EMBO/soda-nel huggingface dataset:
Histological Hallmarks and Role of Slug/PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis (2020)
Figure 5

Figure 5: Our animal model of PF-PH exhibits similar vascular wall cell proliferation and Slug expression to PF-PH patients. (A) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC) in the experimental model. (* vs Bleo, ** p<0.01) Relative expression of Slug mRNA (B) and protein (C) normalized to GAPDH. (D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib, vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, vWF) and smooth muscle cells (SMC, aSMA in white); and quantification. (* vs Bleo, p<0.05) (E) CD68 mRNA quantification (F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in in Bleo and Bleo-MCT rats and quantification. The number of samples per group for each experiment are included within each bar graph. Statistical test: panel A, D, E t-test; panel B, C, F : ANOVA (* vs. Ctrl, † vs. PF, ! vs. PH; ** p<0.01, † p<0.05,†† p<0.01 ! P<0.05, !! P<0.01).
Histological Hallmarks and Role of Slug/PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis (2020)
Figure 6

Figure 6: Increased PIP expression in the lungs of PF-PH compared to PF rats. Relative expression of PIP mRNA (A) and protein (B) normalized to GAPDH. (C) Representative images and PIP quantification. The number of samples per group for each experiment are included within each bar graph. (t-test; * p<0.05)
Histological Hallmarks and Role of Slug/PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis (2020)
Figure 7

Figure 7: Slug inhibition reduces PH severity in our rat model of PF-PH. (A) Experimental schematic of the prevention protocol. All rats received Bleomycin at day 0 and MCT at day 14. Starting at day 17, rats received five intratracheal nebulizations (twice a week for three weeks) of either a small interfering RNA (siRNA) directed against Slug (Si-Slug) or scramble siRNA (Si-Scrm). Relative expression of Slug protein (B) and mRNA (C) normalized to GAPDH. (D) right ventricular systolic pressure (RVSP) and (E) right ventricular hypertrophy index in PF-PH rats treated with Si-Slug compared to Si-Scrm. Relative expression of fibronectin (F), Collagen I (G) and Collagen III (H) normalized to GAPDH. (I) Masson's trichrome representative images and quantification of the Ashcroft score. (J) Masson's trichrome representative images and quantification of vascular wall thickness. (K) Masson's trichrome representative images and quantification of the vascular remodeling between non-fibrotic and fibrotic areas of the lung. (L) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white). The number of samples per group for each experiment are included within each bar graph. Statistical test: panel B to J : t-test; panel K and L ANOVA (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).
Histological Hallmarks and Role of Slug/PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis (2020)
Figure 8

Figure 8: Slug inhibition reduces vascular remodeling and proliferation of vascular wall cells. (A) Representative images and quantification of Slug-positive macrophages (slug, red; CD68 green). (B) Relative expression of CD68 mRNA normalized to GAPDH. (C) Representative images and quantification of Slug-positive macrophages between non-fibrotic and fibrotic areas of the lung. Relative expression of PIP mRNA (D) and protein (E) normalized to GAPDH. (F) Representative images and quantification of PIP immunohistochemistry. (G) Proposed mechanism. In pulmonary fibrosis, vascular wall thickening is mainly seen in fibrotic areas of the lung; while in PH secondary to PF, vascular remodeling is seen both in fibrotic and non-fibrotic areas of the lung. The extension of vascular wall thickening to the non-fibrotic areas of the lung in PF-PH is concomitant with an increased expression of Slug and PIP in the lungs in both fibrotic and non-fibrotic areas. At the cellular level, Slug up-regulation in macrophages in PF-PH leads to increased PIP expression in the extra-cellular space, which in turn triggers SMC proliferation leading to vascular wall thickening observed in fibrotic and non-fibrotic areas of the lung. (ECM: Extracellular Matrix; SMC / EC : smooth muscle cells and endothelial cells). The number of samples per group for each experiment is included within each bar graph. Statistical tests: panel A, B and D to F t-test; panel C ANOVA (* p<0.05, ** p<0.01, **** p<0.0001).
Lysosomal degradation of the maternal dorsal determinant Hwa safeguards dorsal body axis formation (2021)
Figure 1

Figure 1 - Hwa degraded through lysosomal pathway.

A Illustration of experiment design. hwa mRNA (100 pg) was injected into oocytes at stage VI. Embryos were treated with 100 nM Baf-A1 or 20 μM MG132 at 1-cell stage and collected at indicated stages.

B Western blot analysis for Hwa performed with lysates from embryos treated as indicated in (A). 1cs, 1-cell stage; 2cs, 2-cell stage; st6, 32-cell stage; st8.5, blastula stage. Blots are representative of 3 independent experiments.

C Densitometry analysis of Hwa bands from the western blot in (B) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, *p < 0.05, **p < 0.01 by unpaired t test.

D Western blot analysis of exogenous Hwa in HEK293T cells. Treatment of cells with lysosome inhibitors bafilomycin A1 (Baf-A1) and NH4Cl for 24 hours, DMSO was used as control. Dosage of Baf-A1: 10 nM, 50 nM, 100 nM; Dosage of NH4Cl: 2.5 mM, 5 mM, 10 mM. Hwa with modification is indicated by white arrow; Degraded Hwa is indicated by black arrow. Blots are representative of 3 independent experiments.

E Densitometry analysis of Hwa bands from the western blot in (D) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, **p < 0.01 by unpaired t test.
Lysosomal degradation of the maternal dorsal determinant Hwa safeguards dorsal body axis formation (2021)
Figure 2

Figure 2 - Hwa interacts with ZNRF3.

A Schematic illustration of Flag pull-down assay. The empty vector (Flag-control) and Hwa-Flag were purified from HEK293T cells with anti-Flag beads and incubated with oocytes homogenate; associated proteins were identified by mass spectrometry.

B Coomassie brilliant blue staining and MS analysis of Hwa-associated proteins after immunoprecipitation (Flag pull-down). Right (outlined text), thirteen matched peptide sequences that correspond to ZNRF3. Hwa-MF, Myc-Flag-tagged Hwa.

C Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments.

D Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-HA were immunoblotted with Flag and HA antibodies. Blots are representative of 3 independent experiments.

E Colocalization of Hwa-Flag (red) and ZNRF3-HA (green) or ZNRF3ΔRING-HA (green) in Hela cells is indicated by yellow dots. Baf-A1, 50 nM, 12 hrs. Magnification: 100 ×; scale bar: 10 μm.

F Schematic diagram of ZNRF3 truncations. ZNRF3FL, full-length ZNRF3; ZNRF3ΔRING, ZNRF3 lacking the RING domain; ZNRF31-320, amino acids 1-320 of ZNRF3; ZNRF3320-606, amino acids 320-606 of ZNRF3; ZNRF3606-854, amino acids 606-854 of ZNRF3.

G Co-IP of HEK293T cells transfected to express Hwa-Flag and vector only or plasmids encoding ZNRF3 truncation mutants as indicated in (F). Lysates immunoprecipitated with anti-HA were analyzed by immunoblotting with Flag and HA antibodies. Blots are representative of 3 independent experiments.
Lysosomal degradation of the maternal dorsal determinant Hwa safeguards dorsal body axis formation (2021)
Figure 3

Figure 3 - Maternal ZNRF3 is essential for the dorsal ventral axis formation in Xenopus.

A qRT-PCR analysis of the efficacies of znrf3 AS normalized to odc. Data are shown as mean ± SEM. n = 3 biological replicates; ****p < 0.0001 by unpaired t test.

B Whole mount in situ hybridization of Spemann organizer markers chrd, gsc, and xnr3 in gastrula embryos (stage 10.5). Oocytes were injected with AS or mRNAs as indicated, Dosage: znrf3 AS (3 ng AS4 + 3 ng AS9), znrf3 mRNA 100 pg, znrf3ΔRING mRNA 50 pg. embryos were obtained through Host transfer technology. Scale bars: 1 mm.

C Representative phenotypes of tailbud stage Xenopus leavis embryos injected with ctrl or znrf3ΔRING mRNAs as indicated. mRNAs were injected in pre-mature oocytes and embryos were obtained through Host transfer technology. Scale bars: 1 mm.

D Western blot analysis of Axin and β-catenin in blastula stage (st7-8) embryos injected with 500 pg znrf3ΔRING or 500 pg hwa at 1-cell stage. Lysates were immunoblotted with Axin, β-catenin, HA, and GAPDH. Blots are representative of 3 independent experiments.

E Relative levels of β-catenin (left) and Axin (right) in (D) normalized to GAPDH. n = 3 independent experiments, mean ± SEM, *p < 0.05, ***p < 0.005, ****p < 0.0001 by unpaired t test.

F Representative phenotypes of Xenopus embryos at tailbud stage. Embryos were injected with ctrl (20 pg gfp mRNA), 20 pg znrf3ΔRing mRNA at one ventral blastomere of 4-cell stage embryos, or 20 ng β-catenin morpholino (β-cat MO) at 1-cell stage as indicated. Duplicated axes (red arrowhead). Scale bars: 1 mm.

G Representative phenotypes of Xenopus laevis embryos at tailbud stage. 20 ng hwa morpholino (hwa MO) was injected in oocytes at stage VI, embryos were obtained through Host transfer technology. znrf3ΔRING mRNA (20 pg) was injected at one ventral blastomere of 4-cell stage embryos. β-gal mRNA 200 pg was co-injected with znrf3ΔRING mRNA as lineage tracer. Duplicated axes (red arrowhead). Scale bars: 1 mm.

H Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos (stage 10.5). Embryos were injected and obtained as descripted in (G). Scale bars: 1 mm.

I Representative phenotypes of tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl (gfp mRNA), znrf3FL, and znrf3 truncations as indicated at 4-cell stage. Duplicated axes (red arrowhead). znrf3FL, full-length znrf3; znrf3ΔRING, znrf3 lacking the RING domain; znrf31-320, amino acids 1-320 of znrf3; znrf3320-606, amino acids 320-606 of znrf3; znrf3606-854, amino acids 606-854 of znrf3. Scale bars: 1 mm.
Lysosomal degradation of the maternal dorsal determinant Hwa safeguards dorsal body axis formation (2021)
Figure 4

Figure 4 - Hwa undergoes multi-monoubiquitination.

A Hwa is ubiquitinated by ZNRF3, but not ZNRF3ΔRING. HEK293T cells were co-transfected with Hwa-Flag and ZNRF3 or ZNRF3ΔRING. The lysates were immunoblotted with Myc, Flag, and α-tubulin antibodies. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments.

B Hwa ubiquitination was analyzed under denaturing conditions. HEK293T cells co-transfected with wildtype His-HA-Ubiquitin (His-HA-Ub WT) or lysine-less mutant (K0), Hwa-Flag, and ZNRF3-Myc. The lysates were immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. K0, all lysine sites on ubiquitin were mutated. Blots are representative of 3 independent experiments.

C Lysine sites (K) in Hwa protein sequence. Three ubiquitination lysine sites (K178, K179, and K234) identified by MS were indicated in red. Other seven lysine sites were indicated in bold.

D Screen of ZNRF3-mediated Hwa ubiquitination sites under denaturing conditions. HEK293T cells were co-transfected with His-HA-Ub, Hwa-Flag or Hwa10KR-Flag, and ZNRF3 or ZNRF3ΔRING. The lysate was immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Hwa10KR-Flag mutant, all lysine sites in Hwa sequence were replaced with arginine. Denaturing conditions: before immunoprecipitation, lysates were denatured by adding SDS to a final concentration at 1% and boiling at 95℃ for 15 min. Blots are representative of 3 independent experiments.
Lysosomal degradation of the maternal dorsal determinant Hwa safeguards dorsal body axis formation (2021)
Figure 5

Figure 5 - ZNRF3 promotes ubiquitin-mediated lysosome trafficking and degradation of Hwa

A Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag, and ZNRF3-HA (5 μg) or ZNRF3ΔRING (2.5 μg). Lysate was immunoblotted with Flag, HA, and GAPDH antibodies. Blots are representative of 3 independent experiments.

B Densitometry analysis of Hwa bands from the western blot in (A) for corresponding lanes normalized to GAPDH. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01 by unpaired t test.

C Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag and ZNRF3-HA. Cells were treated with or without Baf-A1 (50 nM) or MG132 (20 μM) for about 12 hours. Lysates were immunoblotted with Flag, HA, and α-tub (α-tubulin) antibodies. Blots are representative of 3 independent experiments.

D Densitometry analysis of Hwa bands from the western blot in (C) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01, ****p < 0.0001 by unpaired t test.

E Ubiquitination analysis of Hwa with or without Baf-A1 (50 nM, 12 hours). HEK293T cells were co-transfected with Hwa-Flag, and ZNRF3-HA or ZNRF3ΔRING-HA. Lysate was immunoblotted with Myc, Flag, and α-tub (α-tubulin) antibodies. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments.

F Colocalization by immunofluorescence of Hwa (green signals) with lysosome (LAMP1, red signals) in Hela cells. Cells were co-transfected with Hwa-Flag, and ZNRF3-HA or ZNRF3ΔRING-HA. Magnification, 100 ×; scale bars: 10 μm.

G Colocalization analysis of Hwa-Flag and lysosome by ImageJ software. Data are presented as mean ± SEM; n > 20 cells from each group. *p < 0.05, ****p < 0.0001 by unpaired t test.

H Flow chart of surface biotinylation and streptavidin pull-down.

I Surface biotinylation and streptavidin pulldown of Hwa-Myc-Flag. HEK293T cells were co-transfected with Hwa-MF (Hwa-Myc-Flag), and ZNRF3-Myc or ZNRF3ΔRING-Myc. Lysate was immunoblotted with Myc, Flag, and GAPDH antibodies. Lysates immunoprecipitated with anti-Flag (2nd elution) were immunoblotted with Flag and HA antibodies.
Lysosomal degradation of the maternal dorsal determinant Hwa safeguards dorsal body axis formation (2021)
Figure 6

Figure 6 - ZNRF3 prevents the secondary axis-inducing activity of Hwa

A Morphology of classified embryos at tailbud stage. Scale bars: 1mm.

B Ratios of embryos in different classes. Oocytes were injected with 5 pg hwa mRNA or hwa10KR mRNA and 100 pg znrf3 mRNA as indicated. Embryos were obtained through Host transfer technology.

C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos. Embryos were obtained as descripted in (B). Scale bars: 1mm.

D Induction of the secondary (2nd) axis by hwa or znrf3 as indicated in Xenopus embryos. One ventral blastomere of 4-cell stage embryos was injected with hwa mRNA (5 pg) or hwa10KR mRNA (5 pg) and znrf3 mRNA (100 pg). Up: Classes of embryos. Down: Ratio of classified embryos. The 2nd axis (red arrowhead). Scale bars: 1mm.

E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage. 1-cell stage embryos were co-injected with hwa (100 pg) or hwa10KR (100 pg) and znrf3 (1 ng) or znrf3ΔRING (100 pg) as indicated. Lysates were immunoblotted with HA, Flag, and α-tub (α-tubulin).

Expanded View Figure
Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion (2019)
Figure 2

Figure 2. The nucleus is a limiting factor for host cell invasion.

A. Wild Type RH parasites mid-invasion. Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes, fixed with 4% PFA before labelling the TJ with anti-Ron2 (yellow) and DAPI (magenta) for nuclear staining. White arrow points at invasion direction.

B. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. 100 parasites found mid-invasion were counted in triplicate and compared to extracellular, freshly egressed parasites. One-way ANOVA was used for statistical analysis. **** p value <0.0001.

C. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. Indicated parasites were analysed. For adf cKD induced and non-induced conditions were compared, as indicated. 30 parasites were counted for each condition in triplicate. One-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. *** p value 0.0009.

D. Time-lapse microscopy analysis of indicated parasites invading HFFs cells. Time was determined from the onset of invasion. The time-lapse is presented in Movie EV4. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite.

E. Speed profiles of invading parasites shown in D. The relative displacement of the apical tip over time allowed speed calculations. Individual stages were named in regard to the invasion step (Penetration initiation, Speed decrease and finalisation). Invasion was analysed with Icy Image Processing Software (Pasteur Institut) using the Manual Tracking plugin by analysing each movie 10 times and averaging the measurements (for additional speed profiles see Appendix Fig.S2).

F. Time-lapse analysis demonstrating abortive invasion of indicated parasites. Time was determined from the onset of invasion. The time-lapse is presented in Movie EV5. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite.

Data information: Error bars represent standard deviation. Numbers indicate minutes:seconds; and Scale bars represent 5 µm.
Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion (2019)
Figure 3

Figure 3. F-actin dynamics during Toxoplasma gondii invasion during fixed and live imaging.

A. Time-lapse analysis depicting invading RH adfcKD parasites expressing Cb-EmeraldFP. A F-actin ring can be observed at the TJ in the majority of cases (red arrowheads, first panel). Invasion can also occur without formation of an F-actin ring (second panel). During capped invasion, an F-actin ring is formed (third panel). Invasion of adf cKD parasites occurs without formation of an F-actin ring (fourth panel). F-actin accumulation is observed at the posterior pole of the parasite in all cases (orange arrows). Green segmented lines depict the area that was measured for generation of intensity plot profiles shown in (B). Yellow and purple dotted lines indicate the extra- and intracellular part of the parasite respectively. The time-lapse is presented in Movie EV6.

B. Intensity plot profiles of the movies shown in (A). Depicted plots correlate to half-invaded parasites. In the case of F-actin ring formation, two distinct peaks are detected, correlating to the F-actin ring at the TJ (red arrowhead) and the posterior pole of the parasite (orange arrow). In the case of no F-actin ring, no accumulation at the TJ can be observed; only the peak correlating to posterior F-actin can be detected. For adf cKD, only one peak can be seen corresponding to the posterior pole of the parasite.

C. Quantification of F-actin accumulation at the TJ for RH Cb-EmeraldFP parasites as detected in time lapse analysis. The values are expressed as percentage. 28 total parasite invasion events were captured across three different biological replicates. Two-tailed unpaired t-test analysis was performed. **** p value <0.0001.

D. Representative immunofluorescence assays on indicated parasite strains during host cell penetration. Parasites were labelled with indicated antibodies. SAG1 staining was performed prior to permeabilisation to stain only the extracellular portion of the parasite. Antibodies against Ron2 were used to stain the TJ. Red arrowhead: position of the TJ. Orange arrow: posterior pole of the parasite.

E. Tight Junction assay on fixed samples (as shown in (D)) depicting invasion rates of indicated parasites. While in the majority of control parasites F-actin can be detected at the junction (~80%), no junctional ring can be detected in ~20% of all invasion events. In contrast, adf cKD, act1 cKO or myoA KO only show an overall invasion rate of ~20% compared to controls. In these cases junctional F-actin accumulation could never be detected. Note that the number of total invasion events for adf cKD, act1cKO and myoAKO correlates to the number of invasion events, seen for RH without F-actin ring formation.

F. Quantification of posterior F-actin accumulation during invasion of indicated parasite lines.

Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm. One-way ANOVA analysis was performed for each graph in E and F. ****p value <0.0001.
Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion (2019)
Figure 4

Figure 4. Time lapse analysis of F-actin and nuclear dynamics during host cell invasion

A. Time-lapse analysis of invading RH Cb-EmeraldFP parasites. During penetration, an F-actin ring is formed at the TJ (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) and posterior F-actin appears to be directly connected to the nucleus and TJ (blue arrow). The time-lapse is presented in Movie EV7.

B. Analysis of nucleus deformation during live imaging shown in A. The nucleus constricts while its passing through the TJ (red arrowheads).

C. The posterior end of the parasite deforms during the invasion process. F-actin is accumulated during invasion, with the posterior end contracting (blue arrow).

D. Time-lapse stills depicting RH Cb-EmeraldFP parasites invading in absence of detectable F-actin at the junction (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) during invasion. Actin accumulation at the posterior pole of the parasite is strongly detected in all cases (blue arrow). The time-lapse is presented in Movie EV7.

E. Analysis of nucleus deformation during live imaging shown in (D). The nucleus appears to not show constriction once its passing through the TJ when the F-actin ring is not present.

F. The posterior end of the parasite also deforms during the invasion process.

G. Z-stack gallery depicting mid-invading myoA KO parasites. Note that the nucleus is located located at the back during invasion events.

Data information: Numbers indicate minutes:seconds. Scale bar represents 5 µm. White arrow points to direction of invasion.