EMBO/soda-nel huggingface dataset:
Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth (2018)
Figure 1

Figure 1: The Hippo effector Yap is required for optimal liver development.

A) Fluorescent and histological analysis of liver morphology in WT and yap-/- mutant larvae on a lf:CFP background at 5 dpf. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 200μm (bright field, BF) and 50μm (histology, H+E).

B) Quantitative analysis of fluorescent liver area in WT and lf:Yap larvae at 5 dpf. n>30, ****p<0.0001, two-sided Student's t-test; values represent the mean ± standard error of the mean (s.e.m.).
Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth (2018)
Figure 2

Figure 2: Yap regulates hepatoblast formation and expansion.

A) Whole-mount in situ hybridization (WISH) analysis of progenitor marker prox1, hhex, and foxa3 expression in WT and yap-/- mutant embryos at 36 hpf. Arrow and bracket highlight the hepatic bud. Scale bar: 200μm.

B) qPCR analysis of hhex and prox1 gene expression in WT and yap-/- mutant embryos at 48 hpf. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.

C) WISH analysis of prox1, hhex and foxa3 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. Arrow and bracket highlight the hepatic bud. Scale bar: 200μm.

D) Quantitative analysis of hepatic bud area (prox1 staining) in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. n>10, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.
Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth (2018)
Figure 3

Figure 3: Yap deficient adults are small and exhibit liver hypoplasia.

A) Fluorescent and histological analysis of liver morphology in dissected WT and yap-/- mutant adults on a lf:CFP background. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 2mm (brightfield, BF) and 50μm (histology, H+E).

B) Quantitative determination of body mass in WT and yap-/- adults. n=8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.

C) Quantitative determination of liver size in WT and yap-/- adults as determined by measuring liver mass as a percentage of body mass. n=12, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.

D) qPCR analysis of the hhex expression in adult liver tissue isolated from WT and yap-/- fish. n>3, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m.

E) qPCR analysis of the prox1 expression in adult liver tissue isolated from WT and yap-/- mutant fish. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.
Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth (2018)
Figure 4

Figure 4: Transcriptional profiling reveals that Yap regulates the expression of glut1 and glut2.

A) Heatmap of RNAseq analysis illustrating statistically significant differential gene expression between WT and yap-/- mutant larvae at 3 dpf. Blue indicates decreased expression, while red demonstrates increased expression.

B) Heatmap analysis of RNAseq data highlights the reduction of glucose transporters (glut1 and glut2) in yap-/- mutant larvae at 3 dpf.

C) qPCR analysis of glut1 and glut2 expression in WT and yap-/- mutant larvae at 3 dpf. n>3, *p<0.05, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m.

D) qPCR analysis of glut1 and glut2 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and isolated at 30 hpf. n>3, *p<0.05, **p<0.01, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m.

E) qPCR analysis of glut1 and glut2 expression in liver tissue dissected from WT, yap-/- mutant and lf:Yap transgenic adults. n>3, *p<0.05, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m.
Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth (2018)
Figure 5

Figure 5: Yap regulates glucose metabolism and nucleotide biosynthesis.

A) Glucose tolerance test in 3-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.

B) Glucose tolerance test in 12-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.

C) Clustergram analysis of polar metabolite abundance from WT and yap-/- mutant larvae at 3 dpf as determined by LC-MS/MS via selected reaction monitoring (SRM) analysis. n=4, p<0.05, two-sided Student's t-test.

D) Steady state abundance of glycolytic intermediates in WT and yap-/- mutant larvae as determined by SRM analysis. n=4, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m.

E) Steady state abundance of nucleotides in WT and yap-/- mutant larvae as determined by SRM analysis. n=4, *p<0.05, ***p<0.001, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m.

F) Relative isotopic enrichment of 13C in glycolytic intermediates following incubation of WT and yap-/- mutant larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.

G) Relative isotopic enrichment of 13C in nucleotide precursors following incubation of WT and yap-/- larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m.
Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth (2018)
Figure 6

Figure 6: Metabolic intervention impacts Yap-driven growth phenotypes.

A) Effect of nucleoside (NS) cocktail treatment from 1-3 dpf on the incidence of cardiac edema in WT and yap-/- mutant larvae. Each point represents the percentage of affected embryos in each independent clutch. n>7, **p<0.01, two-sided Student's t-test; values represent the mean s.e.m.

B) Effect of WZB-117 (WZB, 10 μM) treatment from 3-5 dpf in WT and lf:Yap larvae on liver size as determined by fluorescence microscopy at 5 dpf in lf:GFP reporters. Scale bar: 200μm.

C) Quantitative analysis of the effect of WZB treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>21, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m.

D) Quantitative analysis of the effect of Trehalose (100 μM) treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>70, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m.
Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth (2018)
Figure 7

Figure 7: The regulation of glut expression and glucose uptake by Yap is evolutionarily conserved.

A) Dot plots showing the correlation between GLUT1 and YAP1 mRNA expression in the cancer cell line encyclopedia (CCLE, 967 lines). Pearson coefficient is 0.46.

B) Dot plots showing the correlation between GLUT1 and AMOTL2 mRNA expression in the cancer cell line encyclopedia (CCLE, 967 lines). Pearson coefficient is 0.45.

C) ChIPseq tracks at the GLUT1 locus highlighting YAP and TEAD enrichment overlaying H3K27Ac marks at an intragenic enhancer element in different contexts (LATS2 mutant MSTO211H cancer cells and HA-YAP5SA overexpressing HuCCT1 liver cancer cells).

D) Gross morphology of the liver from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 1 cm.

E) Immunohistochemical analysis of GLUT1 expression in liver tissue from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 50μm.

F) FDG PET imaging of coronal views from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Arrow is pointing to the liver.

G) Scintillation analysis of FDG uptake (fraction of injected dose per gram of liver tissue, %ID/g) in dissected liver tissue from WT and TetON Yap transgenic mice, treated with doxycycline for 10 days. n=4, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m.
Functional Compensation Between Hematopoietic Stem Cell Clones In Vivo (2018)
Figure 1

Figure 1: Wildtype (WT) HSCs compensate for the lymphopoietic deficiencies of co-transplanted mutant HSCs in blood production.

A We co-transplanted barcoded WT HSCs with competitor WT, B cell deficient (uMT-/-), or B and T cell deficient (NSG or Rag2-/-γc-/-) HSCs into irradiated recipient mice. Peripheral blood was harvested from recipient mice at 6 to 8 months after transplantation and sorted into granulocytes (Gr), B, CD4 T, and CD8 T cells for population and clonal level analyses.

B-C Competitor donor derived Gr, B, CD4 T, and CD8 T cells in the peripheral blood. In the control group, competitor donor cells are WT HSCs. In the deficient group, competitor donor cells are uMT-/- or NSG HSCs.

D-E WT donor derived Gr, B, CD4 T, and CD8 T cells in the peripheral blood.

F-G Total production of Gr, B, CD4 T, and CD8 T cells in the peripheral blood (derived from WT HSCs, competitor HSCs, helper whole bone marrow cells, and residual host cells) shown as percentages of the total number of white blood cells (WBCs).

Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test).
Functional Compensation Between Hematopoietic Stem Cell Clones In Vivo (2018)
Figure 2

Figure 2: Highly expanded HSC clones increase their differentiation in response to the lymphopoietic deficiencies of other HSCs.

A-B Total numbers of barcoded WT clones that give rise to granulocytes (Gr), B cells, CD4 T cells, and CD8 T cells.

C-J Number of HSC clones that produce different amounts of Gr, B, CD4 T, and CD8 T cells. We combined the sequencing data with the flow cytometry data to calculate clonal abundance for each clone as follows: Clonal abundance =100%*(Each cell population (Gr, B, CD4T or CD8T cells) % WBCs) * (Donor % Each cell population) * (GFP % Donor cells) * (number of reads for each barcode) / (total reads of all barcodes)

K-L Production of Gr, B, CD4 T, and CD8 T cells by expanding WT clones that produce more than 0.1% of WBCs in each lineage.

Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test).
Functional Compensation Between Hematopoietic Stem Cell Clones In Vivo (2018)
Figure 4

Figure 4: Compensation for lymphopoietic deficiency is manifested as an increase in cell numbers at the progenitor level.

A-B Total amount of progenitors as a percentage of ckit and Il7rα enriched bone marrow (BM) cells. Shown are hematopoietic stem cells (HSCs), Flk2- and Flk2+ multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), megakaryocyte-erythroid progenitors (MEP), and granulocyte-monocyte progenitors (GMP).

C-D WT donor derived progenitors as a percentage of ckit and Il7rα enriched BM cells.

Data information: Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test).
Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome (2012)
figf1

(a) Western blot analysis of myosin-VI-depleted HeLa cells untreated or treated with 100 nM bafilomycin A1. (b) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01, ***P0.001 (c) Confocal immunofluorescence microscopy of LC3 punctae (green) in HeLa cells following knockdown of myosin VI. Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images (d) Quantification of LC3- and p62-positive punctae was performed and the results are represented as the average punctae fluorescence intensity per cell (±s.d.; n = 3) from >1,500 cells per experiment. (e) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA-resistant GFP–myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132. (f) Western blot analysis of MEFs cultured from wild-type and Snell’s waltzer (SV) mice treated with 1 μM MG132 or 100 nM bafilomycin A1. (g) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01. (h) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control. (±s.d.; n = 3). (i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1. (j) Quantification of western blot LC3-II intensity was performed (n = 2). (k) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control (n = 2). (l) Western blot (top) and confocal immunofluorescence microscopy (bottom) of mock- or myosin-VI-siRNA-treated HeLa cells stably expressing the RFP–GFP–LC3 reporter. Hoechst labels the nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock- or myosin-VI-siRNA-treated HeLa cells expressing RFP–GFP–LC3. The data are represented as the Pearson’s coefficient of the RFP and GFP signal correlation from >100cells per experiment (±s.d.; n = 3). (n) Confocal immunofluorescence microscopy of myosin-VI-depleted RPE cells stably expressing GFP–LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.
Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome (2012)
figf2

(a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy following 16 h MG132 treatment (zero time point) and 8 h post-washout of inhibitor. Immunolabelling for p62 was performed to visualize aggregates (red) and Hoechst was used to identify nuclei (blue). (b) Top, western blot analysis confirming siRNA depletion. Bottom, quantification of immunofluorescent p62-positive aggregates was evaluated using an automated Cellomics VTi microscope. The results were calculated as the average p62 fluorescence intensity at 8 h post-MG132 washout normalized to the zero time point and are represented as the percentage of the control (±s.d.; n = 3). *P0.05, ***P0.001. See Methods and Supplementary Table S1 for details of the SMARTpool (SP) and single (07) oligonucleotides. (c) Parental HeLa cells or HeLa cells stable expressing siRNA-resistant GFP–myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI and were subsequently treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy at 16 h post-MG132 treatment (t = 0) or allowed to recover following washout of MG132 for 8 h (t = 8). Cells were processed for quantification with the automated Cellomics VTi microscope to evaluate the p62 fluorescence intensity. The results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared with mock control cells following recovery from MG132 washout (t = 8; ±s.d.; n = 3). (d) HeLa cells with stable expression of HttQ72–GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy. Immunolabelling was performed for GFP (green) and p62 (red). Nuclei are labelled with Hoechst (blue). (e) Quantification of HttQ72–GFP aggregates was performed on myosin-VI-siRNA-transfected HeLa cells. The results were calculated as the percentage of GFP-expressing cells with greater than 15 GFP-positive spots per cell. The results represent the mean (±s.d.) from n = 3independent experiments, ***P0.001. The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.
Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome (2012)
figf3

(a) RPE cells transiently co-transfected with GFP–myosin VI and cherry–LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 h (bottom row) were processed for immunofluorescence microscopy. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images. (b) RPE cells treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 co-localization. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images. (c) Image stills acquired from spinning-disc time-lapse video microscopy of RPE cells transiently co-transfected with cherry–LC3 and GFP–myosin VI. The outlined regions highlight areas of interest within the cell that are shown to the right (i–iii) as video image stills captured at 1-min intervals. The arrowheads highlight myosin-VI-positive vesicles (green) coming into contact and co-localizing with LC3-positive vesicles (red). (d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry–LC3 following transient transfection with GFP–myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualized with phalloidin (white). Nuclei were labelled with Hoechst (blue). The arrowheads indicate areas of actin-rich myosin-VI/LC3-positive autophagosomes. Scale bars, 20 μm (a,b,d); 10 μm (c, whole-cell image); 2 μm (c, cropped images).
Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome (2012)
figf4

(a) Cartoon illustration of myosin VI domain structure. Myosin VI contains a catalytic motor domain, a unique insert (green), an IQ calmodulin-binding motif and a cargo-binding tail domain. The cargo-binding tail domain contains multiple protein interaction motifs (RRL and WWY) as well as regions for ubiquitin and PIP2 binding. In addition, two alternative splicing events give rise to two insertions in the cargo-binding tail region. (b) RPE cells with stable expression of cherry–LC3 were transiently transfected with the GFP–myosin VI cargo-binding tail domain containing various mutations in the protein-interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 h to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction. The arrowheads indicate areas of co-localization. Scale bar, 20 μm. (c) A Pearson’s coefficient was calculated on the basis of the degree of co-localization between the different GFP–myosin VI mutant tails and cherry–LC3 from confocal immunofluorescence micrographs. The graph represents data from more than 20 transfected cells from n = 2 independent experiments.
Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome (2012)
figf5

(a) Left, immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h. Immunolabelling was performed against the indicated endogenous proteins optineurin, T6BP and NDP52 (red), and LC3 (green). The insets show higher magnifications of the areas outlined in the main images. Right, from the confocal images, a Pearson’s coefficient was calculated to estimate the degree of co-localization of the different autophagy adaptors with LC3. As a negative control, a Pearson’s coefficient was calculated after rotation of one colour image by 90°. The results represent >20 cells from n = 2 independent experiments. (b) Left, stable cherry–LC3-expressing RPE cells were transiently transfected with GFP–myosin VI, treated with Torin1 for 3 h to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of co-localization are white in the merged three-colour images and the insets show higher magnifications of the areas outlined in the main images. Right, a Pearson’s coefficient was calculated for the degree of co-localization between the three colours. As a negative control, a Pearson’s coefficient was calculated after rotating one colour image by 90°. Results represent >10 cells from n = 2 independent experiments. (c) HeLa cells were either mock transfected or co-transfected with siRNA targeted against TNO. Cells were either left untreated or treated with100 nM bafilomycin A1 (BfnA1) for 4 h. Western blot analysis was performed against the indicated proteins. (d) Quantification of LC3-II intensity (±s.d.) was performed from western blot data of TNO-siRNA treated HeLa cells. The results represent n = 3independent experiments. **P0.01, ***P0.001 (e) Immunofluorescence microscopy of RFP–GFP–LC3-expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. The results are calculated from >100 cells from n = 2 independent experiments represented as the Pearson’s coefficient of GFP/RFP signal overlap. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.