EMBO/soda-nel huggingface dataset:
Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling (2020)
Figure 6

Figure 6. Extracellular Islr attenuates epithelial Hippo signaling to activate Yap signaling in vitro.

(A) Schematics of culture epithelial cells with supernatant from HEK293FT cells transfected with pcDNA3.1 empty vector or pcDNA3.1-hISLR-HA plasmids. Western blotting for HA in the supernatant from HEK293FT cells transfected with the indicated plasmids.

(B) Gene set enrichment for YAP/TAZ/TEAD direct target genes in transcriptome profiles for HEK293FT cells treated by hISLR-HA-enriched supernatant.

(C) Heatmap showing the altered Hippo related genes in the transcriptome profiles. The parameter of the color key indicated the fold changes converted to log2 of gene fragments per kilobase per million mapped reads (FPKM) values.

(D) Western blotting for pMST1/2, MST1, pMOB1 and MOB1, pYAP1 and YAP1 in lysates from HEK293FT cells cultured in the supernatants with or without hISLR-HA. β-actin was used as a loading control.

(E) Western blotting for HA and ISLR in the supernatant from NCM460 colonic epithelial cells with pcDNA3.1 empty vector or pcDNA3.1-hISLR-HA plasmids. Western blotting for pMST1/2, MST1, pMOB1 and MOB1, pYAP1 and YAP1 in lysates from NCM460 CRC cells cultured in the supernatants with or without hISLR-HA. β-actin was used as a loading control.

(F) Immunofluorescence for YAP1 in HEK293FT cells cultured in the supernatant with or without hISLR-HA. Scale bar: 10 μm.

(G) Western blotting for YAP1 in cytoplasmic or nuclear proteins from HEK293FT cells cultured in the supernatant with or without hISLR-HA. Lamin a/c, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins. (H) qRT-PCR for CTGF and CYR61 in HEK293FT cells cultured in the supernatant with or without hISLR-HA. n=3.

Data information: In (H), data are presented as mean ± SD. ***P < 0.001 (Student's t-test).
Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling (2020)
Figure 7

Figure 7. Extracellular Islr activates Yap signaling in vivo.

(A) Western blotting for Islr in the supernatant from primary cultured intestinal epithelial cells (IECs) transfected with pcDNA3.1 or pcDNA3.1-mIslr-HA plasmids, and western blotting for pMst1/2, Mst1, pMob1 and Mob1, pYap1 and Yap1 in lysates from primary IECs cultured in the supernatants with or without mIslr-HA. β-actin was used as a loading control.

(B) Western blotting for Yap1 in nuclear or cytoplasmic proteins from primary intestinal epithelial cells cultured in the supernatant with or without mIslr. Histone H3, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins.

(C) qRT-PCR for Ctgf, Cry61 and Fstl1 in primary intestinal epithelial cells cultured in the supernatant with or without mIslr for 24 hours. n=4.

(D) Activity of TEAD-Luciferase reporter in primary intestinal epithelial cells upon mIslr, Yap1 or mIslr and Yap1. n=4.

(E) The growth curve of primary intestinal epithelial cells upon mIslr or/and Yap1 siRNA over time. n=5.

(F) qRT-PCR for Ctgf and Cry61 in primary intestinal epithelial cells 48 hours after treatments of mIslr or/and Yap1 siRNA (siYap1). n=4.

(G) Representative images of primary intestinal organoids cultured at indicated conditions and timepoints. Scale bar: 100 μm. The organoids were co-cultured in a mixed medium of the supernatant with/without mIslr from CT26 cells and regular organoid culture solution at the ratio of 1:4. Quantification of organoids diameter at indicated timepoints. n>150 organoids at each timepoint.

(H) Western blotting for pMst1/2, Mst1, pMob1 and Mob1, pYap1 and Yap1 24 hours after the treatment. β-actin was used as a loading control.

(I) qRT-PCR for Ctgf, Cry61 and Fstl1 in primary intestinal organoids 24 hours after the treatments. n=3.

(J) Immunofluorescence for Yap1 and β-catenin in intestinal organoids 24 hours after the indicated treatments. The treatments are identical to (G). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm. n=12.

Data information: In (C-G, I and J), data are presented as mean ± SD. **P < 0.01; ***P < 0.001 (Student's t-test).
Architecture of the yeast Elongator complex (2016)
Figure 1

Figure 1 EM reconstruction of endogenous holoElongator.

A. SDS-PAGE gel showing the purified holoElongator complex used as input for gel filtration. Protein bands marked with asterisks presumably correspond to different phosphorylation states of Elp1 that have been previously described [34].

B. Representative negative stain-EM field of the holoElongator complex. Particles in side and tilted views are highlighted. Scale bar, 50 nm.

C. Reference-free class averages and back-projections of the holoElongator model. Scale bar, 20 nm.

D. EM reconstruction of the holoElongator complex at 31 Å resolution.

E. Fitting of the Elp456 crystal structure into the ring density of the holoElongator reconstruction.

F. Fitting of the Elp1 CTD into the holoElongator reconstruction.
Architecture of the yeast Elongator complex (2016)
Figure 2

Figure 2 EM reconstruction of endogenous Elp123 sub-complex.

A. SDS-PAGE gel showing the purified Elp123 sub-complex used as input for gel filtration. Protein bands marked with asterisks presumably correspond to different phosphorylation states of Elp1.

B. Representative negative stain-EM field of the Elp123 sub-complex. Particles in side and top views are highlighted. Scale bar, 50 nm.

C. Reference-free class averages and back-projections of the Elp123 model. Scale bar, 20 nm.

D. EM reconstruction of the Elp123 sub-complex at 27 Å resolution.

E. Fitting of the Elp1 CTD into the Elp123 reconstruction.

F. Fitting of the Elp2 crystal structure in the difference density generated by subtracting the partial Elp123 reconstruction from the complete Elp123 reconstruction.
Architecture of the yeast Elongator complex (2016)
Figure 4

Figure 4 Integrative models of the Elp123 sub-complex and the holoElongator complex. Models are shown in ribbon representation fitted to the corresponding EM maps. The lysine–lysine crosslinks are mapped on the models. The crosslinks are colored blue if they are satisfied (Cα–Cα atoms of the linked positions less than 30 Å apart) or red if violated. The crosslink visualization was created with the Xlink Analyzer plugin for UCSF Chimera [77].

A. The model of the Elp123 sub-complex.

B. Crosslinks supporting the orientation of Elp3 (left) and Elp2 (right). The crosslinks are shown separately to highlight crosslinks linking from Elp3 and Elp2 to other subunits, respectively.

C. The model of holoElongator.

D. Crosslinks supporting the orientation of the Elp456 hexamer.
Architecture of the yeast Elongator complex (2016)
Figure 5

Figure 5 Validation of the Elp123 and holoElongator models.

A. GST-pull down assays of purified GST-tagged Elp3 SAM domain (aa72-389) and KAT domain (aa348-557) with untagged full length Elp1 and Elp451-2706. GSH resin and proteins were used as input controls. Lower panel shows 5% of the input and upper panel shows bound fractions. All samples were analyzed by SDS-PAGE and stained by Coomassie. Identities of respective proteins are indicated on the right.

B. same as A, but using only GST-tagged Elp3 SAM domain with combinations and variants of Elp1, Elp2, and Elp456.

C. same as A using GST-tagged full length Elp1 and truncations and deletions of Elp456.

D. same as A using GST-tagged C-terminally truncated Elp1 in combination with truncated versions of Elp4.
Architecture of the yeast Elongator complex (2016)
Figure 6

Figure 6 Model of Elongator’s tRNA modification reaction.

A. Overview of holoElongator and individual domains important for the interaction of Elp123 with Elp456. Elp1 (orange), Elp2 (yellow), Elp3 (pink), Elp4 (green), Elp5 (blue) and Elp6 (sand) are shown in surface representation to indicate the topological nature of our model. Individual domains are labeled.

B. Same as A but tilted view to highlight the active site cavity (left). Close up view of the active site in which all known tRNA binding sites and a region of Elp2 important for tRNA modification activity are highlighted. Individual structures of the subunits are shown in cartoon and surface representation.

C. Close up view on the localization of Elp3 and the orientation of its KAT and SAM domain in relation to Elp1 and Elp2.

D. Close up view on the relative spatial orientation of the conserved basic patch in Elp2 and the potential interaction site between Elp456 and a region of Elp2 important for tRNA modification activity.
RASSF1A Controls Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma. (2019)
Figure 1

Figure 1. RASSF1A suppresses metastasis in lung adenocarcinoma.

Kaplan-Meier curves for overall survival (OS) in lung adenocarcinoma TCGA_LUAD (RASSF1 mRNA high/low cutoff FKPM 5.85) and squamous cell carcinoma patients TCGA_LUSC (RASSF1 mRNA high/low cutoff FKPM 6.52). Significance derived from log-rank test.

Western blot with indicated antibodies of isogenic H1299 cells stably transfected with either empty vector pcDNA3 (H1299control) or RASSF1A (H1299RASSF1A). Bottom: cell proliferation resazurin assay. (n=2). Error bars represent mean ± S.E.M.

Cartoon of lung adenocarcinoma orthotopic injection, with sites of primary and metastatic tumours indicated (ipsil = ipsilateral, same lung as primary tumour; contra = contralateral in the opposite lung). Mice were euthanized to collect the lungs at day 17 or day 30 after inoculation with tumours cells into the left lung.

Size of lung primary tumours measured at day 30 after lung orthotopic injection either with H1299control (n=14 mice per group; experimental groups of 6 and 8 different shading within the groups represents two independent experiments) or with H1299RASSF1A cells stably re-expressing RASSF1A, (n=12 mice per group; experimental groups of 6 and 6). The size of primary tumours was measured by MRI software ITK-SNAP. Statistical significance via 2-tailed Student's t-test. Error bars represent mean ± S.E.M.

Visual assessment quantification of metastatic events on the ipsilateral (left) and or contralateral (right) lungs, generated by H1299control or H1299RASSF1A at day 30. Result was calculated from 2 independent in vivo experiments (as in D). Graph shows significant decreasing of metastases when lungs were injected with H1299RASSF1A. Statistical significance via 2-tailed Student's t-test. Error bars represent mean ± S.E.M.
RASSF1A Controls Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma. (2019)
Figure 2

Figure 2. RASSF1A methylation is associated increased P4HA2 and collagen I deposition.

Mass-spectrometry analysis of proteins purified from extracellular matrix isolated from H1299control and H1299RASSF1A lung adenocarcinoma cell lines with summary of results. Only proteins identified with two or more peptides were taken into consideration. The resulting list of proteins was restricted to ECM proteins (n=3).

Kaplan Meier plots depicting prognosis in TCGA_LUAD (lung adenocarcinoma) with high and low mRNA expression of ECM proteins revealed by proteomics. Significance derived from log-rank test.

Representative images of H&E and IHC staining for P4HA2, and quantification (bars) of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm.

Hydroxyproline assay (Cell biolabs Inc.) for total hydroxyproline activity of cells.

Western blot indicating levels of Collagen I in H1299control and H1299RASSF1A cells.

Western blot indicating levels of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination.

Quantification of immunofluorescence staining of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination. H1299RASSF1A cell line was used as negative control for comparison. Representative images in EV2C.

Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HOP92 cells after siRNA against RASSF1A. Scale bars 20µm.

Western blot analyses in HOP92 cells showing increased expression of collagen I after siRNA against RASSF1A.

Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HeLa cells after siRNA against RASSF1A. Scale bars 20µm.

Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.
RASSF1A Controls Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma. (2019)
Figure 3

Figure 3. Cells lacking RASSF1A alter ECM and are more invasive.

Invasion of H1299control and H1299RASSF1A cells through three-dimensional collagen matrix coated inserts, over 24 hours (n=3). Scale bars 100µm.

Representative images and quantification of H1299 cells treated with siNT, siRNA against P4HA2 or in the presence of the prolylhydroxylase inhibitor DPCA (4μM), and allowed to invade for 24 hours through a three-dimensional Matrigel matrix Boyden chamber. Scale bars 100µm.

Representative images and quantification of HOP92 cells stably transfected with shcontrol or shRASSF1A and allowed to invade for 24 hours through a three-dimensional Matrigel matrix Boyden chamber. Scale bars 100µm.

3D collagen contraction assay: 5x105/ml cells were embedded into collagen rat tail I matrix (2mg/ml) and analysed after 4 days. White circles indicates diameter of gel plugs at time 0. Representative bright-field images (bottom) with quantification, showing the effect of H1299RASSF1A or H1299control cells on collagen gel remodelling and after cells pre-treatment with siRNA against P4HA2, 4μM DPCA or combination as indicated.

Upper image: Cartoon of hanging drops method for spheroids formation and embedding collagen rat tail I matrix (2mg/ml). Bottom: Representative immunofluorescence images of YAP distribution and its nuclear quantification (right bars) by Pearson's coefficient for correlation of YAP and DAPI co-staining in H1299control and H1299RASSF1A 3D spheroids grown in 3D collagen matrix (2mg/ml). Scale bars 10µm.

Relative mRNA expression levels of P4HA2 and YAP target genes CYR61 and CTGF in H1299control cells in presence of siNT or siRNA targeting YAP1.

Representative immunofluorescence images of YAP distribution (right) and its nuclear quantification by Pearson's coefficient for correlation (left) of YAP and DAPI co-staining in H1299control and H1299RASSF1A plated on extracted ECM from H1299control or H1299RASSF1A cells with or without treatment with siP4HA2 or 4μM DPCA. (Correlation, Pearson's coefficient). (n=3, 300 cells per experiment) Scale bars 10µm.

Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.
RASSF1A Controls Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma. (2019)
Figure 4

Figure 4. RASSF1A expression is associated with disorganized ECM and abrogated stiffness in vitro and in vivo.

Bar graph representing quantification of stiffness measured by atomic force microscopy (Young modulus) of ECM area generated by H1299control or H1299RASSAF1A cells in vitro.

Representative atomic force microscopy (AFM) images showing organization of extracellular matrix generated by H1299control and H1299RASSF1A cells in 3D collagen matrices (2mg/ml).

Bar graph representing quantification of stiffness by atomic force microscopy (Young modulus) in of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours on day 30.

Representative topographic images of stiffness map of H1299control and H1299RASSF1A lung primary tumours provided by AFM.

AFM images of ECM fibre organization within primary lung tumours generated by H1299control and H1299RASSF1A cells on day 30.

Second harmonic generation (SHG) representative Images and Quantification (bars) showing deposition and organization of collagen fibres produced by H1299control and H1299RASSF1A spheroids embedded in collagen I matrix (2mg/ml), treated with siNT, siRNA against P4HA2 or 4μM DPCA to restrict P4HA2 activity. Red arrowheads show highly organized, long collagen fibres. Scale bars 20µm.

SHG images of organization and quantification (bars) of collagen fractions in primary lung tumours and metastases at day 30. Scale bars 10µm.

Representative H&E and quantification (bars) of picrosirius red staining of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm, zoom 20µm.

Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.
RASSF1A Controls Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma. (2019)
Figure 5

Figure 5. ECM stiffness is important for Nanog expression and its nuclear translocation.

Left: Representative images of H1299 cells on 3D collagen wells with defined stiffness with NANOG (green) or DAPI (blue). Scale bars 10µm. Right: Quantification of NANOG:DAPI nuclear co-localization (upper graph) and total NANOG fluorescence intensity (bottom graph). Quantification of nuclear co-localization (n=300cells/experiment) is represented by Pearson's coefficient.

Representative immunofluorescence images show merge of NANOG (green), DAPI (blue) distribution in H1299control and H1299RASSF1A cells cultured on 2D glass. Scale bars 10µm. Right: Quantification of nuclear co-localization (n=200cells/experiment) is represented by Pearson's coefficient.

Representative immunofluorescence images of CD133 in H1299control and H1299RASSF1A cells grown on 3D collagen wells with defined stiffness. Scale bars 10µm. Bottom: Quantification of n=200 cells/experiment.

Representative images of immunofluorescence staining for the pluripotency marker NANOG and β-catenin in three-dimensional spheroids embedded in collagen matrix (2mg/ml). Scale bars 50µm. Quantification in EV4B.

Representative images of H&E and immunohistochemical staining for NANOG and YAP1 in primary lung tumours, (day 17). Scale bars 100µm. Right: Graph bars represent quantification of NANOG and YAP1 staining based on strong (3+, 2+) nuclear intensity (%) for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17.

Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.
RASSF1A Controls Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma. (2019)
Figure 6

Figure 6. YAP regulates Nanog and OCT4 expression via P4HA2 in H1299control cells.

Relative mRNA expression levels of NANOG, OCT4 and SOX2 in H1299control and H1299RASSF1A lung adenocarcinoma cells.

Representative immunofluorescence images of NANOG and YAP in the presence of siNT or siYAP in H1299control and H1299RASSF1A cells embedded and grown in three-dimensional collagen matrix (2mg/ml). Scale bars 10µm.

Western blots of H1299control and H1299RASSF1A lysates for NANOG, SOX2 and OCT4 in the presence of siNT or siYAP.

Relative H1299control mRNA expression levels NANOG, OCT4 and SOX2 in H1299control cells in the presence of siNT or siYAP.

Representative immunofluorescence images of H1299control and H1299RASSF1A spheroids embedded and grown in collagen matrix (2mg/ml) and stained for NANOG (Green) and YAP (Red) with or without 4μM DPCA. Scale bars 10µm.

Western blots of H1299control lysates for NANOG, SOX2 and OCT4 in the presence of siNT, siP4HA2 or 4μM DPCA.

Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.
RASSF1A Controls Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma. (2019)
Figure 7

Figure 7. P4HA2-mediated collagen synthesis attenuates cancer cell differentiation.

Western blot analyses of expression levels of TTF-1 and Mucin 5B in H1299control and H1299RASSF1A cells with band intensity quantification ratio determine by ImageJ analyser.

Representative images of H&E and immunohistochemical staining for lung differentiation markers TTF-1 and Mucin 5B for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17 with quantification (right) of TTF-1 based on nuclear intensity. Scale bars 100µm. Statistical significance was determined by Student's t-test (2-tailed). Error bars represent the mean ± S.E.M

Representative images of immunofluorescence staining of spheroids grown in collagen matrix (2mg/ml) for differentiation markers TTF-1 (Green) and Mucin 5B (Red). Scale bars 50µm, Zoom 10µm.

Bright field images of H1299control and H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 hours when 3D spheroids were seeded on three-dimensional substrate. Scale bars 200µm.
ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major. (2012)
Figure 3

ATG5 puncta in <italic>L. major</italic> promastigotes.

(A) The occurrence of mC-ATG5 puncta in WT L. major promastigotes expressing mC-ATG5 incubated in nutrient-rich medium at log phase at 26°C. (B) The multiplicity of puncta in promastigotes expressing mC-ATG5 after starvation for up to 1 h in PBS at 26°C. The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken. (C) Co-labelling of puncta with mC-ATG5 and GFP-ATG12 co-expressed in promastigotes incubated in PBS for 2 h at 26°C. (D) In nutrient-rich medium, GFP-ATG8 punctum without mC-ATG5 staining is arrowed. (E) Incubated in PBS for 30 min at 26°C. (F) In nutrient-rich medium at 26°C. (G) Incubated in PBS for 30 min at 26°C, the small panels are enlargements of the merged panels. (H–I) Promastigotes in nutrient-rich medium. (J–K) Promastigotes at late log phase in nutrient-rich conditions. (L) Promastigotes at stationary phase in nutrient-rich conditions. Scale bar throughout, 10 µm.
ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major. (2012)
Figure 4

Phenotypic characterisation of Δ<italic>atg5</italic>.

(A) Growth curve of Leishmania promastigotes in HOMEM medium at 26°C. *, Δatg5 differed significantly from WT (p<0.05). (B) The occurrence of GFP-ATG8 puncta in promastigotes after incubation in nutrient-deprived (PBS, ND) and nutrient-rich (HOMEM medium, NR) conditions for 2 h at 26°C. Scale bar, 10 µm. (C) Occurrence of GFP-ATG8 puncta in promastigotes when incubated in the conditions detailed in (B). Means ± SD from four independent experiments. * and **, occurrence of GFP-ATG8 puncta in Δatg5 were significantly different from in WT in nutrient-deprived and nutrient-rich conditions (p<0.05). (D) Western blot analysis of extracts of promastigotes expressing GFP-ATG8 at logarithmic growth under standard conditions and probed with α-GFP antibody. The faster migrating, lipidated band is labelled GFP-ATG8-II while the un-lipidated band migrating more slowly is labelled GFP-ATG8-I.