EMBO/soda-nel huggingface dataset:
MPK3/6-induced degradation of ARR1/10/12 promotes salt tolerance in Arabidopsis (2021)
Figure 8

Figure 8. MPK3/6-mediated ARR10 phosphorylation decreases the ability of ARR10 protein in repressing salt tolerance.

A-D Four-day-old seedlings of 35S:ARR10:YFP, 35S:ARR10S383A:YFP, and 35S:ARR10S383D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) and relative primary root lengths (B) were determined. After ten days of salt treatment, changes in survival rates (C) and fresh weights (D) were examined. Scale Bar, 1 cm.

Data information: In (B-D), data are means of three biological replicates ± SD (n=60 for B, and n=12 for C and D). ns indicates no significant difference to the corresponding controls. **, ***, and **** indicate significant difference to the corresponding controls with P<0.01, P<0.001, and P<0.0001, respectively (student's t-test).
MPK3/6-induced degradation of ARR1/10/12 promotes salt tolerance in Arabidopsis (2021)
Figure 9

Figure 9. MPK3/6-mediated ARR12 phosphorylation decreases the ability of ARR12 protein in repressing salt tolerance.

A-D Four-day-old seedlings of 35S:ARR12:YFP, 35S:ARR12S323A:YFP, and 35S:ARR12S323D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) and relative primary root lengths (B) were determined. After ten days of salt treatment, changes in survival rates (C) and fresh weights (D) were examined. Scale Bar, 1 cm.

Data information: In (B-D), data are means of three biological replicates ± SD (n=60 for B, and n=12 for C and D). ns indicates no significant difference to the corresponding controls. **, ***, and **** indicate significant difference to the corresponding controls with P<0.01, P<0.001, and P<0.0001, respectively (student's t-test).
MPK3/6-induced degradation of ARR1/10/12 promotes salt tolerance in Arabidopsis (2021)
Figure 10

Figure 10. MPK3/6 act upstream of ARR1/10/12 in response to salt stress.

A-D Three-day-old seedlings of Col-0, MPK3SR, MPK3SRarr1/12, and arr1/12 were transferred to 0.5 μM NA-PP1-containing 1/2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1/12 and then the plants were transferred to 0.2 μM NA-PP1-containing 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. Pictures were taken and the elongated root length was determined 3 d later (A-B). Changes in survival rates (C) and fresh weights (D) were examined 10 d later. The fresh weight of NaCl treated seedlings/fresh weight of NaCl untreated seedlings ratios were labelled in the columns in (D). Scale Bar, 1 cm.

Data information: In (B-D), data are means of three biological replicates ± SD (n=60 for B, and n=12 for C and D). ** , ***, and **** indicate significant difference to the corresponding controls with P<0.01, P<0.001, and P<0.0001, respectively (student's t-test).
Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling (2022)
Figure 2

Figure 2. RNAseq-based transcriptional profiling of mouse and human myogenic progenitors upon activation of NOTCH and PDGF signalling pathways.

(A) Principal Component Analysis (PCA) showing mMuSC-derived myoblasts (left), human myoblasts (centre) and hiMPs (right). 4 cell lines were analysed by RNAseq in treated and untreated conditions for each cell population. Each point on the PCA represents a cell population. Additional information in Appendix Table S1,2.

(B) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05 to be considered significant.

(C) Heatmaps showing changes in expression of key myogenic (MYOD, MYOGENIN), perivascular (PDGFRB, NG2, CD146, ALPL) and NOTCH target (HEY1, HES1) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled.

(D) Validation of RNAseq data of panel (C) by real-time PCR analysis of the same myogenic, perivascular and NOTCH target transcripts in treated and untreated hiMPs (experimental replicates = 3; error bars; S.E.M.). Statistical analysis (paired t test) was performed on ΔCt values whilst graphs show fold change relative to untreated controls.

(E) Curated dot plot Gene Ontology (GO; left), Kyoto Encyclopaedia of Genes and Genomes (KEGG; centre) and Reactome (right) enrichment analyses showing shared gene functions amongst the cell groups; numbers in brackets: genes analysed with a p value threshold set at 0.05; full lists in a dedicated spreadsheet available in Supplemental Information.
Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling (2022)
Figure 3

Figure 3. Analysis of morphology, proliferation and differentiation of DLL4 and PDGFBB-treated hiMPs.

(A) P value-adjusted hierarchical clustering heatmap generated from a gene ontology list of genes involved in regulation of cell morphology (GO 0008360; P set at 0.05).

(B) Phase contrast images displaying morphology of untreated and treated hiMPs. Scale bar: 25 μm.

(C) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs. Morphology was quantified as­ circularity ratio, where 1 = perfect circle and 0 = line (experimental replicates = 3). Statistical analysis (paired t test) was performed on the first quartile to enhance detection of morphological changes (error bars: SD).

(D) P value-adjusted hierarchical clustering heatmap generated from a gene ontology list of genes involved in proliferation of stem and myogenic cell types (GO 2000291; 0048660; 0014857; 0072091; P set at 0.05).

(E) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours. Scale bar: 75 μm.

(F) Bar graphs quantifying EdU experiment shown in (E) (experimental replicates = 3; error bars: SEM). Statistical analysis based on an unpaired t-test.

(G) P value-adjusted hierarchical clustering heatmap of NOTCH signalling genes (Kegg pathway 04330).

(H) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week, induced to differentiate for 4 days in the presence or absence of γ-secretase inhibitor L685458 and immunostained for myosin heavy chain (MyHC). Scale bar: 75µm.

(I) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes (experimental replicates = 3; error bars: SEM). Statistical significance based on one-way ANOVA with Tukey's multiple comparison. Scale bar: 75 μm.

(J) Immunofluorescence panels showing human specific LAMIN A/C+ (nuclei) and SPECTRIN (sarcolemma) staining in tibialis anterior muscles of immunodeficient mice (N = 3) transplanted with treated (n = 3 muscles) and untreated (n = 3 muscles) N5 hiMPs.

(K) Quantification of LAMIN A/C+ grafted human cells across each muscle. Data information: full gene list for heatmaps in (A) and (D) available in Appendix Table S4.
Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling (2022)
Figure 5

Figure 5. Modelling and assessing trans-endothelial migration of hiMPs.

(A) P value-adjusted hierarchical clustering heatmap displaying hierarchical clustering of genes associated with leukocyte trans-endothelial migration (KEGG pathway: hsa04670; P set at 0.05). (B) Graphical representation of an individual chip of the OrganoPlate® (produced with BioRender, www.biorender.com). Each chip consists of a top perfusion channel, central ECM channel and bottom perfusion channel. Phase guides between channels allows for generation of surface tension after deposition of collagen-I within the ECM channel so that there is no physical barrier between the collagen gel and perfusion channels. This facilitates generation of a 3D blood vessel that is in direct contact with the ECM channel.

(C) Maximum intensity projections of the top perfusion channel, 48h after seeding HUVECs, immunostained for CD31 and F-actin. Scale bar: 100µm.

(D) 3D projections of blood vessel-like tubules of the top perfusion channel stained for F-actin. Scale bar: 50µm.

(E) Representative fluorescence images of 150 kDa TRITC-conjugated dextran added to the top perfusion channel of OrganoPlate® chips with and without 3D endothelial monolayers generated by HUVECs. Chips were imaged every 3 minutes. See Figure S5 for extended panel and quantification. Scale bar: 100 µm

(F) Representative fluorescence images of CMFDA-stained untreated and DLL4 & PDGF-BB-treated hiMPs within the top perfusion channel, 15 minutes after delivery and kept on the OrganoFlow®. Scale bar: 50µm.

(G) Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t-test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 minutes within a single chip.

(H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm2, that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm2) fields per condition was quantified (error bars: SEM). Scale bar: 250 μm.

(I) Bar graph showing fold-change in trans-endothelial migration (error bars: SEM). Statistical significance based on one-way ANOVA with Bonferroni's multiple comparison.
Mitochondrial Safeguard': a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation (2020)
Figure 1

Figure 1. Increased conversion of long isoforms of Opa1 to short forms in Drp1-KO MEFs.

(A) Proteolytic processing of Opa1. L1 is cleaved by both Oma1 and Yme1L, while L2 is only cleaved by Oma1.

(B) Western blotting of WT and Drp1-KO MEFs with or without FCCP treatment (30 min) using the indicated antibodies.

(C) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.
Mitochondrial Safeguard': a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation (2020)
Figure 2

Figure 2. An experimental system to examine Opa1 processing.

(A) L2 was tagged with HA at the C-terminus and expressed from the doxycycline-inducible promoter.

(B) Western blotting of WT MEFs, Drp1-KO MEFs, and Drp1-KO MEFs carrying Drp1, all of which express L2-HA, using the indicated antibodies. The expression of L2-HA was induced for 16 h (0.1 µg/ml doxycycline). The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(C) Quantification of band intensity. Values are average ± SD (n = 3).

(D) The activation of Oma1. Oma1 is proteolytically activated and then undergoes degradation.

(E) WT and Drp1-KO MEFs, both of which express L2-HA, were transduced with lentiviruses carrying either scramble or Oma1-targeted shRNAs. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(F) Quantification of band intensity. Values are average ± SD (n = 3).

Data information: Significance was calculated using ANOVA with post-hoc Tukey in (C and F): *p<0.05, **p<0.01, ***p<0.001.
Mitochondrial Safeguard': a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation (2020)
Figure 3

Figure 3. Effects of antimycin A (10 nM) and oligomycin (10 ng/ml) on flickering and Opa1 cleavage in Drp1-KO MEFs.

(A) The mitochondrial membrane potential was measured in WT and Drp1-KO MEFs using a membrane potential-dependent dye (MitoLite NIR) and flow cytometry.

(B) Drp1-KO MEFs carrying Su9-GFP were treated with DMSO (control) or 10 nM antimycin A or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1-EV4). Scale bar, 10 µm.

(C-E) The percentage of cells that showed flickering in 30 min. Values are average ± SD (n = 3 experiments). In each experiment, 25-89 cells were analyzed.

(F) Drp1-KO MEFs were treated with DMSO or 10 nM antimycin A or 10 ng/ml oligomycin or 10 µM FCCP for 1 h. The mitochondrial membrane potential was analyzed using MitoLite NIR and flow cytometry.

(G) Drp1-KO MEFs expressing L2-HA (induced for 16 h) were treated with 10 nM antimycin A along with 10 µM FCCP. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(H) Quantification of band intensity. Values are average ± SD (n = 3).

(I) Western blotting of Drp1-KO MEFs with or without 10 ng/ml oligomycin treatment. The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(J) Quantification of band intensity. Values are average ± SD (n = 3).

(K) WT MEFs carrying Su9-GFP were treated with DMSO or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1 and EV5). Scale bar, 10 µm.

(L) WT MEFs expressing L2-HA (induced for 16 h) were treated with 10 ng/ml oligomycin. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(M) Quantification of band intensity. Values are average ± SD (n = 3).

Data information: Significance was calculated using ANOVA with post-hoc Tukey in (C and H) and Student t-test in (D, E, J and M): *p<0.05, **p<0.01, ***p<0.001.
Mitochondrial Safeguard': a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation (2020)
Figure 4

Figure 4. The loss of Opa1 function decreases flickering and Opa1 cleavage in the absence of Drp1.

(A) Western blotting of Drp1flox/floxOpa1flox/flox and Drp1Opa1-KO MEFs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(B) Quantification of band intensity. Values are average ± SD (n = 3).

(C) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs were transduced with the indicated Opa1 constructs. The cells were observed using laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The percentage of cells that exhibited flickering is shown. Values are average ± SD (n = 3 experiments). In each experiment, 48-62 cells were analyzed.

(D) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs carrying the indicated Opa1 constructs were subjected to confocal immunofluorescence microscopy using anti-PDH antibodies. Scale bar, 10 µm.

(E) Quantification of mitochondrial morphology is shown (n = 3 experiments). In each experiment, 30 cells were analyzed.

(F) L2-HA constructs carrying a mutation (K301A or K468D) in the GTPase domain.

(G) Western blotting of Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs, both of which carry the indicated Opa1 constructs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(H) Quantification of band intensity. Values are average ± SD (n = 3).

Data information: Significance was calculated using Student t-test in (B) and ANOVA with post-hoc Tukey in (C, E, and H): ***p<0.001.
Mitochondrial Safeguard': a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation (2020)
Figure 5

Figure 5. Artificial flickering induces Opa1 cleavage.

(A) Experimental design for artificial flickering. WT MEFs expressing Su9-GFP were subjected to repetitive FCCP treatments and observed using confocal microscopy in the presence of TMRE.

(B) Three frames from the time-lapse analysis during artificial flickering are shown. The boxed regions are magnified. Scale bar, 5 µm.

(C) The fluorescence intensity of Su9-GFP and TMRE in mitochondria was quantified.

(D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(E) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.
Mitochondrial Safeguard': a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation (2020)
Figure 6

Figure 6. Restoring Opa1 long forms and Mfn1 induces extreme fusion of mitochondria in Drp1-KO MEFs.

(A) Western blotting of WT MEFs and Drp1-KO MEFs, both of which carry shRNAs (Oma1 or scramble) and/or ectopic Mfn1. The asterisk indicates non-specific bands of anti-Oma1 antibodies.

(B) Quantification of band intensity is shown. Values are average ± SD (n = 3).

(C-F) WT MEFs (C and E) and Drp1-KO MEFs (D and F) were transduced with Su9-Eos, along with Oma1 shRNAs and ectopic Mfn1. Su9-Eos in the small region of mitochondria (indicated by the asterisks) was photoconverted using a 405-nm laser on confocal microscope. Within 1 s after photoconversion, images were obtained for both photoconverted and unconverted Su9-Eos signals. Images before the photoconversion were also taken to detect background signals. Scale bar, 10 µm. (E and F) To determine the connectivity of mitochondria, the area containing photoconverted Su9-Eos signals was divided by the area containing the photoconverted and unconverted Su9-Eos signals (total mitochondria). Values are average ± SD (n = 30 cells).

(G) The number of mitochondria was estimated by reversing the connectivity.

(H) Summary of the data.

Data information: Significance was calculated using ANOVA with post-hoc Tukey in (B) and the Kruskal-Wallis test with post-hoc Dunn in (E, F and G): *p<0.05, **p<0.01, ***p<0.001.
Mitochondrial Safeguard': a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation (2020)
Figure 7

Figure 7. Extreme mitochondrial fusion decreases the membrane potential and respiration.

(A-D) WT (A and B) and Drp1-KO MEFs (C and D), both of which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The arrows indicate cells that lost the mitochondrial membrane potential in (C). Scale bar, 10 µm. (B and D) The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.

(E-H) OCRs were analyzed in the same set of MEFs. (F and H) Quantification of the basal OCRs and respiratory capacity is shown. Values are average ± SD (n = 3).

(I) Summary of the data.

Data information: Significance was calculated using ANOVA with post-hoc Tukey in (B, D, F and H): *p<0.05, **p<0.01, ***p<0.001.
Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components. (2001)
Figure 1.

Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis–removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti–SH-EP, α-amylase, or VmPE-1 antibody.
Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components. (2001)
Figure 2.

Subcellular distribution of α-amylase in V. mungo cotyledon cells. Microsome and PSV fractions were prepared from cotyledons of day 3 seedlings as described in Materials and methods. Extracts from dry seeds and day 3 cotyledons, microsomes, and PSV fractions (20 μg protein each) were separated by SDS-PAGE. Proteins in the gel were analyzed by Coomassie Brilliant blue staining (A), or by immunoblotting with anti–SH-EP antibody (B) or anti– α-amylase antibody (C). Bracket indicates the polypeptides derived from storage globulins. Arrowhead indicates α-amylase.
Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components. (2001)
Figure 3.

Electron micrographs showing the immunogold localization of α-amylase in attached cotyledons cells (A–E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti–α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti– α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti–α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti– α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti–α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.
Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components. (2001)
Figure 4.

Electron micrographs showing ultrastructures of cotyledon cells of germinatedV. mungo seeds (A–G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B–E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti–α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.