Figure 3Quantitative real - time PCR ( RNU1A1 and RNU5G - normalized ) showing blockade of microRNA transfer from CD4 + T cells pre - treated with siRNA targeting the exosome secretion mediator Rab27 . Bar charts represent the mean values ± SEM of at least 4 independent experiments . Quantitative real - time PCR showing mRNA target down - modulation after IS formation between DICER - KO B cells and CD4 + T cells nucleofected with either siRab27 or scrambled control siRNA , with normalization to GAPDH . Bar charts represent the mean values ± SEM of at least 4 independent experiments . Representative dot - plots , showing IgG1 expression of gated B220 + B cells after IS formation as in ( G ) ; the bar chart shows the mean fluorescence Intensity ± SEM and quantification of 3 independent experiments is shown . Representative plot ( left ) and quantification ( right ) of the reconstitution of the B cell and T cell compartments after adoptive transfer . The graph shows the mean percentage of CD19 and CD4 positive cells from n ≥ 5 reconstituted mice ± SEM . Representative flow cytometry plot ( left ) and quantification ( right ) of GL7 and CD95 / Fas expression in B cells . The graph shows the mean percentage of GL7 + Fas + cells from n ≥ 5 reconstituted mice ± SEM . Representative dot - plot analysis showing CXCR4 and CD86 expression on B cells for Dark zone ( DZ ) and Light zone ( LZ ) analysis ( gated on GC GL7 + Fas + B cells ) . The graph shows the mean percentage of DZ ( CXCR4 + CD86 - ) and LZ ( CXCR4 - CD86 + ) populations from n ≥ 5 reconstituted mice ± SEM . ELISA quantification of IgM ( H ) and IgG ( I ) in serum from immunized adoptively transferred mice . The graph shows the mean serum concentration of IgM and IgG from n ≥ 5 reconstituted mice , respectively ± SEM .

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Figure 3Quantitative real-time PCR (RNU1A1 and RNU5G- normalized) showing blockade of microRNA transfer from CD4+ T cells pre-treated with siRNA targeting the exosome secretion mediator Rab27. Bar charts represent the mean values ± SEM of at least 4 independent experiments.Quantitative real-time PCR showing mRNA target down-modulation after IS formation between DICER-KO B cells and CD4+ T cells nucleofected with either siRab27 or scrambled control siRNA, with normalization to GAPDH. Bar charts represent the mean values ± SEM of at least 4 independent experiments.Representative dot-plots, showing IgG1 expression of gated B220+ B cells after IS formation as in (G); the bar chart shows the mean fluorescence Intensity ± SEM and quantification of 3 independent experiments is shown.Representative plot (left) and quantification (right) of the reconstitution of the B cell and T cell compartments after adoptive transfer. The graph shows the mean percentage of CD19 and CD4 positive cells from n ≥ 5 reconstituted mice ± SEM.Representative flow cytometry plot (left) and quantification (right) of GL7 and CD95/Fas expression in B cells. The graph shows the mean percentage of GL7+Fas+ cells from n ≥ 5 reconstituted mice ± SEM.Representative dot-plot analysis showing CXCR4 and CD86 expression on B cells for Dark zone (DZ) and Light zone (LZ) analysis (gated on GC GL7+ Fas+ B cells). The graph shows the mean percentage of DZ (CXCR4+CD86-) and LZ (CXCR4-CD86+) populations from n ≥ 5 reconstituted mice ± SEM.ELISA quantification of IgM (H) and IgG (I) in serum from immunized adoptively transferred mice. The graph shows the mean serum concentration of IgM and IgG from n ≥ 5 reconstituted mice, respectively ± SEM.


Figure 1Domain architecture and structure of Miro . ( A ) Bar diagram of Miro domain structure . Numbering corresponds to dMiro isoform D , domain names are described in the text ( B ) Ribbon diagram of the MiroS structure in the ' side view ' . Note three distinct domains : ELM1 ( blue ) , ELM2 ( green ) , cGTPase ( grey / yellow ) , joined by two linkers : Lnk1 ( orange ) , and Lnk2 ( red ) . The structure shown is MgGDP ‐ MiroS , with the Ca2 + ion from the Ca ‐ MiroS structure overlaid for orientation ( Ca2 + / Mg2 + / GDP as yellow / purple / multicoloured spheres ) . cGTPase , C ‐ terminal GTPase ; dMiro , Drosophila Miro ; ELM , EF hand pair with ligand mimic .

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Figure 1Domain architecture and structure of Miro. (A) Bar diagram of Miro domain structure. Numbering corresponds to dMiro isoform D, domain names are described in the text (B) Ribbon diagram of the MiroS structure in the 'side view'. Note three distinct domains: ELM1 (blue), ELM2 (green), cGTPase (grey/yellow), joined by two linkers: Lnk1 (orange), and Lnk2 (red). The structure shown is MgGDP‐MiroS, with the Ca2+ ion from the Ca‐MiroS structure overlaid for orientation ( Ca2+/Mg2+/GDP as yellow/purple/multicoloured spheres). cGTPase, C‐terminal GTPase; dMiro, Drosophila Miro; ELM, EF hand pair with ligand mimic.


Figure 2 ( A ) Top , MiroS shown in the ' top view ' . ELM1 and ELM2 are related by pseudo twofold symmetry , represented by the dyad between them . Bottom , overlay of the two ELM domains ( rmsd of 1 . 8 Å over 84 Cα positions ) . ELM1 ( blue ) , ELM2 ( green ) and identical or similar conserved residues in identical positions are shown as sticks . ( B ) Comparison of the ELM domain hydrophobic cores . The ELM1 core contains the UNL , modelled here as homoserine shown in spheres , occupying a ∼ 442 Å3 cavity highlighted with mesh . The ELM2 core includes three tryptophans in close proximity ( W367 , W391 , W395 in orange ) . Hydrophobic residues in the LM / EF hand pair interfaces are also shown in sticks . LM , ligand mimic ; UNL , unidentified ligand .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Top, MiroS shown in the 'top view'. ELM1 and ELM2 are related by pseudo twofold symmetry, represented by the dyad between them. Bottom, overlay of the two ELM domains (rmsd of 1.8 Å over 84 Cα positions). ELM1 (blue), ELM2 (green) and identical or similar conserved residues in identical positions are shown as sticks.(B) Comparison of the ELM domain hydrophobic cores. The ELM1 core contains the UNL, modelled here as homoserine shown in spheres, occupying a ∼442 Å3 cavity highlighted with mesh. The ELM2 core includes three tryptophans in close proximity (W367, W391, W395 in orange). Hydrophobic residues in the LM/EF hand pair interfaces are also shown in sticks. LM, ligand mimic; UNL, unidentified ligand.


Figure 3Details of the EF hand loops . All loops are shown in approximately the same orientation , with entering and exiting helices ghosted for clarity . Residues of note are coloured in orange . cEF1 is shown in its apo state . cEF2 is shown in its apo , Ca2 + ‐ bound and Mg2 + ‐ bound states . In the cEF2 apo state , E354 is stabilized by nearby loop residue R344 . In the Ca2 + ‐ bound state , cEF2 loop residues coordinate Ca2 + in a pentagonal , bipyramidal arrangement , with E354 as a bidentate ligand . An ordered water molecule at the apex of one of the pyramids is represented as a small red sphere . In the Mg2 + ‐ bound state , the coordination is octahedral with E354 as a monodentate ligand . The hEF1 and hEF2 loops adopt a conformation quite different from the cEF hands , and also lack the acidic residues that typically coordinate cations . Note the conserved hEF1 272VTL274 and hEF2 386VTL388 residues , which overlay identically in Fig 2A . cEF , canonical EF hand ; hEF , hidden EF hand .

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Figure 3Details of the EF hand loops. All loops are shown in approximately the same orientation, with entering and exiting helices ghosted for clarity. Residues of note are coloured in orange. cEF1 is shown in its apo state. cEF2 is shown in its apo, Ca2+‐bound and Mg2+‐bound states. In the cEF2 apo state, E354 is stabilized by nearby loop residue R344. In the Ca2+‐bound state, cEF2 loop residues coordinate Ca2+ in a pentagonal, bipyramidal arrangement, with E354 as a bidentate ligand. An ordered water molecule at the apex of one of the pyramids is represented as a small red sphere. In the Mg2+‐bound state, the coordination is octahedral with E354 as a monodentate ligand. The hEF1 and hEF2 loops adopt a conformation quite different from the cEF hands, and also lack the acidic residues that typically coordinate cations. Note the conserved hEF1 272VTL274 and hEF2 386VTL388 residues, which overlay identically in Fig 2A. cEF, canonical EF hand; hEF, hidden EF hand.


Figure 4 ( A ) The ELM2 / cGTPase interface can be divided into three regions ( insets 1 - 3 ) . Select residues are shown as sticks , polar contacts as dashed lines , and Mg2 + and MgGDP as spheres . Region 1 : αC2 and the hEF2 loop form both backbone and side ‐ chain contacts with αG1 and Switch I in the cGTPase , including the Switch I threonine T481 . Region 2 : αD2 in hEF2 packs against the twisting cGTPase β ‐ strands Gβ1 ‐ 3 . Region 3 : the candidate Pink1 phosphorylation site residue S324 in Lnk1 hydrogen bonds with R444 in Lnk2 , which also forms a salt bridge with D521 , at the end of the Switch II loop . ( B ) cGTPase rearrangements in MgGDP binding . Displayed is the MiroS ‐ MgGDP structure , with select residues from the aligned apo ‐ MiroS structure shown in orange . GDP is shown as sticks ( 2Fo ‐ Fc map , 1 . 2σ ) , and Mg2 + as a purple sphere . In the apo state , D505 in the Switch II 503DIDV506 motif is far from the nucleotide ‐ binding pocket and is hydrogen bonded to R507 , which is also stabilized by interactions with P512 . In the MgGDP ‐ bound state , the D505 side ‐ chain rotates ∼ 7 Å to interact with the β ‐ phosphate ( arrow ) , locking the Switch II loop in a β ‐ strand conformation and extending Gβ3 . V506 rotates away from the nucleotide ‐ binding pocket , and both R507 and Y537 become solvent exposed . cGTPase , C ‐ terminal GTPase ; Sw I / II , Switch I / II ; P loop , phosphate ‐ binding loop .

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Figure 4(A) The ELM2/cGTPase interface can be divided into three regions (insets 1-3). Select residues are shown as sticks, polar contacts as dashed lines, and Mg2+ and MgGDP as spheres. Region 1: αC2 and the hEF2 loop form both backbone and side‐chain contacts with αG1 and Switch I in the cGTPase, including the Switch I threonine T481. Region 2: αD2 in hEF2 packs against the twisting cGTPase β‐strands Gβ1‐3. Region 3: the candidate Pink1 phosphorylation site residue S324 in Lnk1 hydrogen bonds with R444 in Lnk2, which also forms a salt bridge with D521, at the end of the Switch II loop.(B) cGTPase rearrangements in MgGDP binding. Displayed is the MiroS‐MgGDP structure, with select residues from the aligned apo‐MiroS structure shown in orange. GDP is shown as sticks (2Fo‐Fc map, 1.2σ), and Mg2+ as a purple sphere. In the apo state, D505 in the Switch II 503DIDV506 motif is far from the nucleotide‐binding pocket and is hydrogen bonded to R507, which is also stabilized by interactions with P512. In the MgGDP‐bound state, the D505 side‐chain rotates ∼7 Å to interact with the β‐phosphate (arrow), locking the Switch II loop in a β‐strand conformation and extending Gβ3. V506 rotates away from the nucleotide‐binding pocket, and both R507 and Y537 become solvent exposed. cGTPase, C‐terminal GTPase; Sw I/II, Switch I/II; P loop, phosphate‐binding loop.


Figure 5Location of known Parkin ubiquitination sites within the Miro structure . MiroS is shown in a 180 ° ‐ rotated top view ( relative to Fig 2 ) . Four residues in dMiro correspond in sequence and structure to residues that are ubiquitinated by Parkin in human Miro1 ( hMiro1 ) or human Miro2 ( hMiro2 ) [ 22 ] . These are shown as spheres ( K220 , R240 , K439 and K600 ; labelled with dMiro numbering ) . An alignment of a 10 ‐ aa segment of dMiro , hMiro1 and hMiro2 flanking each ubiquitinated site is shown . The large blue ' K ' within each sequence alignment indicates the ubiquitinated residue in hMiro1 or hMiro2 , and a smaller , underlined K indicates that dMiro has a lysine in the same position . Residue S324 , a candidate Pink1 phosphorylation site , is also shown in spheres . Note the side ‐ chain of K439 is disordered in the MiroS structure , so it is modelled as an alanine . dMiro / Dm Miro , Drosophila Miro ; Hs Miro 1 / 2 , human Miro1 / 2 .

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Figure 5Location of known Parkin ubiquitination sites within the Miro structure. MiroS is shown in a 180°‐rotated top view (relative to Fig 2). Four residues in dMiro correspond in sequence and structure to residues that are ubiquitinated by Parkin in human Miro1 (hMiro1) or human Miro2 (hMiro2) [22]. These are shown as spheres (K220, R240, K439 and K600; labelled with dMiro numbering). An alignment of a 10‐aa segment of dMiro, hMiro1 and hMiro2 flanking each ubiquitinated site is shown. The large blue 'K' within each sequence alignment indicates the ubiquitinated residue in hMiro1 or hMiro2, and a smaller, underlined K indicates that dMiro has a lysine in the same position. Residue S324, a candidate Pink1 phosphorylation site, is also shown in spheres. Note the side‐chain of K439 is disordered in the MiroS structure, so it is modelled as an alanine. dMiro/Dm Miro, Drosophila Miro; Hs Miro 1/2, human Miro1/2.


Figure 1HEK293T cells were transfected for 48 hours with each mitoTev - TALE ( 11 . 5 and 8 . 5 RVDs ) and total cell extracts were used for western - blots with anti - T2A antibody . Molecular weights of the two different mitoTev - TALEs are ~ 85KD and ~ 70KDa , respectivelyImmunocytochemistry using the same antibody along with Mitotracker red was performed in COS7 cells , 24 hours after transfection . Nuclei were counterstained with DAPI ( blue ) 11 . 5RVD and 8 . 5RVD mitoTev - TALEs both co - localized with mitochondria , as seen in the merge image . Scale bar : 10

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Figure 1HEK293T cells were transfected for 48 hours with each mitoTev-TALE (11.5 and 8.5 RVDs) and total cell extracts were used for western-blots with anti-T2A antibody. Molecular weights of the two different mitoTev-TALEs are ~85KD and ~70KDa, respectivelyImmunocytochemistry using the same antibody along with Mitotracker red was performed in COS7 cells, 24 hours after transfection. Nuclei were counterstained with DAPI (blue) 11.5RVD and 8.5RVD mitoTev-TALEs both co-localized with mitochondria, as seen in the merge image. Scale bar: 10 µm


Figure 2MtDNA heteroplasmy analyzed by PCR / RFLP , 24 hours post mitoTev - TALE and mitoTALEN transfection . The RFLP analysis shows increased % WT mtDNA in sorted cells when compared to the the untransfected . mitoTALEN monomers positive for both eGFP and mCherry were isolated as " Yellow " . The " Black " cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry and the " GFP - + " population represents mitoTev - TALE sorted cells with low levels of eGFP fluorescence . GFP + + were cells positive for GFP after mitoTev - TALEs transfectionsQuantification of the % of WT load in cybrid sorted populations after transfectionRepresentative RFLP analysis showing the change in heteroplasmy at 1 day and 15 days , after sortingQuantification of the heteroplasmic shift over timeDetermination of the levels of mtDNA ( ND1 / ACTB ) by qPCR of mutant cybrids transfected with MERRF mitoTev - TALEmtDNA levels in wild - type sorted cells transfected with the MERRF mitoTev - TALE . Data is expressed as percentage of the Untransfected cells ( % UNT )

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Figure 2MtDNA heteroplasmy analyzed by PCR/RFLP, 24 hours post mitoTev-TALE and mitoTALEN transfection. The RFLP analysis shows increased %WT mtDNA in sorted cells when compared to the the untransfected. mitoTALEN monomers positive for both eGFP and mCherry were isolated as "Yellow". The "Black" cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry and the "GFP-+" population represents mitoTev-TALE sorted cells with low levels of eGFP fluorescence. GFP++ were cells positive for GFP after mitoTev-TALEs transfectionsQuantification of the % of WT load in cybrid sorted populations after transfectionRepresentative RFLP analysis showing the change in heteroplasmy at 1 day and 15 days, after sortingQuantification of the heteroplasmic shift over timeDetermination of the levels of mtDNA (ND1/ACTB) by qPCR of mutant cybrids transfected with MERRF mitoTev-TALEmtDNA levels in wild-type sorted cells transfected with the MERRF mitoTev-TALE. Data is expressed as percentage of the Untransfected cells (%UNT)


Figure 3RFLP analyses of high mutant cybrids transfected with the MERRF mitoTev - TALE . The sorted cells were analyzed at different times of growth by PCR / RFLP as previously describedQuantification of WT loads of 6 - 8 independent experiments / sortings , analyzed at different time - pointsTotal mtDNA levels determined by qPCR in 6 - 7 separate sortings / experimentsFlow cytometry diagram of gated sorted cells . The GFP - + represent the cells with low levels of fluorescence , GFP + + showed intermediate levels of fluorescence while GFP + + + had the highest levelsQuantification of mtDNA levels by PrimeTime qPCR probes . The mtDNA levels were analyzed in the different populations of sorted cells . Data are expressed as mean ± SEM of 3 - 4 separate experiments mtDNA levels were also determined in the WT cybrids as described above . Data are expressed as mean ± SEM of 4 - 7 separate

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3RFLP analyses of high mutant cybrids transfected with the MERRF mitoTev-TALE. The sorted cells were analyzed at different times of growth by PCR/RFLP as previously describedQuantification of WT loads of 6-8 independent experiments/sortings, analyzed at different time-pointsTotal mtDNA levels determined by qPCR in 6-7 separate sortings/experimentsFlow cytometry diagram of gated sorted cells. The GFP-+ represent the cells with low levels of fluorescence, GFP++ showed intermediate levels of fluorescence while GFP+++ had the highest levelsQuantification of mtDNA levels by PrimeTime qPCR probes. The mtDNA levels were analyzed in the different populations of sorted cells. Data are expressed as mean±SEM of 3-4 separate experiments mtDNA levels were also determined in the WT cybrids as described above. Data are expressed as mean±SEM of 4-7 separate experiments


Figure 4Oxygen consumption rate ( OCR ) upon sequential injection of oligomycin ( Oligo ) , FCCP and rotenone ( Rot ) + antimycin ( AA ) , in untransfected , sorted " GFP - + " and " GFP + + " . The error bars represent the mean ± SEM of 5 - 7 separate experimentsQuantification of 4 - 6 independent experiments comparing the basal respiration , maximal respiration , spare respiratory capacity and ATP - linked respiration of the UNT versus " GFP - + " , " GFP + + " and WT cells . The cells were analyzed between 25 - 28 days after sortingCoupling efficiency , measured as the ratio of ATP - linked / Basal respiration x 100 . Error bars represent the mean ± SEM of 4 - 6 separate experimentsRepresentative membrane of a mitochondrial protein synthesis experiment . The results were normalized to the gel loadingWestern - blot showing the levels of NDUFB8 and COXI in untransfected and sorted cells . The SDHA and tubulin levels were also analyzed . The panel also shows the quantification of these markers normalized by the stain - free loading of 6 independent sortings /

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Oxygen consumption rate (OCR) upon sequential injection of oligomycin (Oligo), FCCP and rotenone (Rot) + antimycin (AA), in untransfected, sorted "GFP-+" and "GFP++". The error bars represent the mean±SEM of 5-7 separate experimentsQuantification of 4-6 independent experiments comparing the basal respiration, maximal respiration, spare respiratory capacity and ATP-linked respiration of the UNT versus "GFP-+", "GFP++" and WT cells. The cells were analyzed between 25-28 days after sortingCoupling efficiency, measured as the ratio of ATP-linked/Basal respiration x 100. Error bars represent the mean±SEM of 4-6 separate experimentsRepresentative membrane of a mitochondrial protein synthesis experiment. The results were normalized to the gel loadingWestern-blot showing the levels of NDUFB8 and COXI in untransfected and sorted cells. The SDHA and tubulin levels were also analyzed. The panel also shows the quantification of these markers normalized by the stain-free loading of 6 independent sortings/experiments


Figure 1C , Diarylquinoline and SQ31f inhibition of ATP hydrolysis by purified ATP synthase bearing truncated α subunits . Mean relative activity ( filled symbols ) ± s . d from n = 3 separate assays ( empty symbols ) for each of four different inhibitors ( triangles , circles , diamonds , and squares ) . D , Inhibition of ATP hydrolysis activity in M . smegmatis IMVs containing ATP synthase with truncated α subunits . Initial ATP hydrolysis activity of the ATP synthase is defined as the activity that is sensitive to 100 μM DCCD . Mean relative activity from n = 3 separate assays is shown . E , Uncoupling activity of diarylquinoline analogs in M . smegmatis IMVs monitored with ACMA fluorescence . Each compound was assayed separately , and the initial fluorescence was set to 1 . 0 . RFU : relative fluorescence units .

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Figure 1C, Diarylquinoline and SQ31f inhibition of ATP hydrolysis by purified ATP synthase bearing truncated α subunits. Mean relative activity (filled symbols) ± s.d from n=3 separate assays (empty symbols) for each of four different inhibitors (triangles, circles, diamonds, and squares).D, Inhibition of ATP hydrolysis activity in M. smegmatis IMVs containing ATP synthase with truncated α subunits. Initial ATP hydrolysis activity of the ATP synthase is defined as the activity that is sensitive to 100 μM DCCD. Mean relative activity from n=3 separate assays is shown.E, Uncoupling activity of diarylquinoline analogs in M. smegmatis IMVs monitored with ACMA fluorescence. Each compound was assayed separately, and the initial fluorescence was set to 1.0. RFU: relative fluorescence units.


Figure 3A , Uncoupling activity of SQ31f in M . smegmatis IMVs monitored with acridine orange fluorescence . B , SQ31f inhibition of ATP hydrolysis by purified ATP synthase with truncated α subunits ( blue curve ) occurs at higher concentrations than inhibition of ATP synthesis by M . smegmatis IMVs ( purple curve ) . Mean relative activity ( filled symbols ) ± s . d from n = 3 separate assays ( triangle and circles ) .

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Figure 3A, Uncoupling activity of SQ31f in M. smegmatis IMVs monitored with acridine orange fluorescence.B, SQ31f inhibition of ATP hydrolysis by purified ATP synthase with truncated α subunits (blue curve) occurs at higher concentrations than inhibition of ATP synthesis by M. smegmatis IMVs (purple curve). Mean relative activity (filled symbols) ± s.d from n=3 separate assays (triangle and circles).


Figure 1 ( C ) Levels of mucosal vtRNA2 - 1 , vtRNA1 - 1 , vtRNA1 - 2 , and vtRNA1 - 3 in the colon of patients with active ulcerative colitis ( UC ) . Values are the means ± SEM ( n = 6 per group ) . Unpaired , two - tailed Student ' s t test was used . * P < 0 . 05 compared with controls . ( D ) Levels of tissue vtRNAs in the ileal mucosa of patients with Crohn ' s disease ( CD ) . Values are the means ± SEM ( n = 6 per group ) . Unpaired , two - tailed Student ' s t test was used . * P < 0 . 05 compared with controls .

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Figure 1(C) Levels of mucosal vtRNA2-1, vtRNA1-1, vtRNA1-2, and vtRNA1-3 in the colon of patients with active ulcerative colitis (UC). Values are the means ± SEM (n = 6 per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with controls. (D) Levels of tissue vtRNAs in the ileal mucosa of patients with Crohn's disease (CD). Values are the means ± SEM (n = 6 per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with controls.


Figure 2 ( B ) Expression levels of claudin 1 , occludin , E - cadherin , ZO - 1 , JAM - A , and MVP in cells described in A . Hsc70 immunoblotting was performed as an internal control for equal loading .

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Figure 2(B) Expression levels of claudin 1, occludin, E-cadherin, ZO-1, JAM-A, and MVP in cells described in A. Hsc70 immunoblotting was performed as an internal control for equal loading.


Figure 3 ( B ) Immunoblots of claudin 1 , occludin , E - cadherin , ZO - 1 , and JAM - A in cells treated as described in A . Hsc70 immunoblotting served as an internal control for equal loading . Three separate experiments were performed and showed similar results .

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Figure 3(B) Immunoblots of claudin 1, occludin, E-cadherin, ZO-1, and JAM-A in cells treated as described in A. Hsc70 immunoblotting served as an internal control for equal loading. Three separate experiments were performed and showed similar results.


Figure 4 ( E ) Immunostaining ( left ) of Paneth ( lysozyme - positive ) cells and quantitative data ( right ) in intestinal organoids treated as described in A . Lysozyme : green . Scale bars , 100 µm . Values are means ± SEM ( n = 4 biological replicates ) . Unpaired , two - tailed Student ' s t test was used . * P < 0 . 05 compared with control vector .

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Figure 4(E) Immunostaining (left) of Paneth (lysozyme-positive) cells and quantitative data (right) in intestinal organoids treated as described in A. Lysozyme: green. Scale bars, 100 µm. Values are means ± SEM (n = 4 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.


Figure 5 ( B ) Immunoblots of claudin 1 , occludin , JAM - A , ZO - 1 , and E - cadherin in the mucosa from mice treated as described in A . GAPDH immunoblotting served as an internal control for equal loading .

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Figure 5(B) Immunoblots of claudin 1, occludin, JAM-A, ZO-1, and E-cadherin in the mucosa from mice treated as described in A. GAPDH immunoblotting served as an internal control for equal loading.


Figure 6 ( C ) Newly synthesized claudin 1 and occludin proteins in cells treated as described in A . After cells were exposed to L - azidohomoalaine ( AHA ) , cell lysates were incubated with the reaction buffer containing biotin / alkyne reagent ; the biotin - alkyne - azide - modified protein complex was pulled down by paramagnetic streptavidin - conjugated dynabeads . ( D ) Quantitative data of immunoblots shown in C . Values are means ± SEM ( n = 3 biological replicates ) . Unpaired , two - tailed Student ' s t test was used . * P < 0 . 05 compared with control vector .

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Figure 6(C) Newly synthesized claudin 1 and occludin proteins in cells treated as described in A. After cells were exposed to L-azidohomoalaine (AHA), cell lysates were incubated with the reaction buffer containing biotin/alkyne reagent; the biotin-alkyne-azide-modified protein complex was pulled down by paramagnetic streptavidin-conjugated dynabeads. (D) Quantitative data of immunoblots shown in C. Values are means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.


Figure 7 ( A ) Association of vtRNA2 - 1 with HuR in Caco - 2 cells as measured by biotin - labeled RNA pull - down assays . After cytoplasmic lysates were incubated with biotinylated vtRNA2 - 1 , levels of HuR and other proteins in the pull - down material were assessed . Three separate experiments showed similar results . ( D ) Changes in the expression levels of claudin - 1 , occludin , and E - cadherin proteins 48 h after cells were transfected with HuR expression vector alone or co - transfected with HuR and vtRNA2 - 1 expression vectors .

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Figure 7(A) Association of vtRNA2-1 with HuR in Caco-2 cells as measured by biotin-labeled RNA pull-down assays. After cytoplasmic lysates were incubated with biotinylated vtRNA2-1, levels of HuR and other proteins in the pull-down material were assessed. Three separate experiments showed similar results.(D) Changes in the expression levels of claudin-1, occludin, and E-cadherin proteins 48 h after cells were transfected with HuR expression vector alone or co-transfected with HuR and vtRNA2-1 expression vectors.


Figure 1 ( b ) RNAscope analysis of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript ( N = 3 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50µm ( c ) Hematoxylin and Eosin staining of the colonic epithelium following DSS induced damaged . Crypts are dramatically injured by d5 . At d8 through d12 , regenerating crypts are observed . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50µm ( d - e ) Compared to untreated controls , DSS treatment ablates Lgr5 - GFP + stem cells and results in functional loss of tdTomato + lineage tracing as early as d5 . Lgr5 - GFP + stem cells reappear by d12 ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50µm Data are represented as mean ± SD ( e ) analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . ( f ) Effects of DSS colitis injury on RNA expression levels of stem ( Lgr5 and Krt19 ) and progenitor ( Atoh1 and Notch1 ) cell markers . Lgr5 mRNA expression is significantly decreased after exposure to DSS ( N = 6 - 7 per condition ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Mice body weight ( h ) are comparable between three treatments : DSS + DT ( N = 12 ) , only DSS ( N = 8 ) or only DT ( N = 3 ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Mice survival ( i ) are comparable between three treatments : DSS + DT ( N = 12 ) , only DSS ( N = 8 ) or only DT ( N = 3 ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . ( j ) Hematoxylin and Eosin staining of the colonic epithelium shows no morphological difference between DSS alone and DSS + DT treated mice . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . scale bar ( j ) = 500µm . ( k ) The extent of epithelial damage is similar in mice treated with DSS alone versus DSS + DT ( to ablate Lgr5 + stem cells ) ( N = 3 DSS ; N = 6 DSS + DT ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . ( l ) RNAscope for Lgr5 in Lgr5DTR - GFP mice showing loss of Lgr5 transcripts after DT treatment ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars =

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Figure 1(b) RNAscope analysis of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript (N=3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm(c) Hematoxylin and Eosin staining of the colonic epithelium following DSS induced damaged. Crypts are dramatically injured by d5. At d8 through d12, regenerating crypts are observed. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm(d-e) Compared to untreated controls, DSS treatment ablates Lgr5-GFP+ stem cells and results in functional loss of tdTomato+ lineage tracing as early as d5. Lgr5-GFP+ stem cells reappear by d12 (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm Data are represented as mean ± SD (e) analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(f) Effects of DSS colitis injury on RNA expression levels of stem (Lgr5 and Krt19) and progenitor (Atoh1 and Notch1) cell markers. Lgr5 mRNA expression is significantly decreased after exposure to DSS (N=6-7 per condition). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.Mice body weight (h) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.Mice survival (i) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(j) Hematoxylin and Eosin staining of the colonic epithelium shows no morphological difference between DSS alone and DSS+DT treated mice. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bar (j) =500µm.(k) The extent of epithelial damage is similar in mice treated with DSS alone versus DSS+DT (to ablate Lgr5+ stem cells) (N=3 DSS; N=6 DSS+DT). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(l) RNAscope for Lgr5 in Lgr5DTR-GFP mice showing loss of Lgr5 transcripts after DT treatment (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm


Figure 2 ( b - c ) Krt19 + cells lineage trace entire colonic crypts during both normal homeostasis and injury ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines outline the basement membrane . Scale bars ( b ) = 45µm Data are represented as mean ± SEM * P ≤ 0 . 05 , * * P ≤ 0 . 01 . DT ablation of Krt19 + cells ( N ≥ 3 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines outline the basement membrane . scale bar ( e ) = 30µmDT ablation of Krt19 + cells significantly reduced the number of lineage labeled crypts ( N ≥ 3 per condition ) . Data are represented as mean ± SEM using Student ' s t - test * P ≤ 0 . 05 , * * P ≤ 0 . 01 . ( g ) Mice body weight and survival remain unchanged between DSS and DSS + DT treated groups ( N = 6 control ; N = 7 DT ) . Data are represented as mean ± SEM using Student ' s t - test P ≤ 0 . 05 , * * P ≤ 0 . 01 . Hematoxylin and Eosin staining of the colonic epithelium after DSS versus DSS + DT showing more damage is seen in mice treated with both DSS + DT treatment ( N = 6 control ; N = 7 DT ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines outline the basement membrane . scale bar ( h ) = 1mm . quantification of damaged epithelial area ( i ) , showing more damage is seen in mice treated with both DSS + DT treatment ( N = 6 control ; N = 7 DT ) . Data are represented as mean ± SEM using Student ' s t - test * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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Figure 2(b-c) Krt19+ cells lineage trace entire colonic crypts during both normal homeostasis and injury (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. Scale bars (b) =45µm Data are represented as mean ± SEM *P≤0.05, **P≤0.01.DT ablation of Krt19+ cells (N≥3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (e) =30µmDT ablation of Krt19+ cells significantly reduced the number of lineage labeled crypts (N≥3 per condition). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.(g) Mice body weight and survival remain unchanged between DSS and DSS+DT treated groups (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test P≤0.05, **P≤0.01.Hematoxylin and Eosin staining of the colonic epithelium after DSS versus DSS+DT showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (h) =1mm.quantification of damaged epithelial area (i), showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.


Figure 3 ( b - c ) Notch1 + progenitors are able to generate Lgr5 + stem cells and contribute to epithelial renewal in homeostasis ; however , do not contribute to regeneration after colitis ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm . Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons test . * * * * P ≤ 0 . 0001 . Images of whole mount showing Notch1 lineage labeled crypts . No partially labeled crypts were found after Lgr5 + stem cell ablation . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm . Images of tissue sections Notch1CreERT2 ; ROSA26tdTomato ; Lgr5DTR - GFP mice ( f ) showing Notch1 lineage labeled crypts . No partially labeled crypts were found after Lgr5 + stem cell ablation . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm .

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Figure 3(b-c) Notch1+ progenitors are able to generate Lgr5+ stem cells and contribute to epithelial renewal in homeostasis; however, do not contribute to regeneration after colitis (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test. ****P≤0.0001.Images of whole mount showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.Images of tissue sections Notch1CreERT2; ROSA26tdTomato;Lgr5DTR-GFP mice (f) showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.


Figure 4 ( a - b ) Atoh1 + progenitors show stem cell capacity during normal homeostasis and regeneration . At early stages of recovery , d10 - 12 , Atoh1 + cells lineage trace the colonic crypt ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons test * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . ( c - d ) By d30 after DSS , Atoh1 derived stem cells continue to lineage trace complete colonic crypts ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars first two panels of c = 50μm ; scale bars ( last panel of c = 100μm . Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons test * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . Whole mount ( f ) images of Atoh1 lineage labeled crypts . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μmsection ( g ) images of Atoh1 lineage labeled crypts . Majority of crypts are labeled after Lgr5 + stem cell ablation and remain label with the appearance of Lgr5 + stem cells ( inset , arrowheads ) . Atoh1 lineage labeled crypts remain by d30 . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . scale bars = 100μm . ( h ) Significantly increased labeling of crypt base was observed at d8 in DT treated mice ( N = 4 per condition ) . Data are represented as mean ± SD analyzed using Student ' s t - test with Welch ' s correction * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . ( j - k ) Scattered labeling of Atoh1 + cells in control conditions , while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5 + stem cells . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm Data are represented as mean ± SD analyzed using Student ' s t - test with Welch ' s correction * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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Figure 4(a-b) Atoh1+ progenitors show stem cell capacity during normal homeostasis and regeneration. At early stages of recovery, d10-12, Atoh1+ cells lineage trace the colonic crypt (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.(c-d) By d30 after DSS, Atoh1 derived stem cells continue to lineage trace complete colonic crypts (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars first two panels of c =50μm; scale bars (last panel of c =100μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.Whole mount (f) images of Atoh1 lineage labeled crypts. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μmsection (g) images of Atoh1 lineage labeled crypts. Majority of crypts are labeled after Lgr5+ stem cell ablation and remain label with the appearance of Lgr5+ stem cells (inset, arrowheads). Atoh1 lineage labeled crypts remain by d30. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bars =100μm.(h) Significantly increased labeling of crypt base was observed at d8 in DT treated mice (N=4 per condition). Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.(j-k) Scattered labeling of Atoh1+ cells in control conditions, while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5+ stem cells. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.


Figure 5 ( b - c ) Sporadic labeling of Atoh1 + cells after 4 - OHT induction . Following irradiation , entire colonic organoids exhibit full lineage labeling of Atoh1 + cells , characteristic of multipotent stem cells ( N = 3 per condition ) . Data information : Scale bars = 100μm Data are represented as mean ± SD analyzed using Student ' s t - test ( c ) ( d - e ) Flow cytometry for tdTomato + cells in crypt epithelium from DT treated Atoh1CreERT2 ; Lgr5DTR - GFP ; ROSA26tdTomato mice . ( f ) tdTomato + colonic organoids from single Atoh1 - tdTomato + / Lgr5 - negative cells ( N = 1 ; n = 3 technical replicates per condition ) . Data information : Scale bars = 100μm ( g ) Post - passage colonic organoids from RFP + cells of control or DT treated mice continue growing . tdTomato - negative cells of control or DT treated mice also form colonic organoids after passaging . Data information scale bars ( g ) = 50μm . ( h ) tdTomato + colonic organoids established from mice in which Lgr5 + stem cells have been ablated show higher growth efficiency compared to their colonic organoid counterparts ( N = 1 ; n = 3 technical replicates per condition ) . Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons test ( h ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(b-c) Sporadic labeling of Atoh1+ cells after 4-OHT induction. Following irradiation, entire colonic organoids exhibit full lineage labeling of Atoh1+ cells, characteristic of multipotent stem cells (N=3 per condition). Data information: Scale bars =100μm Data are represented as mean ± SD analyzed using Student's t-test (c)(d-e) Flow cytometry for tdTomato+ cells in crypt epithelium from DT treated Atoh1CreERT2;Lgr5DTR-GFP;ROSA26tdTomato mice.(f) tdTomato+ colonic organoids from single Atoh1-tdTomato+/Lgr5-negative cells (N=1; n=3 technical replicates per condition). Data information: Scale bars =100μm(g) Post-passage colonic organoids from RFP+ cells of control or DT treated mice continue growing. tdTomato-negative cells of control or DT treated mice also form colonic organoids after passaging. Data information scale bars (g) =50μm.(h) tdTomato+ colonic organoids established from mice in which Lgr5+ stem cells have been ablated show higher growth efficiency compared to their colonic organoid counterparts (N=1; n=3 technical replicates per condition). Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test (h). *P≤0.05, **P≤0.01, ***P≤0.001.


Figure 6 ( b ) Atoh1 + lineage labelling post - DSS and DSS + DT . Data information : Scale bars ( b ) = 200μm ( c ) DSS + DT treated - mice exhibit significant weight loss and lower survival compared to DSS alone ( N = 4 - 7 control ; N = 3 - 4 DSS ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 . ( d - e ) Hematoxylin and Eosin staining of colonic epithelium after DSS versus DSS + DT ( d ) . Extensive epithelial damage was observed ( d , arrow ) and quantified of in DSS + DT treated mice ( N = 7 control ; N = 4 DSS ) . Data information scale bars ( d ) = 400μ Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 . ( g - h ) DT treatment effectively ablates Atoh1 + cells shown by reduced number of tdTomato + cells per organoid . Data information scale bars = 100μm . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 . ( i - j ) Concurrent irradiation injury and Atoh1 + cell ablation results in significantly reduced organoid survival ( N = 3 ; n = 3 technical replicates per condition ) . Data information : ; scale bars = 100μm . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(b) Atoh1+ lineage labelling post-DSS and DSS+DT. Data information: Scale bars (b)=200μm (c) DSS+DT treated-mice exhibit significant weight loss and lower survival compared to DSS alone (N=4-7 control; N=3-4 DSS). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.(d-e) Hematoxylin and Eosin staining of colonic epithelium after DSS versus DSS+DT (d). Extensive epithelial damage was observed (d, arrow) and quantified of in DSS+DT treated mice (N=7 control; N=4 DSS). Data information scale bars (d)=400μ Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.(g-h) DT treatment effectively ablates Atoh1+ cells shown by reduced number of tdTomato+ cells per organoid. Data information scale bars =100μm. Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.(i-j) Concurrent irradiation injury and Atoh1+ cell ablation results in significantly reduced organoid survival (N=3; n=3 technical replicates per condition). Data information: ; scale bars =100μm. Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.


Figure 1A , B Data were normalised using the control group and are represented as fold - change ( FC ) of the mean fluorescence intensity ( MFI ) . Macrophage cells were obtained from human donors ( n = 4 biological replicas ) and incubated with medium ( CON ) or recombinant human IL - 10 ( hIL - 10r ) , MPN wild - type ( WT ) or MPN expressing IL - 10 ORF ( WT _ IL10 ) . Data are represented as mean ± Standard Error of the Mean ( SEM ) . Statistical analysis was performed using one - way ANOVA + post - hoc Tukey multiple comparison test ( * P < 0 . 05 ; * * P < 0 . 001 , * * * P < 0 . 0001 ) .

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Figure 1A, B Data were normalised using the control group and are represented as fold-change (FC) of the mean fluorescence intensity (MFI). Macrophage cells were obtained from human donors (n = 4 biological replicas) and incubated with medium (CON) or recombinant human IL-10 (hIL-10r), MPN wild-type (WT) or MPN expressing IL-10 ORF (WT_IL10). Data are represented as mean ± Standard Error of the Mean (SEM). Statistical analysis was performed using one-way ANOVA + post-hoc Tukey multiple comparison test (*P < 0.05; **P < 0.001, ***P < 0.0001).


Figure 4 ( A ) EC - 50 ( molar , M ) for human IL - 10 recombinant protein ( hIL - 10r ) and IL - 10 WT ( IL - 10 ORF ) and different variants ( MutM , hIL - 10r , Mut1 , Mut2 , Mut3 , MutSC1 , and MutSC2 ) expressed by M . pneumoniae . Data are represented as mean ± SD . Statistical comparison was done by One - way ANOVA + post - hoc Bonferroni multiple comparison test , using the IL - 10 ORF condition as a reference ( * P < 0 . 05 ) . ( B ) Fold - change ( FC ) in expression levels ( fg / CFU ) of IL - 10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL - 10 ORF ( ORF ) . Statistical comparison of mean ± SD was performed by One - way ANOVA + Tukey post - hoc test ( * P < 0 . 05 ) . ( C , D ) Average and SD values for the FC in the relative EC - 50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20 - min exposure of the BlaER1 ( C ) or HAFTL ( D ) cell lines to IL - 10 ORF ( reference ) or the mutant MutSC1 or MutSC2 Numbers indicate the average FC ± SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) EC-50 (molar, M) for human IL-10 recombinant protein (hIL-10r) and IL-10 WT (IL-10 ORF) and different variants (MutM, hIL-10r, Mut1, Mut2, Mut3, MutSC1, and MutSC2) expressed by M. pneumoniae. Data are represented as mean ± SD. Statistical comparison was done by One-way ANOVA + post-hoc Bonferroni multiple comparison test, using the IL-10 ORF condition as a reference (*P < 0.05).(B) Fold-change (FC) in expression levels (fg/CFU) of IL-10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL-10 ORF (ORF). Statistical comparison of mean ± SD was performed by One-way ANOVA + Tukey post-hoc test (*P < 0.05).(C, D) Average and SD values for the FC in the relative EC-50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20-min exposure of the BlaER1 (C) or HAFTL (D) cell lines to IL-10 ORF (reference) or the mutant MutSC1 or MutSC2 Numbers indicate the average FC ± SD.


Figure 5C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( A ) Average ± SD bacterial loads recovered of the WT ( circle ) and CV8 ( square ) strains in mouse lungs at 2 - dpi or 4 - dpi . Data are shown as log10 CFU / lung homogenate . C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and ( B ) Inflammatory profile of mouse lungs inoculated with WT ( dark blue ) , CV8 ( light blue ) , or PBS ( grey ) . Gene expression was analysed by RT - qPCR using gapdh as an endogenous control ( see Materials and Methods ) . Data are shown as average ± SD of fold - change ( FC ) in mRNA expression ( One - way ANOVA + Tukey post - hoc test ; * P < 0 . 05 ) . C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( C ) Histological findings of mouse lung samples at the analysed time points . Left , plots representing the quantitative evaluation ( score : 0 - 5 ) of alveolar infiltrate ( top panel ) and peribronquial / olar infiltrate ( FC ) ( bottom panel ) ( see Materials and Methods ) . Parameters were normalised using the average of the PBS group ( FC = sample value / average PBS ; FC control group , ~ 1 ) . Data are shown as average ± SD of FC ( one - way ANOVA + Tukey post - hoc test ) ( * P < 0 . 05 ) . Right , representative images of lungs stained with hematoxylin - eosin ( the line represents 250 μm ) . C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and ( D ) IL - 10 secretion by the MPN WT or CV8 strain coding for hIL - 10 WT ( ORF ) , MutSC1 , or MutSC2 . Data are expressed as the mean ± SD of IL - 10 supernatant concentration ( μg / mL ) by biomass ( protein content in μg / mL ) ( n = 3 biological replicas ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group). (A) Average ± SD bacterial loads recovered of the WT (circle) and CV8 (square) strains in mouse lungs at 2-dpi or 4-dpi. Data are shown as log10 CFU/ lung homogenate.C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and (B) Inflammatory profile of mouse lungs inoculated with WT (dark blue), CV8 (light blue), or PBS (grey). Gene expression was analysed by RT-qPCR using gapdh as an endogenous control (see Materials and Methods). Data are shown as average ± SD of fold-change (FC) in mRNA expression (One-way ANOVA + Tukey post-hoc test; *P < 0.05).C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group). (C) Histological findings of mouse lung samples at the analysed time points. Left, plots representing the quantitative evaluation (score: 0-5) of alveolar infiltrate (top panel) and peribronquial/olar infiltrate (FC) (bottom panel) (see Materials and Methods). Parameters were normalised using the average of the PBS group (FC = sample value/average PBS; FC control group, ~1). Data are shown as average ± SD of FC (one-way ANOVA + Tukey post-hoc test) (*P < 0.05). Right, representative images of lungs stained with hematoxylin-eosin (the line represents 250 μm).C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and (D) IL-10 secretion by the MPN WT or CV8 strain coding for hIL-10 WT (ORF), MutSC1, or MutSC2. Data are expressed as the mean ± SD of IL-10 supernatant concentration (μg/mL) by biomass (protein content in μg/mL) (n=3 biological replicas).


Figure 6 ( B ) CFUs of PAO1 ( left ) or Mycoplasma strains ( right ) recovered from lung samples . Data are shown as mean ± SD of Log10 CFU / lung homogenate . Two replicates were performed and individual symbols are shown ( experiment 1 , circle ; experiment 2 , square ) . ( C ) Fold - change ( FC ) of mRNA gene expression of inflammatory markers determined by qPCR . The 2 - ΔΔCt method was used to normalise the values , using gapdh as endogenous control Data are shown as average ± SD of FC . Statistical analyses were performed using unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 * * * ; P < 0 . 001 , * * * * P < 0 . 0001 ) . Two replicates were performed and individual symbols are shown ( Experiment 1 , circle ; Experiment 2 , square ) . ( D ) Quantitative analysis of immunochemistry ( IHC ) against neutrophil elastase ( NE ) of lung samples . Data are represented as the mean ± SD of percentage of positive cells , calculated as follows : % = 100 × ( positive cells / positive cells + negative cells ) ( One - Way ANOVA + Tukey post - hoc test , * P < 0 . 05 ) . ( E ) Representative images of IHC staining of lung samples against NE are shown . Arrows indicate NE - positive cells . Scale bar size : 50 µm .

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Figure 6(B) CFUs of PAO1 (left) or Mycoplasma strains (right) recovered from lung samples. Data are shown as mean ± SD of Log10 CFU/lung homogenate. Two replicates were performed and individual symbols are shown (experiment 1, circle; experiment 2, square).(C) Fold-change (FC) of mRNA gene expression of inflammatory markers determined by qPCR. The 2-ΔΔCt method was used to normalise the values, using gapdh as endogenous control Data are shown as average ± SD of FC. Statistical analyses were performed using unpaired t-test (* P < 0.05; ** P <0.01***; P <0.001, **** P <0.0001). Two replicates were performed and individual symbols are shown (Experiment 1, circle; Experiment 2, square).(D) Quantitative analysis of immunochemistry (IHC) against neutrophil elastase (NE) of lung samples. Data are represented as the mean ± SD of percentage of positive cells, calculated as follows: % = 100 × (positive cells/positive cells + negative cells) (One-Way ANOVA + Tukey post-hoc test, * P <0.05). (E) Representative images of IHC staining of lung samples against NE are shown. Arrows indicate NE-positive cells. Scale bar size: 50 µm.


Figure 1A . Relative levels of OMA1 mRNA in colorectal cancer tissues ( n = 105 ) compared with adjacent normal tissues ( n = 43 ) from colorectal cancer patients were shown ( using the GEO dataset GSE21510 ) . The data are presented as mean ± SEM , and statistical significance was determined by a Mann - Whitney test . * * * p < 0 . 001 . B . Kaplan - Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537 . " Low " indicates patients with OMA1 mRNA levels less than the median . " High " indicates patients with OMA1 mRNA levels greater than the median . Statistical analysis was performed using log - rank tests , n = 55 , p = 0 . 0180 . C . The lysates of tumors ( T ) and adjacent normal ( N ) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin . The " c , d and e " bands of OPA1 indicate cleaved OPA1 bands . Tubulin was used as a loading control . D . Control and OMA1 KO HCT116 cells were cultured in hypoxia ( 1 % O2 ) for 0 ( normoxia ) , 24 , or 48 hours . Cell lysates were then assessed by Western blotting with antibodies against OPA1 , OMA1 , or β - Actin . β - Actin was used as a loading control . Representative immunoblots were from n = 3 independent experiments . E . OPA1 - null mouse embryonic fibroblasts ( MEFs ) expressing control ( empty vector ) or OPA1 isoform 1 were cultured in normoxia or hypoxia ( 1 % O2 ) for 24 hours . Cell lysates were analyzed by Western blotting with anti - OPA1 or anti - Tubulin antibody . Representative immunoblots were from n = 3 independent experiments . F . WT , OMA1 KO and OMA1 KO HCT116 cells expressing WT - OMA1 ( OMA1 - Flag ) or proteolytic inactive OMA1 ( E324Q - Flag ) were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours , and the cell lysates were assessed by western blot with antibodies against OMA1 , and GAPDH . β - Actin was used as a loading control ( representative data from three independent experiments ) . The asterisk indicates a nonspecific band . G . The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1 - Flag / β - Actin or E324Q - Flag / β - Actin in hypoxia for 0 , 24 or 48 hours ( n = 3 independent experiments ) . The data are presented as mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Relative levels of OMA1 mRNA in colorectal cancer tissues (n=105) compared with adjacent normal tissues (n=43) from colorectal cancer patients were shown (using the GEO dataset GSE21510). The data are presented as mean ± SEM, and statistical significance was determined by a Mann-Whitney test. ***p < 0.001.B. Kaplan-Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537. "Low" indicates patients with OMA1 mRNA levels less than the median. "High" indicates patients with OMA1 mRNA levels greater than the median. Statistical analysis was performed using log-rank tests, n = 55, p=0.0180.C. The lysates of tumors (T) and adjacent normal (N) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin. The "c, d and e" bands of OPA1 indicate cleaved OPA1 bands. Tubulin was used as a loading control.D. Control and OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0 (normoxia), 24, or 48 hours. Cell lysates were then assessed by Western blotting with antibodies against OPA1, OMA1, or β-Actin. β-Actin was used as a loading control. Representative immunoblots were from n = 3 independent experiments.E. OPA1-null mouse embryonic fibroblasts (MEFs) expressing control (empty vector) or OPA1 isoform 1 were cultured in normoxia or hypoxia (1% O2) for 24 hours. Cell lysates were analyzed by Western blotting with anti-OPA1 or anti-Tubulin antibody. Representative immunoblots were from n = 3 independent experiments.F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were cultured in hypoxia (1% O2) for 0, 24, or 48 hours, and the cell lysates were assessed by western blot with antibodies against OMA1, and GAPDH. β-Actin was used as a loading control (representative data from three independent experiments). The asterisk indicates a nonspecific band. G. The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1-Flag/β-Actin or E324Q-Flag/β-Actin in hypoxia for 0, 24 or 48 hours (n = 3 independent experiments). The data are presented as mean ± SD.


Figure 2B . Body weight changes of WT ( n = 11 ) and OMA1 - / - ( n = 12 ) mice during AOM / DSS treatment . Data are presented as mean ± SEM . Statistical significance was assessed by a two - way ANOVA , * * p < 0 . 01 . C . Typical intestine images on day 105 after mice were euthanized . D . Colorectal lengths ( C ) of WT ( n = 11 biological replicates ) and OMA1 - / - ( n = 12 biological replicates ) mice were measured . Data with error bars are presented as mean ± SEM . Statistical significance was assessed by unpaired student ' s t - test , * p < 0 . 05 . E . Representative images of colorectal tumors from WT ( left ) and OMA1 - / - ( right ) mice . F . The number of tumors in WT ( n = 11 ) and OMA1 - / - ( n = 12 ) mice was measured . Each dot represents the tumor number of one individual mouse . Data with error bars are presented as mean ± SEM . Statistical significance was assessed by unpaired student ' s t - test , * * * p < 0 . 001 . G , H . The number of tumors of different sizes ( diameter ) in each mouse ( G ) and the distribution of different tumor sizes ( H ) were shown ( WT n = 11 biological replicates , and OMA1 - / - n = 12 biological replicates ) . Results are shown as mean ± SEM . p - values were calculated by unpaired student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 . I . Histological analysis of colorectal tumors of WT and OMA1 - / - mice was shown by hematoxylin and eosin ( H & E ) staining . J . Relative mRNA expression levels of pro - inflammatory genes ( TNFα , IL - 6 , COX - 2 , CCL2 ) in colorectal homogenates of WT and OMA1 _ KO mice treated with AOM / DSS ( n = 4 - 5 per genotype ) . The data represent the mean ± SEM . , n = 3 independent experiments , and statistical significance was determined by a two - tailed student ' s t - test . * p < 0 . 05 , * * p < 0 . 01 . K . The colorectal tissues of WT and OMA1 - / - mice were stained with Ki67 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O

Figure 2B. Body weight changes of WT (n=11) and OMA1-/- (n=12) mice during AOM/DSS treatment. Data are presented as mean ± SEM. Statistical significance was assessed by a two-way ANOVA, **p < 0.01.C. Typical intestine images on day 105 after mice were euthanized. D. Colorectal lengths (C) of WT (n=11 biological replicates) and OMA1-/- (n=12 biological replicates) mice were measured. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, *p < 0.05. E. Representative images of colorectal tumors from WT (left) and OMA1-/- (right) mice. F. The number of tumors in WT (n=11) and OMA1-/- (n=12) mice was measured. Each dot represents the tumor number of one individual mouse. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, ***p < 0.001. G, H. The number of tumors of different sizes (diameter) in each mouse (G) and the distribution of different tumor sizes (H) were shown (WT n=11 biological replicates, and OMA1-/- n=12 biological replicates). Results are shown as mean ± SEM. p-values were calculated by unpaired student's t-test, **p < 0.01, ***p < 0.001.I. Histological analysis of colorectal tumors of WT and OMA1-/- mice was shown by hematoxylin and eosin (H&E) staining.J. Relative mRNA expression levels of pro-inflammatory genes (TNFα, IL-6, COX-2, CCL2) in colorectal homogenates of WT and OMA1_KO mice treated with AOM/DSS (n =4-5 per genotype). The data represent the mean ± SEM., n = 3 independent experiments, and statistical significance was determined by a two-tailed student's t-test. *p < 0.05, **p < 0.01.K. The colorectal tissues of WT and OMA1-/- mice were stained with Ki67.


Figure 3A , B . Control or OMA1 KO HCT116 cells expressing control ( empty vector ) or OMA1 - Flag were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . The glucose uptake ( A ) and lactate production ( B ) were measured by using microplate reader , and the values were normalized to the protein concentration . Error bars indicate the mean ± SD of three independent experiments , statistical significance was assessed by a two - way ANOVA , * P < 0 . 05 . Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH ( A ) . The asterisk indicates a nonspecific band . C . Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h . Subsequent injections of glucose , oligomycin ( Oligo ) , and 2 - deoxy - glucose ( 2 - DG ) were performed as indicated . The extracellular acidification rates ( ECAR ) were measured with a Seahorse Extracellular Flux Analyzer XF96 . The data are presented as mean ± SD ( n = 3 independent experiments ) . D . Quantitative analysis of basal and maximal glycolytic capacity . Error bars are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a two - way ANOVA . N . S . , not significant , * * * p < 0 . 001 . E , F . Lactate production ( E ) and ATP production ( F ) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2 - DG treatment . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . N . S . , not significant , * * p < 0 . 01 , * * * p < 0 . 001 . G , H . Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . The glucose uptake ( G ) and lactate production ( H ) were measured , and the values were normalized to the protein concentration . Statistical significance was assessed by a two - way ANOVA ; error bars are presented as mean ± SD of three independent experiments ; N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, B. Control or OMA1 KO HCT116 cells expressing control (empty vector) or OMA1-Flag were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (A) and lactate production (B) were measured by using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD of three independent experiments, statistical significance was assessed by a two-way ANOVA, *P < 0.05. Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH (A). The asterisk indicates a nonspecific band.C. Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h. Subsequent injections of glucose, oligomycin (Oligo), and 2-deoxy-glucose (2-DG) were performed as indicated. The extracellular acidification rates (ECAR) were measured with a Seahorse Extracellular Flux Analyzer XF96. The data are presented as mean ± SD (n = 3 independent experiments).D. Quantitative analysis of basal and maximal glycolytic capacity. Error bars are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-way ANOVA. N.S., not significant, ***p < 0.001.E, F. Lactate production (E) and ATP production (F) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2-DG treatment. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, **p < 0.01, ***p < 0.001.G, H. Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (G) and lactate production (H) were measured, and the values were normalized to the protein concentration. Statistical significance was assessed by a two-way ANOVA; error bars are presented as mean ± SD of three independent experiments; N.S., not significant, *p < 0.05, **p < 0.01.


Figure 4A . Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia ( 1 % O2 ) for 24 or 48 hours . The whole cell lysates were analyzed by Western blotting with antibodies against HIF - 1α , HK2 , LDHA , PKM2 , GPI , OMA1 , or β - Actin . The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B . Control or OMA1 KO Hela cells were untreated ( - ) or CoCl2 - treated ( + ) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours . Cell lysates were assessed by Western blotting with antibodies against HIF - 1α or β - Actin . Relative protein levels were further evaluated by densitometry analysis . Error bars indicate the mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 . C . Dihydroethidium ( DHE ) staining of optimum cutting temperature ( OCT ) covered tissues from the colons of WT and OMA1 - / - mice ; DAPI was used as counterstain . DHE was shown in red color , while DAPI in blue color ( representative data from three independent experiments ) . D . Quantification of DHE staining . Error bars are presented as mean ± SD ( n = 3 biological replicates ) , statistical significance was assessed by a two - way ANOVA , * * p < 0 . 01 , * * * P < 0 . 001 . E . Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours were stained with mitoSOX , and then analyzed by confocal microscopy ( representative data from three independent experiments ) . F . The fluorescence intensity of mitoSOX was further analyzed by ImageJ software . Bars indicate the mean ± SD of three independent experiments by a two - way ANOVA , * * * p < 0 . 001 . G - I . Control or OMA1 _ KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours , and were treated with DMSO , NAC ( 10 mM ) or Rotenone ( 1 μM ) for 12 hours at the same time . The whole cell lysates were analyzed by Western blotting with antibodies against HIF - 1α , and Tubulin ( G ) . The glucose uptake ( H ) and lactate production ( I ) were measured using microplate reader , and the values were normalized to the protein concentration . Error bars indicate the mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by two - way ANOVA . N . S . , not significant , * P < 0 . 05 , * * * P < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia (1% O2) for 24 or 48 hours. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, HK2, LDHA, PKM2, GPI, OMA1, or β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.B. Control or OMA1 KO Hela cells were untreated (-) or CoCl2-treated (+) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours. Cell lysates were assessed by Western blotting with antibodies against HIF-1α or β-Actin. Relative protein levels were further evaluated by densitometry analysis. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01.C. Dihydroethidium (DHE) staining of optimum cutting temperature (OCT) covered tissues from the colons of WT and OMA1-/- mice; DAPI was used as counterstain. DHE was shown in red color, while DAPI in blue color (representative data from three independent experiments). D. Quantification of DHE staining. Error bars are presented as mean ± SD (n=3 biological replicates), statistical significance was assessed by a two-way ANOVA, **p < 0.01, ***P< 0.001. E. Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours were stained with mitoSOX, and then analyzed by confocal microscopy (representative data from three independent experiments). F. The fluorescence intensity of mitoSOX was further analyzed by ImageJ software. Bars indicate the mean ± SD of three independent experiments by a two-way ANOVA, ***p < 0.001. G-I. Control or OMA1_KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours, and were treated with DMSO, NAC (10 mM) or Rotenone (1 μM) for 12 hours at the same time. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, and Tubulin (G). The glucose uptake (H) and lactate production (I) were measured using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-way ANOVA. N.S., not significant, *P < 0.05, ***P< 0.001.


Figure 5A . 293T cells were transiently co - transfected with empty vector and NDUFB5 - MYC , or OMA1 - E324Q - Flag and NDUFB5 - MYC . Cell lysates were used for co - immunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with anti - Flag or anti - MYC antibodies ( n = 3 independent experiments ) . B . 293T cells were transiently co - transfected with empty vector and NDUFB6 - GFP , or OMA1 - E324Q - Flag and NDUFB6 - GFP . Cell lysates were used for co - immunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with antibodies against Flag or GFP ( n = 3 independent experiments ) . C . 293T cells were transiently co - transfected with empty vector and COX4L1 - GFP , or OMA1 - E324Q - Flag and COX4L1 - GFP . Cell lysates were used for coimmunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with anti - Flag or anti - GFP antibodies ( n = 3 independent experiments ) . D . 293T cells were transiently co - transfected with empty vector and NDUFA4 - GFP , or OMA1 - E324Q - Flag and NDUFA4 - GFP . Cell lysates were used for coimmunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with antibodies against Flag or GFP ( n = 3 independent experiments ) . E . HCT116 cells stably expressing control ( empty vector ) or OMA1 - Flag , and HCT116 cells infected with control or shOMA1 ( OMA1 knockdown ) lentiviral particles and further cultured for 5 days , were then treated with DMSO or 20 μm CCCP for 4 hours . Cell lysates were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , MT - CO2 , OMA1 or Tubulin . The black arrowhead indicated the full - length COX4L1 or NDUFB6 , and the red arrowhead pointed to the cleaved band . The asterisk indicates a nonspecific band . F . WT , OMA1 KO and OMA1 KO HCT116 cells expressing WT - OMA1 ( OMA1 - Flag ) or proteolytic inactive OMA1 ( E324Q - Flag ) were treated with CHX ( 100 µg / ml ) plus CCCP ( 20 µM ) for 0 , 2 , 4 , 6 hours . The cell lysates were then assessed by western blot with antibodies against NDUFA4 , COX4L1 , NDUFB5 , NDUFB6 , OMA1 or HSPD1 ( representative data from three independent experiments ) . The asterisk indicates a nonspecific band . G . Control or OMA1 KO HCT116 cells were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours . Cell lysates were assessed by Western blotting with anti - NDUFB5 , anti - NDUFB6 , anti - COX4L1 , anti - NDUFA4 antibodies , or anti - β - Actin . The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 293T cells were transiently co-transfected with empty vector and NDUFB5-MYC, or OMA1-E324Q-Flag and NDUFB5-MYC. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-MYC antibodies (n = 3 independent experiments).B. 293T cells were transiently co-transfected with empty vector and NDUFB6-GFP, or OMA1-E324Q-Flag and NDUFB6-GFP. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).C. 293T cells were transiently co-transfected with empty vector and COX4L1-GFP, or OMA1-E324Q-Flag and COX4L1-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-GFP antibodies (n = 3 independent experiments).D. 293T cells were transiently co-transfected with empty vector and NDUFA4-GFP, or OMA1-E324Q-Flag and NDUFA4-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).E. HCT116 cells stably expressing control (empty vector) or OMA1-Flag, and HCT116 cells infected with control or shOMA1 (OMA1 knockdown) lentiviral particles and further cultured for 5 days, were then treated with DMSO or 20 μm CCCP for 4 hours. Cell lysates were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, MT-CO2, OMA1 or Tubulin. The black arrowhead indicated the full-length COX4L1 or NDUFB6, and the red arrowhead pointed to the cleaved band. The asterisk indicates a nonspecific band.F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were treated with CHX (100 µg/ml) plus CCCP (20 µM) for 0, 2, 4, 6 hours. The cell lysates were then assessed by western blot with antibodies against NDUFA4, COX4L1, NDUFB5, NDUFB6, OMA1 or HSPD1 (representative data from three independent experiments). The asterisk indicates a nonspecific band.G. Control or OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0, 24, or 48 hours. Cell lysates were assessed by Western blotting with anti-NDUFB5, anti-NDUFB6, anti-COX4L1, anti-NDUFA4 antibodies, or anti-β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.