ATPase (2020)
Figure 2

Figure 2. Substrate movements by Walker B SecA mutants.

Schematic showing replacement of wild-type SecA in a translocation intermediate by Walker B (WB) SecA mutants (green).

WB SecA with additional mutations (WT, Y794G, R342E) was added to preformed intermediates (IM) and incubated for the indicated times before proteolysis. The gel is representative of two replicates.

Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to WB SecA carrying additional mutations, as shown in the inset box.
Protease protection assays show polypeptide movement into the SecY channel by power-strokes of the SecA ATPase (2020)
Figure 3

Figure 3. The roles of the THF and clamp in substrate movement.

Schematic showing the generation of a translocation intermediate with SecA dN20::H6 (purple) recruited to SecYEG liposomes containing Ni-modified lipids (cyan circles). Addition of imidazole dissociates SecA dN20::H6, which can then be replaced by WT or mutant SecA (tan).

Imidazole (imi) and either WT or mutant SecA were added to translocation intermediates (IM) pre-formed with SecA dN20::H6. The samples were incubated for the indicated times before proteolysis with proteinase K (Prot. K). The gel is representative of two replicates, except for the experiment with the R792G/K797G mutant and the 30-min time points.

Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to different SecA mutants as shown in the inset box.

Schematic of the residues at the tip of the SecA two-helix finger. The conserved tyrosine Y794 is indicated in blue, and the conserved basic residues R792 and K797 are in red.

As in (B), but ATPγS was added together with imidazole. The gel is representative of two replicates.

Quantification of (E) as in (C).

As in (D), but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA. The gel is representative of two replicates.

Quantification of (G) as in (F), except that both the intensities of the IM (darker colors) and FL (lighter colors) bands were measured.

Expanded View Figure Legends
Antithetical NFATc1–Sox2 and p53–miR200 signaling networks govern pancreatic cancer cell plasticity (2014)
Figure 1

Loss of p53 tumor suppressor function and constitutive activation of NFATc1 correlates with highly metastatic and dedifferentiated PDAC

Statistical analysis of NFATc1 expression with respect to tumor grading in KPC mice tissues.
Immunohistochemical detection of NFATc1 and p53, illustrating G1‐G2 and G3 in human PDAC tissues. Scale bars, 100 μm.
Statistical illustration of TMA analysis demonstrating NFATc1 strong nuclear positive tumors (n = 161 patients) with respect to functional status of p53 expression in human PDAC tissues.
Representative H&E‐stained sections as well as immunohistochemical detection of HA‐NFATc1 and p53 in well‐ and poorly differentiated pancreatic tissues of KNC mice. Scale bars, 100 μm.
Quantification of p53 expression with respect to the tumor grading by evaluating both the intensity of immunostaining and the number of cells with overexpression by means of an immunoreactivity score (IRS) in KNC mice (every dot represents one mouse).
Incidence of liver metastases in KNC mice (mean values ± SD).
Antithetical NFATc1–Sox2 and p53–miR200 signaling networks govern pancreatic cancer cell plasticity (2014)
Figure 2

Loss of p53 hastens EMT in NFATc1‐driven pancreatic cancer models

Genome‐wide expression and GSEA analysis show p53‐enriched signatures with “EMT” and “stem cell transcript” in KNC tumor cells.
Heat map showing p53‐dependent regulation of “EMT”‐ and “stemness”‐related genes in KNC tumor cells. Fold change relative to control cells is displayed in a green red color scheme for selected genes with FClog2 1.0 or FClog2 > −1.0.
Statistical analysis of wound healing assay performed in primary KNC tumor cells following p53 depletion. Time‐lapse analyzer software was used to quantify the wound closure rate. Means ± SD are shown from one out of three independent experiments.
Western blot analysis revealing protein expression levels upon p53 depletion in KNC tumor cells.
X‐fold mRNA expression of EMT‐related marker genes after p53 depletion in KNC tumor cells. Means ± SD from at least three independent experiments are shown. Asterisks display significance (*P 0.05).
Representative protein expression of EMT inducers in pancreatic lysates prepared from KNC mice with different tumor gradings.

Source data are available online for this figure.
Antithetical NFATc1–Sox2 and p53–miR200 signaling networks govern pancreatic cancer cell plasticity (2014)
Figure 4

NFATc1 is required for the promotion of EMT in PDAC

Quantitative RT‐PCR analysis of EMT‐related markers obtained from tumors derived from mice of the indicated genotypes (n = 3, means ± SD).
Relative mRNA expression of NFATc1‐ and EMT‐related marker genes on administration of CsA (1 μM) in KPNC cells. Data represents means ± SD from at least three independent experiments. Asterisks show significance (*P 0.05).
Western blot analysis revealing protein expression levels in KPNC cells from the indicated proteins following transfection of siRNA control or siRNA toward NFATc1, respectively.

Source data are available online for this figure.