A A representation of the pairwise degree of overlap in targets ( size of the circle ) and correlation in gene expression changes ( colour ) among overlapping targets for indicated TF perturbations . Red indicates positive correlations and blue negative correlations .

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A A representation of the pairwise degree of overlap in targets (size of the circle) and correlation in gene expression changes (colour) among overlapping targets for indicated TF perturbations. Red indicates positive correlations and blue negative correlations.


D Identification of target gene modules - groups of genes with common regulatory patterns by TFs . Colour indicates the fold change ( adjusted for significance ) of gene expression following each TF perturbation . Rows ( perturbed TFs ) and columns ( target genes ) are hierarchically clustered . 44 modules for target genes are shown . Below examples . Selected modules with highlighted overall regulation pattern and example genes listed below .

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D Identification of target gene modules - groups of genes with common regulatory patterns by TFs. Colour indicates the fold change (adjusted for significance) of gene expression following each TF perturbation. Rows (perturbed TFs) and columns (target genes) are hierarchically clustered. 44 modules for target genes are shown. Below examples. Selected modules with highlighted overall regulation pattern and example genes listed below.


D Changes in expression for genes co - regulated by separate Cebpa / Meis1 / Spi1 and Hoxb8 perturbations ( FDR < 0 . 1 and | log2 ( FoldChange ) | > 0 . 2 ) . The interaction row indicates changes beyond simple additive effect ( white = additive effect ) . Each gene was annotated with an interaction class ( int . class ) as explained in ( C ) , using the | log2 ( FoldChange ) | > 0 . 2 ) threshold to assign change direction . E Fractions of genes in each interaction class showed in ( D ) .

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D Changes in expression for genes co-regulated by separate Cebpa/Meis1/Spi1 and Hoxb8 perturbations (FDR <0.1 and |log2(FoldChange)| > 0.2). The interaction row indicates changes beyond simple additive effect (white = additive effect). Each gene was annotated with an interaction class (int. class) as explained in (C), using the |log2(FoldChange)| > 0.2) threshold to assign change direction. E Fractions of genes in each interaction class showed in (D).


A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8 - FL cells .

A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8-FL cells.


B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment . Enrichment over a random value and the p - value ( based on the hypergeometric test ) are shown ( bars corresponding to non - significant enrichment values are semi - transparent ) . Analysis was performed separately for genes up - and down - regulated .

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B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment. Enrichment over a random value and the p-value (based on the hypergeometric test) are shown (bars corresponding to non-significant enrichment values are semi-transparent). Analysis was performed separately for genes up- and down-regulated.


C , D Genomic views of relevant TF binding to Cebpa and Gata3 loci ( supporting direct regulatory interactions identified in Figs 3A , B , C ) . The red arrow indicates a putative enhancer element , which is bound by Ebf1 and flanked by AcK27 - rich regions .

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C, D Genomic views of relevant TF binding to Cebpa and Gata3 loci (supporting direct regulatory interactions identified in Figs 3A,B,C). The red arrow indicates a putative enhancer element, which is bound by Ebf1 and flanked by AcK27-rich regions.


C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8 - FL cells . Each quadrant corresponds to a set of target genes with a common regulation by Gata3 / Ebf1 - I - inhibition / inhibition , II - activation / inhibition , III - activation / activation , IV - inhibition / activation . For each group the sums of scaled expression values in mouse LK / LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression . For landscape annotation see Fig 7B andE . . Changes in expression for example genes are provided in Appendix Fig 2G - J . Blue line indicates the linear fit with shaded areas as confidence intervals .

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C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8-FL cells. Each quadrant corresponds to a set of target genes with a common regulation by Gata3/Ebf1 - I - inhibition/inhibition, II - activation/inhibition, III - activation/activation, IV - inhibition/activation. For each group the sums of scaled expression values in mouse LK/LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression. For landscape annotation see Fig 7B andE.. Changes in expression for example genes are provided in Appendix Fig 2G-J. Blue line indicates the linear fit with shaded areas as confidence intervals.


E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8 - FL cells and re - expression of Ebf1 in Ebf1 - / - pre - pro B cells ( Li et al , 2018 ) . Blue line indicates the linear fit with shaded areas as confidence intervals .

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E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8-FL cells and re-expression of Ebf1 in Ebf1-/- pre-pro B cells (Li et al, 2018). Blue line indicates the linear fit with shaded areas as confidence intervals.


F , G Expression of the key marker genes ( F ) and Ebf1 , Pax5 , Gata3 , Cebpa factors ( G ) along pseudotime corresponding to the B - cell differentiation trajectory in human foetal liver cells , data from ( Popescu et al , 2019 ) .

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F, G Expression of the key marker genes (F) and Ebf1, Pax5, Gata3, Cebpa factors (G) along pseudotime corresponding to the B-cell differentiation trajectory in human foetal liver cells, data from (Popescu et al, 2019).


D . Relative FCR activities in the roots of wild - type plants and photoreceptor mutants grown under white light with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

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D. Relative FCR activities in the roots of wild-type plants and photoreceptor mutants grown under white light with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


D - F . Fe contents of whole plants , FER expression in roots and relative FCR activities in the roots of plants grown under Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . Plants were exposed to high ( H - , 2 . 0 ) or low ( L - , 0 . 5 ) red ( R ) to far red ( FR ) ratios .

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D-F. Fe contents of whole plants, FER expression in roots and relative FCR activities in the roots of plants grown under Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d. Plants were exposed to high (H-, 2.0) or low (L-, 0.5) red (R) to far red (FR) ratios.


C . Accumulation of HY5 protein in leaves and roots of reciprocal grafts of phyB and WT plants grown under white light ( 200 µmol m - 2s - 1 ) for 7 d . Rubisco and β - Actin were used as loading controls for leaves and roots in the western blot analysis , respectively . The values shown above each lane indicate the relative abundance of the HY5 protein . D . Accumulation of HY5 protein in the roots of WT and phyB plants under high ( H , 2 . 0 ) or low ratio of ( L , 0 . 5 ) red ( R ) to far red ( FR ) light conditions . β - Actin was used as loading controls in the western blot analysis . The values shown above each lane indicate the relative abundance of the HY5 protein .

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C. Accumulation of HY5 protein in leaves and roots of reciprocal grafts of phyB and WT plants grown under white light (200 µmol m-2s-1) for 7 d. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively. The values shown above each lane indicate the relative abundance of the HY5 protein. D. Accumulation of HY5 protein in the roots of WT and phyB plants under high (H, 2.0) or low ratio of (L, 0.5) red (R) to far red (FR) light conditions. β-Actin was used as loading controls in the western blot analysis. The values shown above each lane indicate the relative abundance of the HY5 protein.


D . Fe content of whole plants . WT and hy5 plants were grown under high ( H ) or low ratios of ( L ) red ( R ) to far red ( FR ) light conditions with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

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D. Fe content of whole plants. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


E . FER expression in roots . WT and hy5 plants were grown under high ( H ) or low ratios of ( L ) red ( R ) to far red ( FR ) light conditions with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

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E. FER expression in roots. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


E . Direct binding of HY5 to the FER promoter analyzed using ChIP - qPCR in 35S - HY5 - 3HA - overexpressing ( HY5 - OE ) tomato plants . WT and HY5 - OE plants were exposed to white light ( 200 µmol m - 2s - 1 ) grown under Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . Input chromatin was isolated from root samples at 7 d . The epitope - tagged HY5 - chromatin complex was immunoprecipitated with an anti - HA antibody . A control reaction was processed side - by - side using mouse IgG . Input - and ChIP - DNA samples were quantified by RT - qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in ( D ) . The ChIP results are presented as percentage of the input DNA .

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E. Direct binding of HY5 to the FER promoter analyzed using ChIP-qPCR in 35S-HY5-3HA-overexpressing (HY5-OE) tomato plants. WT and HY5-OE plants were exposed to white light (200 µmol m-2s-1) grown under Fe-deficient (2 µM, -Fe) conditions for 7 d. Input chromatin was isolated from root samples at 7 d. The epitope-tagged HY5-chromatin complex was immunoprecipitated with an anti-HA antibody. A control reaction was processed side-by-side using mouse IgG. Input- and ChIP-DNA samples were quantified by RT-qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in (D). The ChIP results are presented as percentage of the input DNA.


A - C . Fe content of whole plants ( A ) and the photochemical efficiencies of PSI and PSII [ Y ( I ) and Y ( II ) ] ( B and C ) in young leaves of grafted tomato plants with grafts between WT / WT and WT / fer grown under H - R / FR ( 2 . 0 ) or L - R / FR ( 0 . 5 ) conditions for 7 d .

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A-C. Fe content of whole plants (A) and the photochemical efficiencies of PSI and PSII [Y(I) and Y(II)] (B and C) in young leaves of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.


D - F . Transcript levels of FER , FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT / WT and WT / fer grown under H - R / FR ( 2 . 0 ) or L - R / FR ( 0 . 5 ) conditions for 7 d .

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D-F. Transcript levels of FER, FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.


B . The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe - EDTA in nutrient solution in a growth room .

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B. The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room.


D . Fe content in the fruits of WT and phyB plants . Plants were grown in soil in a greenhouse .

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D. Fe content in the fruits of WT and phyB plants. Plants were grown in soil in a greenhouse.


1D 1H - NMR spectra of a metabolite mix and the individual metabolites contained therein . Identification of single compound peaks from 1D 1H - NMR spectra of a metabolite mix . Compound detection is exemplified by showing sections of the 1D 1H - NMR spectra of ATP , IMP , proline ( PRO ) , malate ( MAL ) , shikimate ( SKM ) and methionine ( MET ) .

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1D 1H-NMR spectra of a metabolite mix and the individual metabolites contained therein. Identification of single compound peaks from 1D 1H-NMR spectra of a metabolite mix. Compound detection is exemplified by showing sections of the 1D 1H-NMR spectra of ATP, IMP, proline (PRO), malate (MAL), shikimate (SKM) and methionine (MET).


Relative activity of NADP + - dependent glucose - 6 - phosphate dehydrogenase in the presence of potential regulators , error bars represent the s . e . m . . All regulators were tested at two concentrations in four distinct replicates ( n = 4 , ATP : 5mM , 18 mM ; GTP : 2 mM , 10 mM ; IMP and AMP : 1 mM , 5 mM ) , the asterisk denotes significant inhibition ( 18 mM ATP : p - value = 0 . 029 , 10 mM GTP : p - value = 0 . 025 , one - tailed t - test ) .

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Relative activity of NADP+-dependent glucose-6-phosphate dehydrogenase in the presence of potential regulators, error bars represent the s.e.m.. All regulators were tested at two concentrations in four distinct replicates (n=4, ATP: 5mM, 18 mM; GTP: 2 mM, 10 mM; IMP and AMP: 1 mM, 5 mM), the asterisk denotes significant inhibition (18 mM ATP: p-value = 0.029, 10 mM GTP: p-value = 0.025, one-tailed t-test).


Relative activity of phosphate acetyltransferase in the presence of phenylpyruvate and tryptophan , error bars represent the s . e . m . . All regulators were tested at three concentrations in three distinct replicates ( n = 3 , phenylpyruvate : 0 . 1 , 1 , 5 mM , L - tryptophan : 0 . 1 , 1 , 4 . 4 mM ) .

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Relative activity of phosphate acetyltransferase in the presence of phenylpyruvate and tryptophan, error bars represent the s.e.m.. All regulators were tested at three concentrations in three distinct replicates (n=3, phenylpyruvate: 0.1, 1, 5 mM, L-tryptophan: 0.1, 1, 4.4 mM).


Relative activity of fructose - bisphosphate aldolase class II in the presence of 5 mM of potential regulators , except hypoxanthine , which was tested at 3 . 22 mM . All regulators were tested in three distinct replicates ( n = 3 ) , error bars represent the s . e . m . . The asterisk denotes significant activation ( 5 mM 3PG : p - value = 0 . 025 , 5 mM ATP : p - value = 0 . 024 , 5 mM PEP : p - value = 0 . 046 , one - tailed t - test ) .

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Relative activity of fructose-bisphosphate aldolase class II in the presence of 5 mM of potential regulators, except hypoxanthine, which was tested at 3.22 mM. All regulators were tested in three distinct replicates (n=3), error bars represent the s.e.m.. The asterisk denotes significant activation (5 mM 3PG: p-value = 0.025, 5 mM ATP: p-value = 0.024, 5 mM PEP: p-value = 0.046, one-tailed t-test).


B C99 - Bpa constructs remain essentially cleavage - competent . Numbers denote the relative γ - secretase cleavage efficiencies of C99 - Bpa substrates as calculated from the AICD / C99 - Bpa ratios relative to that of WT C99 , which was set to 1 . Inefficiently cleaved substrates ( cleavage efficiency below 20 % ) were mostly found for Bpa substitutions in extramembraneous domains . Specificity of substrate cleavage was confirmed by inhibition of AICD formation in the presence of L - 685 , 458 . Residues of the TMD are highlighted in orange here and elsewhere where appropriate . C Values obtained in ( B ) were additionally plotted . Yellow line indicates the cleavage efficiency of WT C99 , which was set to 1 . ECD , extracellular domain ; ICD , intracellular domain .

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B C99-Bpa constructs remain essentially cleavage-competent. Numbers denote the relative γ-secretase cleavage efficiencies of C99-Bpa substrates as calculated from the AICD/C99-Bpa ratios relative to that of WT C99, which was set to 1. Inefficiently cleaved substrates (cleavage efficiency below 20%) were mostly found for Bpa substitutions in extramembraneous domains. Specificity of substrate cleavage was confirmed by inhibition of AICD formation in the presence of L-685,458. Residues of the TMD are highlighted in orange here and elsewhere where appropriate.C Values obtained in (B) were additionally plotted. Yellow line indicates the cleavage efficiency of WT C99, which was set to 1. ECD, extracellular domain; ICD, intracellular domain.


B Identification of C99 interaction sites with γ - secretase . C99 - Bpa substrates were irradiated with UV light in the presence of CHAPSO - solubilized γ - secretase . Crosslinked substrates were captured by Ni - NTA affinity pulldown and bound subunits were identified by a ~ 10 kDa molecular weight increase compared to the input . Results are shown for the major substrate crosslinking residues . Crosslink formation was not observed in the absence of UV irradiation or in the presence of Triton X - 100 , which dissociates the γ - secretase complex , proving crosslink specificity . Bracket indicates the molecular weight range of putative C99 - APH - 1 crosslink bands . APH - 1aL , long splice variant of APH - 1a .

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B Identification of C99 interaction sites with γ-secretase. C99-Bpa substrates were irradiated with UV light in the presence of CHAPSO-solubilized γ-secretase. Crosslinked substrates were captured by Ni-NTA affinity pulldown and bound subunits were identified by a ~10 kDa molecular weight increase compared to the input. Results are shown for the major substrate crosslinking residues. Crosslink formation was not observed in the absence of UV irradiation or in the presence of Triton X-100, which dissociates the γ-secretase complex, proving crosslink specificity. Bracket indicates the molecular weight range of putative C99-APH-1 crosslink bands. APH-1aL, long splice variant of APH-1a.


C Excess amounts of parental C99 WT substrate compete crosslinking of C99 - Bpa substrates to γ - secretase . Single and double asterisks indicate antibody crossreactivities to C99 monomer and dimer , respectively .

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C Excess amounts of parental C99 WT substrate compete crosslinking of C99-Bpa substrates to γ-secretase. Single and double asterisks indicate antibody crossreactivities to C99 monomer and dimer, respectively.


Quantitative crosslinking analysis revealed V44 , L49 , M51 and L52 as major interactions sites of C99 with γ - secretase with high crosslinking efficiencies ranging from ~ 2 - 8 % of input . While the PS1 NTF was bound by C99 over nearly the whole range of residues , only very few residues bound to PS1 CTF . Several residues in the substrate N - terminal extracellular domain bound to NCT . PEN - 2 - binding residues clustered in the membrane - flanking extracellular and intracellular domains of C99 .

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Quantitative crosslinking analysis revealed V44, L49, M51 and L52 as major interactions sites of C99 with γ-secretase with high crosslinking efficiencies ranging from ~2-8% of input. While the PS1 NTF was bound by C99 over nearly the whole range of residues, only very few residues bound to PS1 CTF. Several residues in the substrate N-terminal extracellular domain bound to NCT. PEN-2-binding residues clustered in the membrane-flanking extracellular and intracellular domains of C99.


A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site . L , L - 685 , 458 ; D , DAPT .

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A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site. L, L-685,458; D, DAPT.


B Preventing access to the active site by GSIs identifies substrate residues interacting with the exosites by their increased labeling intensity .

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B Preventing access to the active site by GSIs identifies substrate residues interacting with the exosites by their increased labeling intensity.


A PS1 FAD mutations display little or no effect on substrate interactions at the exosites . Asterisks indicate antibody crossreactivities to C99 monomer .

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A PS1 FAD mutations display little or no effect on substrate interactions at the exosites. Asterisks indicate antibody crossreactivities to C99 monomer.


B PS1 FAD mutations alter substrate binding to the PS1 NTF in the γ - secretase cleavage domain . The weak PS1 A246E FAD mutation alters substrate interactions predominantly at V49 and L52 , whereas the highly pathogenic PS1 L166P FAD mutation causes more broad changes over the whole cleavage domain . Arrows denote changes in binding of individual residues . Corresponding quantitation of substrate binding is shown below the immunoblots . Bars denote the mean ± S . E . ( n = 3 ) . Yellow line indicates level of substrate binding to PS1 WT , which was set to 1 . Blue and red bars indicate increases or decreases in binding , respectively .

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B PS1 FAD mutations alter substrate binding to the PS1 NTF in the γ-secretase cleavage domain. The weak PS1 A246E FAD mutation alters substrate interactions predominantly at V49 and L52, whereas the highly pathogenic PS1 L166P FAD mutation causes more broad changes over the whole cleavage domain. Arrows denote changes in binding of individual residues. Corresponding quantitation of substrate binding is shown below the immunoblots. Bars denote the mean ± S.E. (n = 3). Yellow line indicates level of substrate binding to PS1 WT, which was set to 1. Blue and red bars indicate increases or decreases in binding, respectively.


C Both mutants decrease crosslinking to the PS1 CTF .

C Both mutants decrease crosslinking to the PS1 CTF.


B Substrates bound to exosites in the PS1 NTF at 4 ° C can be cleaved in the 37 ° C chase incubation in a L - 685 , 458 - dependent manner . Note that , although many of these substrates crosslinked only weakly at the exosites , their cleavage in the 37 ° C chase proved that these reflected functional substrate - binding states . C Substrates crosslinking in the exosites to NCT and PEN - 2 cannot be cleaved .

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B Substrates bound to exosites in the PS1 NTF at 4°C can be cleaved in the 37°C chase incubation in a L-685,458-dependent manner. Note that, although many of these substrates crosslinked only weakly at the exosites, their cleavage in the 37°C chase proved that these reflected functional substrate-binding states.C Substrates crosslinking in the exosites to NCT and PEN-2 cannot be cleaved.


D , E Substrates can be cleaved when crosslinked to PS1 NTF but not when crosslinked to NCT ( D ) or PEN - 2 ( E ) .

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D, E Substrates can be cleaved when crosslinked to PS1 NTF but not when crosslinked to NCT (D) or PEN-2 (E).


F Excess amounts of Notch1 substrate N102 - FmH compete with exosite - binding of C99 . See Fig EV4 for competition of C99 binding in the active site region .

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F Excess amounts of Notch1 substrate N102-FmH compete with exosite-binding of C99. See Fig EV4 for competition of C99 binding in the active site region.


B Representative confocal microscopy of human PBMCs stained with DAPI ( white arrows ) co - cultured with previously labeled UC - MSC with MitoTracker - Green ( top image ) and specific antibody for human CD90 ( bottom image ) .

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B Representative confocal microscopy of human PBMCs stained with DAPI (white arrows) co-cultured with previously labeled UC-MSC with MitoTracker-Green (top image) and specific antibody for human CD90 (bottom image).


C Percentage of mitochondrial transfer ( MitoT ) on total human CD45 + cells after 24 hrs co - culture with MitoTracker - Green labeled bone marrow ( BM ) , menstrual ( Mens ) or umbilical cord ( UC ) - MSCs ( MSC : PBMC ratio 1 : 25 ) ( n = 3 biological replicates , with 3 different donors of BM , Mens and UC - MSCs ) .

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C Percentage of mitochondrial transfer (MitoT) on total human CD45+ cells after 24 hrs co-culture with MitoTracker-Green labeled bone marrow (BM), menstrual (Mens) or umbilical cord (UC)-MSCs (MSC:PBMC ratio 1:25) (n=3 biological replicates, with 3 different donors of BM, Mens and UC-MSCs).


D FACS analysis of MitoT on T CD3 + ( n = 4 ) , B CD19 + ( n = 4 ) and natural killer ( NK ) CD56 + ( n = 3 ) cells after 24 hrs co - culture with MitoGreen labeled UC - MSCs ( ratio 1 : 25 ) ( Biological replicates , with different donors of UC - MSCs ) .

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D FACS analysis of MitoT on T CD3+ (n=4), B CD19+ (n=4) and natural killer (NK) CD56+ (n=3) cells after 24 hrs co-culture with MitoGreen labeled UC-MSCs (ratio 1:25) (Biological replicates, with different donors of UC-MSCs).


E FACS analysis of MitoT on CD3 + CD4 + or CD3 + CD8 + T cell populations after 24 hrs co - culture with labeled UC - MSCs ( ratio 1 : 25 ) ( n = 3 biological replicates , with 3 different donors of UC - MSCs ) .

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E FACS analysis of MitoT on CD3+CD4+ or CD3+CD8+ T cell populations after 24 hrs co-culture with labeled UC-MSCs (ratio 1:25) (n=3 biological replicates, with 3 different donors of UC-MSCs).


F Representative FACS plots of MitoT on CD45 + CD3 + T cells at increasing amounts of UC - MSC .

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F Representative FACS plots of MitoT on CD45+CD3+ T cells at increasing amounts of UC-MSC.


G Percentage of MitoT on CD45 + CD3 + human cells after 24 hrs co - culture with increasing ratios of UC - MSC : PBMCs ( n = 5 biological replicates , with 5 different UC - MSCs and 5 different PBMCs ) .

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G Percentage of MitoT on CD45+CD3+ human cells after 24 hrs co-culture with increasing ratios of UC-MSC:PBMCs (n=5 biological replicates, with 5 different UC-MSCs and 5 different PBMCs).


H FACS analysis of MitoT on CD45 + CD3 + T cells after short times of co - culture with UC - MSCs at two different temperatures , at a ratio 1 : 25 ( n = 3 biological replicates , with 3 different UC - MSCs and 3 different PBMCs ) .

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H FACS analysis of MitoT on CD45+CD3+ T cells after short times of co-culture with UC-MSCs at two different temperatures, at a ratio 1:25 (n=3 biological replicates, with 3 different UC-MSCs and 3 different PBMCs).


I Mean fluorescence intensity ( MFI ) of MitoTracker - Green + UC - MSCs at baseline or after 72 hours PBMC co - culture . Representative FACS plot ( left panel ) and average of MFI ( n = 4 biological replicates , with 4 different UC - MSCs ) ( right panel ) .

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I Mean fluorescence intensity (MFI) of MitoTracker-Green+ UC-MSCs at baseline or after 72 hours PBMC co-culture. Representative FACS plot (left panel) and average of MFI (n=4 biological replicates, with 4 different UC-MSCs) (right panel).


J Confocal microscopy of human PBMCs after co - culture with MitoTracker - Green stained UC - MSCs . Images showed MSC - mitochondria ( green ) inside the CD3 + T cells ( red ) .

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J Confocal microscopy of human PBMCs after co-culture with MitoTracker-Green stained UC-MSCs. Images showed MSC-mitochondria (green) inside the CD3+ T cells (red).


K qPCR analysis of human β2 - microglobulin ( B2M ) , human specific mitochondrial ( Mito ) and mouse specific ( β - actin ) gene expression levels in UC - MSCs , mouse derived spleen cells or FACS sorted CD45 + mouse cells after 24 hrs co - culture with UC - MSCs ( n = 3 biological replicates ) . n / d = not detected .

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K qPCR analysis of human β2-microglobulin (B2M), human specific mitochondrial (Mito) and mouse specific (β-actin) gene expression levels in UC-MSCs, mouse derived spleen cells or FACS sorted CD45+ mouse cells after 24 hrs co-culture with UC-MSCs (n=3 biological replicates). n/d = not detected.


M FACS analysis of in vivo MitoT from UC - MSCs to Balb / c mouse mesenteric lymph node and spleen cells , 24 hours after intraperitoneal transplantation of 5 * 106 MitoTracker - labeled MSCs or without cells ( control ) ( n = 1 ) .

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M FACS analysis of in vivo MitoT from UC-MSCs to Balb/c mouse mesenteric lymph node and spleen cells, 24 hours after intraperitoneal transplantation of 5*106 MitoTracker-labeled MSCs or without cells (control) (n=1).


B Confocal microscopy images of human CD3 + T cells with endogenous MT - labeled with MitoTracker Red CMXRos ( white arrows , left panel ) or after mitoception with MitoTracker Green labeled - MT from UC - MSCs ( white asterisk , right panel ) .

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B Confocal microscopy images of human CD3+ T cells with endogenous MT-labeled with MitoTracker Red CMXRos (white arrows, left panel) or after mitoception with MitoTracker Green labeled-MT from UC-MSCs (white asterisk, right panel).


C FACS analysis of MitoT on CD3 + human T cells after mitoception with increasing amounts of UC - MSC MT corresponding to the same cell number ratios used in co - culture experiments ( n = 4 biological replicates , with 4 different donors of UC - MSCs ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

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C FACS analysis of MitoT on CD3+ human T cells after mitoception with increasing amounts of UC-MSC MT corresponding to the same cell number ratios used in co-culture experiments (n=4 biological replicates, with 4 different donors of UC-MSCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.


D Representative transmission electron microscopy images of FACS - sorted human CD3 + MitoTneg and CD3 + MitoTpos cells following mitoception with UC - MSC MT . Red arrows show human mitochondria .

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D Representative transmission electron microscopy images of FACS-sorted human CD3+MitoTneg and CD3+MitoTpos cells following mitoception with UC-MSC MT. Red arrows show human mitochondria.


Quantification of human MT in sorted CD3 + MitoTneg and MitoTpos cells after mitoception with UC - MSCs , by transmission electron microscopy ( n = 25 independent cells analyzed per group ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

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Quantification of human MT in sorted CD3+ MitoTneg and MitoTpos cells after mitoception with UC-MSCs, by transmission electron microscopy (n=25 independent cells analyzed per group). Graph shows mean ± SEM and statistical analysis by Student's t-test.


B Volcano plot analysis derived from FPKM values showing DE genes ( p < 0 . 05 , Student ' s t test ) in sorted MitoTneg and MitoTpos CD3 + cells ( from 4 different donors of PBMCs ) .

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B Volcano plot analysis derived from FPKM values showing DE genes (p<0.05, Student's t test) in sorted MitoTneg and MitoTpos CD3+ cells (from 4 different donors of PBMCs).


C Heatmap depiction of RNA - seq analysis of significantly ( p < 0 . 01 , Student ' s t test ) DE coding genes from sorted MitoTpos and MitoTneg CD3 + cells ( from 4 different donors of PBMCs ) .

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C Heatmap depiction of RNA-seq analysis of significantly (p<0.01, Student's t test) DE coding genes from sorted MitoTpos and MitoTneg CD3+ cells (from 4 different donors of PBMCs).


E Differentially expressed genes from the significantly enriched pathways listed in figure 2D . Highlighted in red are key markers of Treg phenotype .

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E Differentially expressed genes from the significantly enriched pathways listed in figure 2D. Highlighted in red are key markers of Treg phenotype.


F qRT - PCR analysis of genes related to T cell activation after mitoception with UC - MSC MT ( n = 4 biological replicates , with 4 different donors of PBMCs ) . The graph depicts fold expression in CD3 + MitoTpos cells relative to CD3 + MitoTneg cells , which are set at 1 . Graph shows mean ± SEM and statistical analysis by Student ' s t - test ( * p < 0 . 05 ) .

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F qRT-PCR analysis of genes related to T cell activation after mitoception with UC-MSC MT (n=4 biological replicates, with 4 different donors of PBMCs). The graph depicts fold expression in CD3+MitoTpos cells relative to CD3+MitoTneg cells, which are set at 1. Graph shows mean ± SEM and statistical analysis by Student's t-test (*p<0.05).


A Representative bright - field microscopy images of FACS - sorted CD4 + naïve MitoTneg ( upper panel ) and MitoTpos ( lower panel ) cells after 5 days in culture with plain media ( no cytokines added ) . Red arrows are indicative of activation / proliferation clusters .

A Representative bright-field microscopy images of FACS-sorted CD4+ naïve MitoTneg (upper panel) and MitoTpos (lower panel) cells after 5 days in culture with plain media (no cytokines added). Red arrows are indicative of activation/proliferation clusters.


B Representative FACS plots of Treg ( CD127lowCD25 + FoxP3 + ) populations after T cell differentiation on media with or without cytokines and antibody supplements ( anti - CD3 , IL - 2 and TGF - b1 ) .

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B Representative FACS plots of Treg (CD127lowCD25+FoxP3+) populations after T cell differentiation on media with or without cytokines and antibody supplements (anti-CD3, IL-2 and TGF-b1).


C FACS analysis of Treg induction after 5 - 7 days differentiation of FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells in plain ( n = 5 ) or differentiation media ( n = 6 ) ( Biological replicates , with different donors of PBMCs ) .

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C FACS analysis of Treg induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain (n=5) or differentiation media (n=6) (Biological replicates, with different donors of PBMCs).


D ICOS expression on FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells after 5 - 7 days in culture with differentiation media . Representative FACS plot ( left panel ) and bar graph ( right panel ) of ICOS high population ( n = 4 biological replicates ) .

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D ICOS expression on FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of ICOS high population (n=4 biological replicates).


E CCR7 expression on sorted CD4 + naïve MitoTneg and MitoTpos cells after 5 - 7 days in culture with differentiation media . Representative FACS plot ( left panel ) and bar graph ( right panel ) of CCR7 + population ( n = 4 biological replicates ) .

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E CCR7 expression on sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of CCR7+ population (n=4 biological replicates).


F FACS analysis of Th1 ( CD4 + IFNγ + ) induction after 5 - 7 days differentiation of FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells in plain media ( n = 3 biological replicates ) . n / s = not significant .

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F FACS analysis of Th1 (CD4+IFNγ+) induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain media (n=3 biological replicates). n/s= not significant.


Representative proliferation FACS histogram of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos cells ( ratio 1 : 1 ) after 5 - 7 days of CD4 + naïve cell activation with Treg induction / differentiation media ( anti - CD3 , IL - 2 and TGF - b1 ) . PBMC alone represents positive control of proliferation and PBMC co - culture with MitoTneg correspond to the inhibition control with induced Treg cells .

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Representative proliferation FACS histogram of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells (ratio 1:1) after 5-7 days of CD4+ naïve cell activation with Treg induction/differentiation media (anti-CD3, IL-2 and TGF-b1). PBMC alone represents positive control of proliferation and PBMC co-culture with MitoTneg correspond to the inhibition control with induced Treg cells.


C Percentage of immunosuppression of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos cells after 5 - 7 days of CD4 + naïve cell activation with differentiation media ( ratio MitoT cells : PBMC ) ( n = 3 biological replicates , with different donors of PBMCs ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

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C Percentage of immunosuppression of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells after 5-7 days of CD4+ naïve cell activation with differentiation media (ratio MitoT cells:PBMC) (n=3 biological replicates, with different donors of PBMCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.


B Representative FACS plots of mitoception ( % of MitoTracker + ) in CD4 + or CD8 + T cells from mitocepted PBMC compared to control PBMC , before intravenous injection .

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B Representative FACS plots of mitoception (% of MitoTracker+) in CD4+ or CD8+T cells from mitocepted PBMC compared to control PBMC, before intravenous injection.


C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 mice per group ) . Control group in grey , were untreated mice ( n = 6 mice ) . * p < 0 . 05 by Mann Whitney t - test .

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C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group). Control group in grey, were untreated mice (n=6 mice). * p<0.05 by Mann Whitney t-test.


D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount ( 12x106 cells ) of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 mice per group ) ( p = 0 . 0218 by log - rank test ) .

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D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount (12x106 cells) of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group) (p=0.0218 by log-rank test).


E FACS analysis of human Treg ( CD4 + CD25 + FoxP3 + ) cell engraftment in mouse spleen following transplantation of PBMC - MitoT or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) .

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E FACS analysis of human Treg (CD4+CD25+FoxP3+) cell engraftment in mouse spleen following transplantation of PBMC-MitoT or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


F FACS analysis of human pro - inflammatory T helper1 ( CD4 + IFNγ + , left panel ) and T cytotoxic ( CD8 + IFNγ + , right panel ) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) .

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F FACS analysis of human pro-inflammatory T helper1 (CD4+IFNγ+, left panel) and T cytotoxic (CD8+IFNγ+, right panel) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC - MitoT , PBMC and non - transplanted control samples , stained with Masson ' s trichrome staining . Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts . Arrowheads represents a reduced number of Paneth cells .

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G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC-MitoT, PBMC and non-transplanted control samples, stained with Masson's trichrome staining. Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts. Arrowheads represents a reduced number of Paneth cells.


H - I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC ( n = 8 independent mice ) or control PBMC transplanted mice ( n = 5 independent mice ) .

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H-I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice).


J Quantification of Paneth cells per crypt , from at least 50 crypts per PBMC - MitoT ( n = 8 mice ) , PBMC transplanted ( n = 5 mice ) or non - transplanted control ( n = 3 ) mouse .

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J Quantification of Paneth cells per crypt, from at least 50 crypts per PBMC-MitoT (n=8 mice), PBMC transplanted (n=5 mice) or non-transplanted control (n=3) mouse.


Representative histopathologic images of GVHD in lung sections obtained from PBMC - MitoT , PBMC and non - transplanted control , stained with Masson ' s trichrome staining .

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Representative histopathologic images of GVHD in lung sections obtained from PBMC-MitoT, PBMC and non-transplanted control, stained with Masson's trichrome staining.


L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC ( n = 8 independent mice ) or control PBMC transplanted mice ( n = 5 independent mice ) .

L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice).


M FACS analysis of total human CD4 + cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) .

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M FACS analysis of total human CD4+ cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


a , b , Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells ( a ) ( n = 5 / 4 ) and for autophagy proteins in primary intestinal epithelial cell scrapings ( b ) ( n = 3 ) .

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a, b, Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells (a) (n = 5/4) and for autophagy proteins in primary intestinal epithelial cell scrapings (b) (n = 3).


c , Transmission electron microscopy ( TEM ) of crypts . Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr ; IEC hypomorphic Paneth cells .

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c, Transmission electron microscopy (TEM) of crypts. Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr;IEC hypomorphic Paneth cells.


d , e , Crypt showing GFP - LC3 punctae ( d ) , quantified in e ( n = 10 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . Scale bar , 5 µm .

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d, e, Crypt showing GFP-LC3 punctae (d), quantified in e (n = 10; unpaired Student's t-test; mean ± s.e.m.). Scale bar, 5 µm.


f , g , Immunoblot of silenced MODE - K cells ( f ) and primary intestinal epithelial cell scrapings ( g ) for the PERK - eIF2α branch ( n = 3 ) .

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f, g, Immunoblot of silenced MODE-K cells (f) and primary intestinal epithelial cell scrapings (g) for the PERK-eIF2α branch (n = 3).


j , GFP - LC3 punctae per crypt after treatment with tamoxifen , and vehicle or salubrinal ( n = 10 ; one - way ANOVA with post - hoc Bonferroni ; mean ± s . e . m . ) .

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j, GFP-LC3 punctae per crypt after treatment with tamoxifen, and vehicle or salubrinal (n = 10; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).


k , Enteritis histology score after salubrinal and tamoxifen co - treatment ( n = 12 / 14 / 13 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . Results represent three ( a , f , g ) or two ( c , e , h , i ) independent experiments . * P 0 . 05 , * * * P 0 . 001 .

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k, Enteritis histology score after salubrinal and tamoxifen co-treatment (n = 12/14/13; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent three (a, f, g) or two (c, e, h, i) independent experiments. *P 0.05, ***P 0.001.


a , TEM of crypts of Lieberkühn ( n = 2 ) . Scale bars , 2 µm .

a, TEM of crypts of Lieberkühn (n = 2). Scale bars, 2 µm.


b , Representative haematoxylin and eosin stainings . Note transmural inflammation extending through muscularis propria ( white arrow ) into serosa ( black arrow ) in Atg7 / Xbp1Dgr ; IEC and Atg16l1 / Xbp1Dgr ; IEC mice scored in c and e . Scale bars , 50 µm , except for lower two panels ( 10 µm ) . c , Enteritis histology score ( n = 26 / 12 / 18 / 27 ; 10 - 18 weeks ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . e , Enteritis histology score ( n = 11 ; 18 weeks ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

b, Representative haematoxylin and eosin stainings. Note transmural inflammation extending through muscularis propria (white arrow) into serosa (black arrow) in Atg7/Xbp1Dgr;IEC and Atg16l1/Xbp1Dgr;IEC mice scored in c and e. Scale bars, 50 µm, except for lower two panels (10 µm). c, Enteritis histology score (n = 26/12/18/27; 10-18 weeks; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).e, Enteritis histology score (n = 11; 18 weeks; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). NS, not significant.


d , CryptTEM in indicated genotypes ( n = 2 ) . Scale bars , 2 µm .

d, CryptTEM in indicated genotypes (n = 2). Scale bars, 2 µm.


f , Xbp1 mRNA splicing ( Xbp1u , unspliced ; Xbp1s , spliced ) of crypts ; densitometry in g ( n = 7 ; unpaired Student ' s t - test ; mean ± s . e . m . ) .

O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f, Xbp1 mRNA splicing (Xbp1u, unspliced; Xbp1s, spliced) of crypts; densitometry in g (n = 7; unpaired Student's t-test; mean ± s.e.m.).


h , GRP78 ( green ) immunofluorescence , white arrows indicate GRP78 + crypts ( DAPI , blue ; n = 5 ) . Scale bars , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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h, GRP78 (green) immunofluorescence, white arrows indicate GRP78+ crypts (DAPI, blue; n = 5). Scale bars, 10 µm. *P 0.05, **P 0.01, ***P 0.001.


a , shCtrl or shXbp1 MODE - K cells were co - silenced for Atg16l1 ( siAtg16l1 ) or with scrambled siRNA ( siCtrl ) , and analysed by flow cytometry for annexin V and propidium iodide ( PI ; n = 3 ; one - way ANOVA with post - hoc Bonferroni ; mean ± s . e . m . ) .

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a, shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analysed by flow cytometry for annexin V and propidium iodide (PI; n = 3; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).


b , Immunoblot of primary intestinal epithelial cell scrapings ( n = 3 ) .

b, Immunoblot of primary intestinal epithelial cell scrapings (n = 3).


c , Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE - K cells after TNF stimulation .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O

c, Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE-K cells after TNF stimulation.


d , TUNEL + intestinal epithelial cells ( IECs ) per 100 crypts after BAY11 - 7082 or vehicle treatment ( n = 3 / 4 / 4 ; one - way ANOVA with post - hoc Holm ' s - corrected unpaired Student ' s t - test ; mean ± s . e . m . ) . e , Enteritis histology score of mice treated with BAY11 - 7082 or vehicle ( n = 10 / 10 / 9 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) .

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d, TUNEL+ intestinal epithelial cells (IECs) per 100 crypts after BAY11-7082 or vehicle treatment (n = 3/4/4; one-way ANOVA with post-hoc Holm's-corrected unpaired Student's t-test; mean ± s.e.m.). e, Enteritis histology score of mice treated with BAY11-7082 or vehicle (n = 10/10/9; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).


f , Immunoblot of intestinal epithelial cell scrapings for ( p - ) IRE1α ; after IRE1α ; immunoprecipitation ( IP ) . beta - actin , loading control of whole lysates .

O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

f, Immunoblot of intestinal epithelial cell scrapings for (p-)IRE1α; after IRE1α; immunoprecipitation (IP). beta-actin, loading control of whole lysates.


g , Immunoblot of primary intestinal epithelial cell scrapings ( n = 4 ) .

g, Immunoblot of primary intestinal epithelial cell scrapings (n = 4).


h , i , Enteritis histology score of indicated genotypes ( h , n = 15 / 16 / 14 / 15 ; i , n = 5 / 10 / 12 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) .

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h, i, Enteritis histology score of indicated genotypes (h, n = 15/16/14/15; i, n = 5/10/12; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).


j , Enteritis histology score of specific pathogen - free ( SPF ) and germ free ( GF ) housed mice ( n = 10 / 9 / 7 / 7 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . k , Representative images of p - IκBα immunoreactivity under conditions as in j ( n = 4 ) . Scale bars , 20 µm . Results represent four ( f ) , three ( c ) or two ( a , b ) independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

j, Enteritis histology score of specific pathogen-free (SPF) and germ free (GF) housed mice (n = 10/9/7/7; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). k, Representative images of p-IκBα immunoreactivity under conditions as in j (n = 4). Scale bars, 20 µm. Results represent four (f), three (c) or two (a, b) independent experiments. *P  0.05, **P  0.01, ***P  0.001.


a , Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days ( n = 3 ) . b , Enteritis histology score for experiment as in a ( n = 4 ; median shown ; Mann - Whitney U - test ) .

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a, Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days (n = 3). b, Enteritis histology score for experiment as in a (n = 4; median shown; Mann-Whitney U-test).


c , Representative images of EYFP - Rosa26 / D6 - cre + / - reporter mice and EYFP - Rosa26 ( controls ) . Co - localization of Defa6 Cre - driven EYFP expression ( yellow ) with lysozyme - expressing Paneth cells ( red ; n = 3 ) . DAPI , blue ; scale bars , 50 µm .

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c, Representative images of EYFP-Rosa26/D6-cre+/- reporter mice and EYFP-Rosa26 (controls). Co-localization of Defa6 Cre-driven EYFP expression (yellow) with lysozyme-expressing Paneth cells (red; n = 3). DAPI, blue; scale bars, 50 µm.


d , Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr ; PC and wild - type controls ( n = 2 ) .

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d, Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr;PC and wild-type controls (n = 2).


e , f , Representative confocal images of lysozyme ( green ) expressing Paneth cells ( e ) with quantification of crypts with indicated number of lysozyme + granulated dots in f ( n = 5 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . DAPI , blue ; scale bars , 10 µm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, f, Representative confocal images of lysozyme (green) expressing Paneth cells (e) with quantification of crypts with indicated number of lysozyme+ granulated dots in f (n = 5; unpaired Student's t-test; mean ± s.e.m.). DAPI, blue; scale bars, 10 µm.


g , Immunohistochemistry for p - eIF2α ( n = 3 ) . Scale bars , 20 µm .

g, Immunohistochemistry for p-eIF2α (n = 3). Scale bars, 20 µm.


h , Representative haematoxylin and eosin images of Xbp1Dgr ; PC and wild - type mice scored in i . Scale bars , 50 µm . i , Enteritis scoring in Xbp1fl / fl ( WTfl ) , Defa6 - cre + ( WTcre ) and Defa6 - cre + ; Xbp1fl / fl ( Xbp1Dgr ; PC ) mice ( n = 21 / 26 / 29 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . Results represent two ( b , d ) independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

h, Representative haematoxylin and eosin images of Xbp1Dgr;PC and wild-type mice scored in i. Scale bars, 50 µm. i, Enteritis scoring in Xbp1fl/fl (WTfl), Defa6-cre+ (WTcre) and Defa6-cre+;Xbp1fl/fl (Xbp1Dgr;PC) mice (n = 21/26/29; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent two (b, d) independent experiments. *P 0.05, **P 0.01, ***P 0.001.


The syntheses of the ligands PR - 924 , 9 , 14 , 16 , 17 and 18 have been described previously ( de Bruin et al , 2014 ) . All ligands share the peptide scaffold shown in the upper left corner with P1 , P2 and P3 side chains and an N - cap ; they are equipped with a l - Ala ( stereochemistry ( S ) ) or d - Ala ( stereochemistry ( R ) ) residue at P3 ( red ) and either an N - acylmorpholine or a 3 - methyl - 1H - indene group ( green ) . Furthermore , compounds differ in their P2 residue ( either methoxytyrosine or tryptophane ; blue ) . Inhibitor 9 , also termed LU - 015i , is endowed with an exceptional cyclohexyl residue ( gray ) , which is associated with increased selectivity for human β5i .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

The syntheses of the ligands PR-924, 9, 14, 16, 17 and 18 have been described previously (de Bruin et al, 2014). All ligands share the peptide scaffold shown in the upper left corner with P1, P2 and P3 side chains and an N-cap; they are equipped with a l-Ala (stereochemistry (S)) or d-Ala (stereochemistry (R)) residue at P3 (red) and either an N-acylmorpholine or a 3-methyl-1H-indene group (green). Furthermore, compounds differ in their P2 residue (either methoxytyrosine or tryptophane; blue). Inhibitor 9, also termed LU-015i, is endowed with an exceptional cyclohexyl residue (gray), which is associated with increased selectivity for human β5i.


A . Inhibition of human β5c and β5i by ONX 0914 and PR - 924 . IC50 values are deduced from the fitted data .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

A. Inhibition of human β5c and β5i by ONX 0914 and PR-924. IC50 values are deduced from the fitted data.


B . IC50 values [ µM ] for all compounds towards the yeast , human and mouse β5 subunits .

B. IC50 values [µM] for all compounds towards the yeast, human and mouse β5 subunits.


E . Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 ° C or 37 ° C . Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 °C or 37 °C. Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity.


F . Surface illustration of the yCP . The β5 and β6 subunits are depicted in dark gray , with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue , respectively . The β5 ' and β6 ' subunits of the second β - ring are hidden on the back of 20S proteasome .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

F. Surface illustration of the yCP. The β5 and β6 subunits are depicted in dark gray, with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue, respectively. The β5' and β6' subunits of the second β-ring are hidden on the back of 20S proteasome.


A . Superposition of the β5i / β6 active sites of genuine mouse iCP and the hβ5i / hβ6 human / yeast chimera illustrates that Met45 adopts the same ' open ' conformation in both structures . Amino acid side chains important for substrate binding are depicted as sticks . The catalytic Thr1 is colored in black . Met31 , the single amino acid difference between mouse and human β5i active sites , is highlighted in green .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

A. Superposition of the β5i/β6 active sites of genuine mouse iCP and the hβ5i/hβ6 human/yeast chimera illustrates that Met45 adopts the same 'open' conformation in both structures. Amino acid side chains important for substrate binding are depicted as sticks. The catalytic Thr1 is colored in black. Met31, the single amino acid difference between mouse and human β5i active sites, is highlighted in green.


B . Superposition of the genuine mouse and the human chimeric β5c / β6 active sites according to panel A proves the proper orientation of Met45 in the ' closed ' conformation .

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

B. Superposition of the genuine mouse and the human chimeric β5c/β6 active sites according to panel A proves the proper orientation of Met45 in the 'closed' conformation.


C . Superposition of the β5c / β6 and β5i / β6 active sites of the natural mouse cCP and iCP according to panel A . Note the distinct conformations of Met45 ( Huber et al , 2012 ) .

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C. Superposition of the β5c/β6 and β5i/β6 active sites of the natural mouse cCP and iCP according to panel A. Note the distinct conformations of Met45 (Huber et al, 2012).


D . Superposition of the hβ5c / hβ6 and the hβ5i / hβ6 chimeric proteasomes confirm the distinct positions of Met45 analogously to the natural mouse subunits shown in panel C .

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D. Superposition of the hβ5c/hβ6 and the hβ5i/hβ6 chimeric proteasomes confirm the distinct positions of Met45 analogously to the natural mouse subunits shown in panel C.


A . Complex structures of the human β5c / β6 ( left ) and β5i / β6 ( right ) chimeric proteasomes with PR - 924 . The ligand binds in a linear ( l ) manner to the β5c / β6 substrate binding channel , but in a kinked ( k ) conformation to the β5i / β6 active site . The 2FO - FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are shown as blue meshes contoured at 1σ . The inhibitor and Thr1 have been omitted for phasing .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

A. Complex structures of the human β5c/β6 (left) and β5i/β6 (right) chimeric proteasomes with PR-924. The ligand binds in a linear (l) manner to the β5c/β6 substrate binding channel, but in a kinked (k) conformation to the β5i/β6 active site. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are shown as blue meshes contoured at 1σ. The inhibitor and Thr1 have been omitted for phasing.


B . Comparison of the conformations of ONX 0914 , PR - 924 , 14 , 16 , 17 and 18 bound to the chimeric hβ5 / hβ6 substrate binding channels . The l amino acid compounds ONX 0914 and 14 display a linear ( l ) binding mode to the hβ5c / hβ6 ( left ) and the hβ5i / hβ6 ( right ) chimeras , whereas the ligands 16 and 17 ( P3 - d - Ala and morpholine cap ) adopt a kinked ( k ) conformation in both β5 substrate binding channels . Remarkably , the orientation of PR - 924 and its analogue 18 ( P3 - d - Ala and 3 - methyl - 1H - indene cap ) in the hβ5c / hβ6 chimera ( and in WT yβ5 ; see Appendix Figure S5 ) ) differs from that observed with the hβ5i / hβ6 chimera . Note that the inhibitors are rotated by about 45 ° compared to panel A .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

B. Comparison of the conformations of ONX 0914, PR-924, 14, 16, 17 and 18 bound to the chimeric hβ5/hβ6 substrate binding channels. The l amino acid compounds ONX 0914 and 14 display a linear (l) binding mode to the hβ5c/hβ6 (left) and the hβ5i/hβ6 (right) chimeras, whereas the ligands 16 and 17 (P3-d-Ala and morpholine cap) adopt a kinked (k) conformation in both β5 substrate binding channels. Remarkably, the orientation of PR-924 and its analogue 18 (P3-d-Ala and 3-methyl-1H-indene cap) in the hβ5c/hβ6 chimera (and in WT yβ5; see Appendix Figure S5)) differs from that observed with the hβ5i/hβ6 chimera. Note that the inhibitors are rotated by about 45 ° compared to panel A.


A . Complex structures of the mouse chimeric proteasome ( left ) and the hβ5i - V31M / hβ6 mutant ( right ) with PR - 924 visualize the kinked ( k ) orientation . The 2FO - FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are depicted according to Fig 5A . Steric hindrance of Met31 ( green ) with the bulky 3 - methyl - 1H - indene cap is indicated by black double arrows .

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A. Complex structures of the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right) with PR-924 visualize the kinked (k) orientation. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are depicted according to Fig 5A. Steric hindrance of Met31 (green) with the bulky 3-methyl-1H-indene cap is indicated by black double arrows.


B . Superposition of inhibitors bound to the mouse chimeric proteasome ( left ) and the hβ5i - V31M / hβ6 mutant ( right ) . Compounds display the same binding modes ( kinked ( k ) or linear ( l ) ) to the mβ5i / mβ6 active site as to the corresponding human β5i / β6 subunit ( Fig . 5 ) and to the hβ5i - V31M / hβ6 mutant . Note that the inhibitors are rotated by about 45 ° compared to panel A .

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B. Superposition of inhibitors bound to the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right). Compounds display the same binding modes (kinked (k) or linear (l)) to the mβ5i/mβ6 active site as to the corresponding human β5i/β6 subunit (Fig. 5) and to the hβ5i-V31M/hβ6 mutant. Note that the inhibitors are rotated by about 45° compared to panel A.


C . Superposition of chimeric mammalian and WT yeast β5 active sites bound to the P3 - d - Ala compounds PR - 924 and 18 . The nucleophilic Thr1 is depicted as well as the S1 pocket forming residues 31 and 45 . Residue 31 impacts on the position of the 3 - methyl - 1H - indene cap of PR - 924 and 18 . Due to steric hindrance , the N - cap is shifted in the mouse and in the hβ5i - V31M / hβ6 chimera compared to the hβ5i / hβ6 mutant proteasome crystal structures ( black double arrow ) . Note that the structures are rotated by 90 ° compared to panel B .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

C. Superposition of chimeric mammalian and WT yeast β5 active sites bound to the P3-d-Ala compounds PR-924 and 18. The nucleophilic Thr1 is depicted as well as the S1 pocket forming residues 31 and 45. Residue 31 impacts on the position of the 3-methyl-1H-indene cap of PR-924 and 18. Due to steric hindrance, the N-cap is shifted in the mouse and in the hβ5i-V31M/hβ6 chimera compared to the hβ5i/hβ6 mutant proteasome crystal structures (black double arrow). Note that the structures are rotated by 90° compared to panel B.


ONX 0914 binds in a linear mode to all active sites . Changing the stereochemistry at the P3 site from l - Ala ( S ) to d - Ala ( R ) leads to a kinked conformation in the β5 substrate binding channels ( compound 16 ) . Substitution of the morpholine cap of 16 by a larger 3 - metyhl - 1H - indene moiety yields PR - 924 and results in different inhibitor conformations at the yβ5 / hβ5c and the mammalian β5i active sites . Hereby , the selectivity for hβ5i over hβ5c is drastically increased ( IC50 ratio hβ5c / hβ5i : 255 ) compared to ONX 0914 ( IC50 ratio hβ5c / hβ5i : 20 ) and compound 16 ( IC50 ratio hβ5c / hβ5i : 5 . 2 , Fig 2 ) . Residue Met31 of mβ5i sterically hinders the kinked binding mode of PR - 924 , thereby creating 7 - fold selectivity for human β5i .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

ONX 0914 binds in a linear mode to all active sites. Changing the stereochemistry at the P3 site from l-Ala (S) to d-Ala (R) leads to a kinked conformation in the β5 substrate binding channels (compound 16). Substitution of the morpholine cap of 16 by a larger 3-metyhl-1H-indene moiety yields PR-924 and results in different inhibitor conformations at the yβ5/hβ5c and the mammalian β5i active sites. Hereby, the selectivity for hβ5i over hβ5c is drastically increased (IC50 ratio hβ5c/hβ5i: 255) compared to ONX 0914 (IC50 ratio hβ5c/hβ5i: 20) and compound 16 (IC50 ratio hβ5c/hβ5i: 5.2, Fig 2). Residue Met31 of mβ5i sterically hinders the kinked binding mode of PR-924, thereby creating 7-fold selectivity for human β5i.


A : Snapshots of LimE - GFP , GFP - myo , and corresponding kymographs for type 1 cells . Corresponding speeds are 10 . 8µm / min and 9 . 4µm / min . White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint , as extracted from the corresponding stress kymographs . C : Snapshots of LimE - GFP , GFP - myo , and corresponding kymographs for type 2 cells . Corresponding speeds are 5 . 6µm / min and 8 . 7µm / min . White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint .

O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 1 cells. Corresponding speeds are 10.8µm/min and 9.4µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint, as extracted from the corresponding stress kymographs. C: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 2 cells. Corresponding speeds are 5.6µm/min and 8.7µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint.


B : Stress maps quantifying the magnitude of the force per area using a color scale with blue / red corresponding to small / large stresses and the direction of forces using vectors . Fan - shaped cells were rotated so that the vertical ( x ) axis is the direction of motion ( see Methods ) . Shown are the overall stress T , the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker , and the corresponding Tx and Ty kymographs along the cells outlines . D : Stress maps of T , Tx and Ty for the type 2 cell with the LimE marker , and corresponding Tx and Ty kymographs along the cells outlines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

B: Stress maps quantifying the magnitude of the force per area using a color scale with blue/red corresponding to small/large stresses and the direction of forces using vectors. Fan-shaped cells were rotated so that the vertical (x) axis is the direction of motion (see Methods). Shown are the overall stress T, the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker, and the corresponding Tx and Ty kymographs along the cells outlines. D: Stress maps of T, Tx and Ty for the type 2 cell with the LimE marker, and corresponding Tx and Ty kymographs along the cells outlines.


A : Sequence of snapshots of the LimE - GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP - myo distribution for a separate oscillatory cell . Corresponding speeds are 7 . 0µm / min and 4 . 6µm / min . The sequences show an entire cycle of oscillation ( spreading and contraction ) . White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A: Sequence of snapshots of the LimE-GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP-myo distribution for a separate oscillatory cell. Corresponding speeds are 7.0µm/min and 4.6µm/min. The sequences show an entire cycle of oscillation (spreading and contraction). White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists.


A : Sequence of snapshots of the LimE - GFP distribution and stress T for an amoeboid cell and the GFP - myo distribution for a separate amoeboid cell . Corresponding speeds are 7 . 4µm / min and 4 . 5µm / min . The sequences show both the phases of protrusion and retraction . White arrows represent the direction of motion .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A: Sequence of snapshots of the LimE-GFP distribution and stress T for an amoeboid cell and the GFP-myo distribution for a separate amoeboid cell. Corresponding speeds are 7.4µm/min and 4.5µm/min. The sequences show both the phases of protrusion and retraction. White arrows represent the direction of motion.


A : Protrusion and retraction speed for amoeboid , oscillatory and fan - shaped cells , defined as the average of the pixels with the 20 % lowest and highest membrane speed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A: Protrusion and retraction speed for amoeboid, oscillatory and fan-shaped cells, defined as the average of the pixels with the 20% lowest and highest membrane speed.


C : Average edge velocity in regions of low and high LimE - GFP and GFP - myo fluorescence . High fluorescence was defined as the 20 % brightest LimE - GFP and GFP - myo pixels in the kymographs while low fluorescence consisted of the remaining 80 % pixels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C: Average edge velocity in regions of low and high LimE-GFP and GFP-myo fluorescence. High fluorescence was defined as the 20% brightest LimE-GFP and GFP-myo pixels in the kymographs while low fluorescence consisted of the remaining 80% pixels.


( C - G ) Resembling the phenotype caused by Notch overexpression ( D ) or dSir2 mutation ( E ) , HP1c knockdown ( F ) and mutant ( G ) flies also exhibit supernumerary anterior scutellar ( aSC ) bristles on the notum , as indicated by the enlarged views in the left bottom panes . Scale bars , 100 μm .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-G) Resembling the phenotype caused by Notch overexpression (D) or dSir2 mutation (E), HP1c knockdown (F) and mutant (G) flies also exhibit supernumerary anterior scutellar (aSC) bristles on the notum, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm.


( H - M ) Compared with control flies ( H and K ) , HP1c knockdown ( J ) and mutant ( M ) flies exhibit an ectopic vein at the distal of L4 and L5 ( arrows ) and a broken posterior cross vein ( arrowhead ) , which is similar to the phenotype caused by Notch overexpression ( I ) and dSir2 mutant ( L ) . Scale bars , 500 μm .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(H-M) Compared with control flies (H and K), HP1c knockdown (J) and mutant (M) flies exhibit an ectopic vein at the distal of L4 and L5 (arrows) and a broken posterior cross vein (arrowhead), which is similar to the phenotype caused by Notch overexpression (I) and dSir2 mutant (L). Scale bars, 500 μm.


( A - D ) The posterior midguts of 10 - day - old control ( A ) , HP1c knockdown ( B ) , HP1c null mutant ( C ) and HP1c overexpression flies ( D ) . Arrows mark ISCs , arrowheads mark ee cells , circles mark ISC - EB , EB - EB or ISC - ISC nests . ISCs were visualized with esg - GFP alone , EBs were stained with both esg - GFP and Su ( H ) - lacZ , while ee cells were labeled by Pros and DNA was marked by DAPI . Scale bars , 30 μm .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(A-D) The posterior midguts of 10-day-old control (A), HP1c knockdown (B), HP1c null mutant (C) and HP1c overexpression flies (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-EB, EB-EB or ISC-ISC nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.


( E - I ) Confocal images of the posterior midgut clones of the indicated genotypes , marked by GFP ( dotted lines ) . Arrows mark ISCs / EBs , arrowheads mark ee cells , and asterisks mark ECs . DNA was visualized with DAPI and ee cells were stained with Pros . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

(E-I) Confocal images of the posterior midgut clones of the indicated genotypes, marked by GFP (dotted lines). Arrows mark ISCs/EBs, arrowheads mark ee cells, and asterisks mark ECs. DNA was visualized with DAPI and ee cells were stained with Pros. Scale bars, 10 μm.


( A - D ) Compared with the control ( A ) flies , posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells ( B ) , while HP1c OE flies show increased ISCs and ee cells ( C ) , and the phenotype of Notch OE can be rescued by HP1c OE ( D ) . Arrows mark ISCs , arrowheads mark ee cells , circles mark ISC - ISC , ISC - EB or EB - EB nests . ISCs were visualized with esg - GFP alone , EBs were stained with both esg - GFP and Su ( H ) - lacZ , while ee cells were labeled by Pros and DNA was marked by DAPI . Scale bars , 30 μm .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

(A-D) Compared with the control (A) flies, posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells (B), while HP1c OE flies show increased ISCs and ee cells (C), and the phenotype of Notch OE can be rescued by HP1c OE (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-ISC, ISC-EB or EB-EB nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.


ChIP - qPCR results show the association of HP1c with Notch target genes compared to RFP control . The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin ( equivalent to 1 ) . Error bars indicate SEM . n = 3 technical replicates . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ChIP-qPCR results show the association of HP1c with Notch target genes compared to RFP control. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). Error bars indicate SEM. n=3 technical replicates. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).


( B ) RT - qPCR analysis determined the expression of Notch target genes when HP1c was depleted ( n = 3 technical replicates , , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) RT-qPCR analysis determined the expression of Notch target genes when HP1c was depleted (n=3 technical replicates,, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, ***p < 0.001).


NRE - GFP signals of S2 cells carrying NICD OE , Hairless KD , Su ( H ) KD , dSir2 KD and HP1c KD , and the statistical results ( F ) ( n = 4 biological replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

NRE-GFP signals of S2 cells carrying NICD OE, Hairless KD, Su(H) KD, dSir2 KD and HP1c KD , and the statistical results (F) (n=4 biological replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).


( D , E ) The interaction between HP1c and Su ( H ) is further confirmed through co - immunoprecipitation ( Co - IP ) . Plasmids expressing HP1c - eGFP and Su ( H ) - flag were transfected into S2 cells . Immunoprecipitation and western blotting were performed with the indicated antibodies .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(D, E) The interaction between HP1c and Su(H) is further confirmed through co-immunoprecipitation (Co-IP). Plasmids expressing HP1c-eGFP and Su(H)-flag were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies.


( B ) The relative binding of HP1c with Notch target genes when Su ( H ) KD using ChIP - qPCR analysis ( n = 3 technical replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) . ( C ) The relative binding of HP1c on Notch target genes when NICD OE using ChIP - qPCR analysis . ( n = 3 technical replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * * * p < 0 . 001 ) .

O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The relative binding of HP1c with Notch target genes when Su(H) KD using ChIP-qPCR analysis (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01). (C) The relative binding of HP1c on Notch target genes when NICD OE using ChIP-qPCR analysis. (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (***p < 0.001).


( M ) Statistical results of the number of ISCs and ee cells per unit area ( 200 μm × 200 μm ) in ( D - F ) . More than twenty - five midguts were examined for each group . Error bars indicate SEM . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Statistical results of the number of ISCs and ee cells per unit area (200 μm × 200 μm) in (D-F). More than twenty-five midguts were examined for each group. Error bars indicate SEM. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01).


( A ) Representative images showing the myogenic differentiation , as assessed by immunostaining for MyHC ( green ) , of human MuSCs ( hMuSCs ) isolated from muscle biopsies of DMD patients , either before the beginning of the clinical trial ( hMuSCs ( - ) ) or after 1 year of treatment with Givinostat ( hMuSCs Giv ( - ) ) . Cells were cultured alone or ( only for hMuSCs - ) in transwell co - culture with FAPs isolated from the same patients and exposed in vitro to Giv ( + ehFAPs Giv Vitro ) or control vehicle ( + ehFAPs ) . The upper panels represent myogenic differentiation of DMD - 1 patient ; bottom panels represent myogenic differentiation of DMD - 2 patient . Dose and timing of Giv treatment as described in Bettica et al . , 2016 ( 23 ) . Scale bar = 50 μm .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images showing the myogenic differentiation, as assessed by immunostaining for MyHC (green), of human MuSCs (hMuSCs) isolated from muscle biopsies of DMD patients, either before the beginning of the clinical trial (hMuSCs (-)) or after 1 year of treatment with Givinostat (hMuSCs Giv (-)). Cells were cultured alone or (only for hMuSCs -) in transwell co-culture with FAPs isolated from the same patients and exposed in vitro to Giv (+ehFAPs Giv Vitro) or control vehicle (+ehFAPs). The upper panels represent myogenic differentiation of DMD-1 patient; bottom panels represent myogenic differentiation of DMD-2 patient. Dose and timing of Giv treatment as described in Bettica et al., 2016 (23). Scale bar = 50 μm.


( C ) Representative images showing the myogenic differentiation of MuSCs isolated from 1 . 5 month old mdx mice , as assessed by immunostaining for MyHC ( green ) . MuSCs were cultured alone ( - ) or in transwell co - culture with mdx FAPs , which were pretreated with DMSO or GW4869 ( GW 10mM added to FAPs 30 minutes before starting the co - culture with MuSCs ) . Scale bar = 50 μm . ( D ) Graph showing the fusion index of MuSCs in the conditions described in ( C ) , ( n = 5 , biological replicates ) . Star ( * ) indicates statistical analysis by Tukey test relative to MuSCs cultured alone ( - ) , * * p < 0 . 01 ; Hash ( # ) indicates statistical analysis by Tukey test relative to MuSCs in co - cultured with FAPs not treated ( DMSO ) , # p < 0 . 05 ; § represents statistical analysis by 2Way Anova test ; § § § p < 0 , 001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images showing the myogenic differentiation of MuSCs isolated from 1.5 month old mdx mice, as assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with mdx FAPs, which were pretreated with DMSO or GW4869 (GW 10mM added to FAPs 30 minutes before starting the co-culture with MuSCs). Scale bar = 50 μm. (D) Graph showing the fusion index of MuSCs in the conditions described in (C), (n=5, biological replicates). Star (*) indicates statistical analysis by Tukey test relative to MuSCs cultured alone (-), ** p < 0.01; Hash (#) indicates statistical analysis by Tukey test relative to MuSCs in co-cultured with FAPs not treated (DMSO), # p<0.05; § represents statistical analysis by 2Way Anova test; §§§ p < 0,001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.