EMBO/soda-nel huggingface dataset:
E-cadherin mediates apical membrane initiation site localisation during de novo polarisation of epithelial cavities (2022)
Figure 1

Figure 1 - Cell division is dispensable for AMIS localisation in mESC 3D cultures in Matrigel.

A Stages of polarisation and lumen formation in mESCs cultured in Matrigel.

B Timeline of experiment setups to assess AMIS formation.

C Immunofluorescence of PAR-3 and E-cadherin. PAR-3 localisation was concentrated at the centre of 2-cell mESC doublets and 3- or 4-cell mESC clusters, whilst E-cadherin was localised along the whole length of cell-cell interfaces in both control and mitomycin C treated conditions.

D Quantification of the frequency of cell clusters with a strong polarised PAR-3 centre. See the representative mESC clusters without the strong polarised PAR-3 in Appendix Fig S1A,B.

E Illustrations of PAR-3 analysis in 2-cell (a) and >2-cell (b) clusters. The line-scan analysis was performed from the yellow arrow to the orange arrow at the cell-cell interface between two cells in (a). The average pixel intensity was analysed at central (red region, inner dotted line) and surrounding non-central regions (orange region, between the inner and outer dotted lines) in (b).

F Line-scan profiles of PAR-3 at the cell-cell interface of 2-cell mESC doublets. Line scans were sectioned and fitted to each 5% along the cell-cell interface length.

G Ratio of PAR-3 pixel intensity values at central and surrounding regions in >2-cell mESC clusters.

H, I Representative images of centralised ZO-1 puncta and polarised Golgi apparatus, labelled by GM130 (H) and quantification of the frequency of cell doublets with a polarised ZO-1 or Golgi apparatus centre (I). Also see the spilt channels in Appendix Fig S1C.

J, K Representative images of polarised centrosomes (J), labelled with γ-tubulin and distance between centrosomes (K) normalised to the length of the long axis of the doublets in 2-cell clusters.

Data information: Data are presented as means ± SEM in (D) & (I); individual line-scans and mean valued line-scans in (F); individual cell cluster values (small dots), mean experimental values (large dots) and means of 3 experiments (bars) in (G); individual values in dots and median values in bars in (K). n = 3 experiments in (D) & (I), 15-32 cell clusters were analysed for each column in every experiment; 15 cell doublets for each condition from one experiment in (F); 15-22 cell clusters for each condition in every experiment in (G); 35 doublets for each condition in (K). Two-way ANOVA analysis in (D); student's t-test analysis in (G), (I), (K). P values were listed in the graphs. All scale bars: 10 µm.
E-cadherin mediates apical membrane initiation site localisation during de novo polarisation of epithelial cavities (2022)
Figure 2

Figure 2 - Polarised PAR6B in mESC 3D cultures.

A Representative images of mCherry-PAR-6B mESC live cells cultured from 24 - 96 hours in Matrigel. See the bight-filed images in Appendix Fig S2A.

B, C Representative images (B) and quantification of the frequency of cell clusters with polarised mCherry-PAR-6B (C) in control and mitomycin division-blocked mESC live cells after 24 hours in Matrigel.

D Movie stills of mCherry-PAR-6B in control and mitomycin C treated mESCs cultured in Matrigel (also Movie EV2). Control cells divided (i) and two mitomycin treated cells touched (ii) to form 2-cell doublets. Control cells divided twice (iii) and two mitomycin treated cell clusters touched (iv) to form 4-cell clusters. (v), Kymograph of mCherry-PAR6B in the arrow-head cell in (ii) along the path between the arrows; each pixel is the fluorescence values averaged over 0.2 µm sections. See more examples in Appendix Fig S2D,E.

Data information: Data in (C) are presented as means ± SEM. n = 3 experiments. At least 20 clusters were analysed for each column in every experiment. Two-way ANOVA analysis; P values were listed in the graphs. All scale bars: 10 µm.
E-cadherin mediates apical membrane initiation site localisation during de novo polarisation of epithelial cavities (2022)
Figure 3

Figure 3 - E-cadherin junctions are important for polarisation.

A, B Immunofluorescence of PAR-3 (A) and proportions of mESC clusters with a strong PAR-3 centre (B) in wild-type (W4) and E-cadherin knock-out (Cdh1 KO) mESCs at 24 hrs in Matrigel. See E-cadherin in Appendix Fig S3A.

C Line-scan profiles of PAR-3 at the cell-cell interface in wild-type control, mitomycin C treated and Cdh1 KO control, mitomycin C treated 2-cell doublets.

D Ratio of PAR-3 pixel intensity values at central and surrounding regions in >2-cell mESC clusters.

E, F Representative images of PAR-3 immunofluorescence (E) and line-scan profiles of PAR-3 at the cell-cell interface (F) in WT homogeneous or WT (ES-E14) /Cdh1 KO chimeric mESC 2-cell doublets. *, WT mESCs.

G, H Representative images of ZO-1 puncta and Golgi apparatus (G) and proportions of mESC doublets with central ZO-1 puncta or polarised Golgi apparatus (H) in WT and Cdh1 KO mESC doublets.

I Distance between centrosomes in cell doublets. The distance was normalised to the length of the long axis of the doubles. See the representative images in Appendix Fig S3D.

J Fluorescence recovery after photobleaching (FRAP) of E-cadherin-eGFP at the centre-most or side 2 µm regions of division-blocked mESC doublets' cell-cell interfaces. See methods in Fig EV2C.

K Mobile fraction of E-cadherin-eGFP calculated from the plotting of (J).

Data information: Data are presented as means ± SEM in (B), (H); individual and mean-valued line-scans in (C) & (F); individual cell cluster values (small dots), mean experimental values (large dots) and means of 3 experiments (bars) ± SEM in (D); individual values in dots and median values in bars in (I); exponential association fitting curves ± SD in (J); means ± SD in (K). n = 3 experiments in (B) & (H), 17-50 cell clusters were analysed for each column in every experiment; 15 doublets for each condition in (C); 17-30 cell clusters were analysed for each condition in every experiment in (D); 15 doublets for each condition in (F); 35-40 doublets in each condition in (I); 15-18 doublets for each condition in (J) & (K). Two-way ANOVA analysis in (B), (D), (I) & (K); student's t-test analysis in (H); P values were listed in the graphs. All scale bars: 10 µm.
E-cadherin mediates apical membrane initiation site localisation during de novo polarisation of epithelial cavities (2022)
Figure 4

Figure 4 - Adhesion molecule P-cadherin, JAM-A and Nectin-2 do not aid PAR-3 in the AMIS.

A-C Immunofluorescence of PAR-3 and P-cadherin (A), proportions of mESC clusters with a positive PAR-3 centre (B) and line-scans of PAR-3 at the cell-cell interface (C) in control, P-cadherin knock-down by RNAi, mitomycin treated and P-cadherin knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel.

D-F Immunofluorescence of PAR-3 and JAM-A (D), proportions of mESC clusters with a positive PAR-3 centre (E) and line-scans of PAR-3 at the cell-cell interface (F) in control, JAM-A knock-down by RNAi, mitomycin treated and JAM-A knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel.

G-I Immunofluorescence of PAR-3 and Nectin-2 (G), proportions of mESC clusters with a positive PAR-3 centre (H) and line-scans of PAR-3 at the cell-cell interface (I) in control, Nectin-2 knock-down by RNAi, mitomycin treated and Nectin-2 knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel.

Data information: Data are presented as means ± SEM in (B), (E), (H); individual and mean-valued line-scans in (C), (F), (I). n = 3 experiments in (B), (E), (H), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (C), (F), (I). Two-way ANOVA analysis in (B), (E), (H); P values were listed in the graphs. All scale bars: 5 µm.
E-cadherin mediates apical membrane initiation site localisation during de novo polarisation of epithelial cavities (2022)
Figure 5

Figure 5 - Cell-ECM interactions in regulating AMIS seeding.

A, B Immunofluorescence of PAR-3 (A) and proportions of mESC doublets with polarised PAR-3 (B) in wild-type (ES-E14) and E-cadherin knock-out (Cdh1 KO) cells at 30 hours in 0.5% agarose.

C Immunofluorescence of PAR-3 in cell division blocked mESCs cultured on E-cadherin or Fibronectin coated glass topped with or without Matrigel for 24 hours.

D Heatmap of PAR-3 in cells from one experiment of (C). Squared frames were fitted to the main bodies of the cells. A pixel was the average value in each 10% along the width of the frames. The heatmaps were stacks of 15 cells in one experiment.

E PAR-3 levels at the central 20% of the heatmaps from three experiments of (C).

F Ratios between PAR-3 in the 2.5 µm diameter central core and whole cell surface from three experiments of (C).

Data information: Data are presented as means ± SEM in (B); individual cell values (small dots), mean experimental values (large dots) and means of experiments ± SEM (bars) in (E) & (F). n = 3 experiments in (B), (E), (F); at least 15 clusters were analysed for each column in every experiment; 15 - 20 cells for each column in every experiment of (E) & (F). Two-way ANOVA analysis in (B), (E), (F); P values were listed in the graphs. All scale bars: 5 µm.
E-cadherin mediates apical membrane initiation site localisation during de novo polarisation of epithelial cavities (2022)
Figure 6

Figure 6 - Lumenogenesis in wild-type and E-cadherin knock-out mESC cultures.

A PAR-3 immunofluorescence in wild-type (ES-E14) and E-cadherin knock-out (Cdh1 KO) mESCs cultured in Matrigel for 48 and 72 hours. At 48 hours, most WT cell clusters had formed a polarised rosette or small lumen (i). However, most Cdh1 KO cells had not formed a central AMIS (ii). A small percentage of Cdh1 KO cells had formed an open 'cup-shape', with apically localised PAR-3 (iii). By 72 hours, a significant proportion of Cdh1 KO cells had formed polarised 'cavity-like' structures, about half of which were configured in an open 'cup-shape' and half as closed 'lumen-like' structures (iv).

B Podocalyxin immunofluorescence in WT and Cdh1 KO mESCs cultured in Matrigel from 1-4 days. See Fig EV5A for ZO-1 staining.

C Examples of masked Cdh1 KO cell cluster surfaces from 'open' and 'closed' cavities. The cavities were categorised based on Podocalyxin signals.

D Percentage of cell clusters with different cavities relative to total cell clusters at different time points. The analysis was compared between the closed/rosette category among the conditions.

E Percentage of cell clusters with closed cavities relative to total cell clusters with cavities calculated from (D).

F Movies stills of LifeAct-mRuby labelled cell clusters. The images are whole cell cluster z-projections, overlaid with 3D rendering of the cluster surfaces to show the forming lumens. See Movie EV3 for z-stack movies and Movie EV4 for 3D rotations.

Data information: Data are presented as means ± SEM in (D) & (E). n = 20 (48 hours WT & Cdh1 KO), 30 (72 hours WT) and 32 (72 hours Cdh1 KO) images in (A); 3 experiments in (D) & (E), at least 25 clusters were analysed for each column in every experiment. Two-way ANOVA analysis in (D) & (E); P values were listed in the graphs. All scale bars: 25 µm.
Inhibiting MARSs reduces hyperhomocysteinemia-associated neural tube and congenital heart defects (2019)
Figure 1

Figure 1. MARS expression levels were increased in samples with gains in copy numbers and developing/proliferating cells

A, B MARS (A) and MARS2 (B) mRNA expression levels were measured by real-time PCR in blood samples with 2 and 3-4 copies. Relative expression levels were normalized to GAPDH levels, and data were quantified relative to the mRNA expression levels of 2 copy samples.

C, D Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3-4 copies. Protein-level intensity shown in (D) was quantified using ImageJ and divided by the protein-level intensity of actin. The results were then graphed relative to samples with 2 copies.

E, F MARS2 protein expression levels were detected (E) and quantified (F). Actin was used as a loading control.

G Cell proliferation was assessed by Ki67 (red) immunofluorescent staining in mice neural tube sections at E10. MARS (green) was co-stained. Scale bar: 150 μm.

H Double immunofluorescent staining for MARS (green) and Ki67 (red) in developing heart sections at E10. Scale bar: 150 μm.

I MARS expression was detected in E80 rhesus monkey brain sections (n=3) by IHC. Local large magnification is shown as framed. Red arrow indicates germinar layer, blue indicates immature neurons. Scale bar: 1mm.

J, K MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. Each D/C lane pair was quantified relative to D as shown at the bottom. The mean MARS levels in all D and C groups were quantified by ImageJ and shown in (K) (n=6).

L MARS2 mRNA expression in embryonic chorion (C) and maternal deciduae (D) were detected by qPCR (n=6).

M MARS protein quantification in the brains of foetal rats (F) and their corresponding mothers (M) from experiment (O) (n=6).

N MARS mRNA levels (n=6) in the brains of foetal rats (F) and their corresponding mothers (M) were determined by qPCR (n=6).

O MARS protein levels in foetal rats (F) and their corresponding mothers (M) were determined by western blot. Each M/F lane pair was quantified relative to M as shown at the bottom.

P Representative result of MARS expression was detected by IHC in tumor tissues (T) and adjacent noncancer tissues (N) of UCC samples. Scale bar: 50 μm.

Q Ki67 and MARS expression was analysed by IHC in HCC samples. Scale bar: 20 μm.

Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test was used for A, B, D, and F. Wilcoxon matched pairs test was used for K, L, M and N.
Inhibiting MARSs reduces hyperhomocysteinemia-associated neural tube and congenital heart defects (2019)
Figure 2

Figure 2. MARSs potentiate Hcy-induced apoptosis and growth inhibition

A Schematic representation of MARS functions.

B Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1-Flag-Bcl-2 plasmids (n=3). 5μg plasmids were transfected in each 1×106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved caspase 3 (c-caspase3) levels were detected to determine apoptosis levels (right).

C, D Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)-, and MARS2 (D)-over-expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n=4).

E Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n=4).

F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n=4).

G Metabolic characterization of untreated and MARS-over-expressing NE4C cells based on pathway analysis of significantly changed metabolites. Columns show significantly enriched pathways, and yellow columns indicate oxidative stress-associated pathways.

Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.
Inhibiting MARSs reduces hyperhomocysteinemia-associated neural tube and congenital heart defects (2019)
Figure 3

Figure 3. MARS/MARS2 results of ROS accumulation by inducing N-Hcy

A, B HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector, MARS (A), or MARS2 (B) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.

C MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by western blot. HTL and superoxide levels were quantified (n=4) relative to vector-transfected NE4C cells. Cells were cultured in 20 μM Hcy-containing media.

D Relative superoxide levels were determined by DHE staining assay and compared (n=4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy-containing media. MARS2 knockdown efficiencies were confirmed by western blot.

E NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n=4).

F The cellular ROS (superoxide) levels were determined (n=4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells.

G Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS-expressing and control cells were determined relative to the untreated NE4C cells.

H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N-Hcy levels in HTL-treated and control cells were detected by western blot.

I Protein N-Hcy levels in response to MARS knockdown (by shRNA) and over-expression were detected by western blot.

J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and the N-Hcy (J) and superoxide (K) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.
Inhibiting MARSs reduces hyperhomocysteinemia-associated neural tube and congenital heart defects (2019)
Figure 4

Figure 4. HTL inactivates SOD1 and SOD2 through N-Hcy

A Compartments of N-Hcy substrates identified by proteomic survey were predicted by WoLF PSORT.

B WebLogo visualization of the amino acids spanning N-Hcy-modified lysine sites.

C SOD activities were down-regulated by in vitro N-homocysteinylation. Flag-tagged SOD1, SOD1K23W,K122W,K128W (3KW) mutant, SOD2, and SOD2K44W, K51W,K98W,K106W,K178W (5KW) mutant were expressed in HEK293T cells, affinity purified, and incubated with or without 10 μM HTL in solution (in vitro). After 12 h, the reaction mixture was desalted and concentrated with filter tubes. N-Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2, respectively (n=4).

D Upon switching N-Hcy substrate lysine (K) sites to nonmodifiable tryptophan (W), the N-Hcy unmodifiable mutants displayed lower specific activity than the wide-type (WT) SOD. SOD1 (WT, K23W, K122W, K128W, and 3KW) and SOD2 (WT, K44W, K51W, K98W, K106W, K178W, and 5KW) were expressed in HEK293T cells. The catalytic activity of affinity-purified SOD proteins was determined and normalized to protein levels. Wild-type SOD1 and SOD2 activities were set at 100% (n=4).

E, F Hcy and HTL treatment in NE4C cells increased N-homocysteinylation of SODs and decreased their activities. Flag-tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells. The cells were transferred to serum-free DMEM with or without 20 μM Hcy (E) or 10 μM HTL (F) 6 h before harvesting. After affinity purification, the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells.

G N-Hcy SOD levels were down-regulated in MARS/MARS2 knockdown cells. Flag-tagged SOD1 and SOD2 were each over-expressed in untreated and MARS/MARS2 knockdown NE4C cells. After affinity purification, the N-Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells (n=4).

Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.
Inhibiting MARSs reduces hyperhomocysteinemia-associated neural tube and congenital heart defects (2019)
Figure 5

Figure 5. N-Hcy activates Wnt signalling through ROS-induced disruption of DVL1-NRX interactions

A Levels of β-catenin in response to Hcy treatment in NE4C cells were detected by western blot (n=3). Representative western blots are shown, and band intensities were quantified relative to the untreated group.

B Levels of β-catenin in response to HTL treatment in NE4C cells were detected. Band intensities were normalized to that of untreated cells.

C TCF activities were measured with a Topflash/Fopflash luciferase reporter system (n=4). TCF activities responding to either 20 μM Hcy- or 10 μM HTL-treated were measured by quantifying Topflash/Fopflash relative to the untreated groups.

D Levels of β-catenin in untreated and MARS knockdown NE4C cells were detected (n=3) with the presence or absence of 20 μM Hcy in the culture media.

E, F NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of Hcy (E) or HTL (F). The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3).

G NRX-Flag and DVL1-Myc were co-transfected in untreated or MARS knockdown NE4C cells. The relative DVL1 levels that co-IPed with NRX-Flag were determined and quantified (n=3) for treated and control cells cultured with or without 20 μM Hcy.

H, I Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL. (H) The levels of β-catenin in cells with and without SOD1/2 expression were determined (n=3). (I) Relative levels of DVL1 that co-IPed with NRX-Flag were quantified in SOD-expressing and control cells cultured with or without 10 μM HTL (n=3).

Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.
Inhibiting MARSs reduces hyperhomocysteinemia-associated neural tube and congenital heart defects (2019)
Figure 6

Figure 6. AHT inhibits Hcy sensing

A Chemical structures of Hcy and its structural analogue acetyl Hcy thioether (AHT) are shown. The analogous parts are circled with dashed lines.

B MARS- or MARS2-catalysed HTL production from Hcy (20 μM) was carried out in vitro in the presence of different AHT concentrations. The amount of HTL produced in each reaction was determined by thin layer chromatography.

C MARS and MARS2 activities were determined (n=4) in the presence of the indicated AHT concentrations in the reactions. AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy.

D Relative HTL levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h.

E AHT (71 or 110 μg/kg per day) was injected into C57BL/6 mice for 6 days. Plasma HTL levels were detected by LC-MS.

F Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments.

G, H NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; N-homocysteinylation levels were detected by western blot. Band intensities were quantified relative to the untreated group using Image J (H).

I Cellular superoxide levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Superoxide levels were normalized to those of untreated NE4C cells.

J Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT-treated NE4C cells (n=4).

K Levels of β-catenin in untreated, Hcy-, or HTL-treated NE4C cells were detected (n=3) with the presence or absence of 70 μM AHT in the culture media. Mean β-catenin levels in untreated NE4C cells were set at 100%.

Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.