( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


Details 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Details 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.


( 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 .

O O O O O O B-SUBCELLULAR O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O 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) 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 .

O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR 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) 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.


Location 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 .

O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O

Location 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.


( A - D ) Cells expressing Rpl5 - GFP ( A and B ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( A and C , left ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts . ( right ) The ratio between cleaved GFP and full - length protein was quantified for every time point in four independent experiments . a . u . , arbitrary unit .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD 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-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit.


( A - D ) Cells expressing Rpl5 - GFP ( A and B ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( B and D ) Cells before starvation or starved for 24 h ( starvation ) were examined both by fluorescence microscopy and differential interferential contrast ( DIC ) . Note that 100 % of wild - type ( WT ) and mutant cells expressed GFP - tagged proteins in the cytoplasm before starvation . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and vacuole .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O

(A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.


( A - D ) Cells expressing Rpl25 - GFP ( C and D ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( A and C , left ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts . ( right ) The ratio between cleaved GFP and full - length protein was quantified for every time point in four independent experiments . a . u . , arbitrary unit .

(A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit.


( A - D ) Cells expressing Rpl25 - GFP ( C and D ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( B and D ) Cells before starvation or starved for 24 h ( starvation ) were examined both by fluorescence microscopy and differential interferential contrast ( DIC ) . Note that 100 % of wild - type ( WT ) and mutant cells expressed GFP - tagged proteins in the cytoplasm before starvation . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and vacuole .

(A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.


( A ) GFP or Rpl25 - GFP were expressed in cells transformed ( + ) or not transformed ( − ) with a plasmid encoding 6His - ubiquitin . ( top ) Expression levels of GFP and Rpl25 - GFP in whole cell lysates were verified with anti - GFP blotting . ( middle ) Purified 6His - ubiquitin conjugates were examined by anti - GFP blotting . ( bottom ) 6His - ubiquitin expression and efficiency of purification was controlled using an anti - 6His antibody . White lines indicate that intervening lanes have been spliced out .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O 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) GFP or Rpl25-GFP were expressed in cells transformed (+) or not transformed (−) with a plasmid encoding 6His-ubiquitin. (top) Expression levels of GFP and Rpl25-GFP in whole cell lysates were verified with anti-GFP blotting. (middle) Purified 6His-ubiquitin conjugates were examined by anti-GFP blotting. (bottom) 6His-ubiquitin expression and efficiency of purification was controlled using an anti-6His antibody. White lines indicate that intervening lanes have been spliced out.


( B ) Analysis of ubiquitylated forms of Rpl25 - GFP in wild - type and the indicated mutant cells as in A .

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(B) Analysis of ubiquitylated forms of Rpl25-GFP in wild-type and the indicated mutant cells as in A.


( C ) Expression of Rpl25 - GFP and its modified form were analyzed by anti - GFP blot of extracts from wild - type and mutant cells starved for the indicated periods . Ub , ubiquitin ; WT , wild type .

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(C) Expression of Rpl25-GFP and its modified form were analyzed by anti-GFP blot of extracts from wild-type and mutant cells starved for the indicated periods. Ub, ubiquitin; WT, wild type.


( A ) Indicated strains were transformed with plasmids encoding either Rpl25 - GFP ( Rpl25 ) or rpl25 K74 , 75R - GFP ( rpl25KR ) before the deletion of a genomic copy of RPL25 . ( top ) Comparable expression levels of Rpl25 - GFP or rpl25 K74 , 75R - GFP in whole cell lysates were confirmed with anti - GFP blotting . Purified 6His - ubiquitin - conjugated forms of Rpl25 - GFP or rpl25 K74 , 75R - GFP ( middle ) and 6His - ubiquitin expression ( bottom ) were analyzed as in Fig . 2 . Ub , ubiquitin .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O

(A) Indicated strains were transformed with plasmids encoding either Rpl25-GFP (Rpl25) or rpl25 K74,75R-GFP (rpl25KR) before the deletion of a genomic copy of RPL25. (top) Comparable expression levels of Rpl25-GFP or rpl25 K74,75R-GFP in whole cell lysates were confirmed with anti-GFP blotting. Purified 6His-ubiquitin-conjugated forms of Rpl25-GFP or rpl25 K74,75R-GFP (middle) and 6His-ubiquitin expression (bottom) were analyzed as in Fig. 2. Ub, ubiquitin.


( B ) Wild - type and ubp3Δ cells expressing Rpl25 - GFP or rpl25 K74 , 75R - GFP were starved for the indicated period of time . ( C ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts , and the ratio between cleaved GFP and full - length protein was quantified for all time points in four independent experiments . a . u . , arbitrary units . ( D ) Localization of GFP - tagged proteins . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and

O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O

(B) Wild-type and ubp3Δ cells expressing Rpl25-GFP or rpl25 K74,75R-GFP were starved for the indicated period of time. (C) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts, and the ratio between cleaved GFP and full-length protein was quantified for all time points in four independent experiments. a.u., arbitrary units. (D) Localization of GFP-tagged proteins. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole


( E ) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti - Rpl3 ( gift from V . Albanese , Institut Jacques Monod , Paris , France ) or anti - Mex67 ( as a control ) antibodies . Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0 . Significance of the differences observed for the ribophagy efficiency was evaluated using Student ' s t test . * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 . White lanes indicate that intervening lanes have been spliced out . The errors bars correspond to standard deviations .

O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti-Rpl3 (gift from V. Albanese, Institut Jacques Monod, Paris, France) or anti-Mex67 (as a control) antibodies. Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0. Significance of the differences observed for the ribophagy efficiency was evaluated using Student's t test. *, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001. White lanes indicate that intervening lanes have been spliced out. The errors bars correspond to standard deviations.


( A ) Wild - type cells expressing Bre5 - GFP , Ltn1 - Flag , or ltn1 - ΔRing - Flag were starved for the indicated periods of time .

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(A) Wild-type cells expressing Bre5-GFP, Ltn1-Flag, or ltn1-ΔRing-Flag were starved for the indicated periods of time.


( B ) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132 . Expression of tagged proteins was analyzed by anti - GFP or anti - Flag blotting of whole cell extracts . Five times less extracts were analyzed for ltn1 - ΔRing - Flag cells . A nonspecific band served as a loading control . White lanes indicate that intervening lanes have been spliced out . WT , wild type .

O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O 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) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132. Expression of tagged proteins was analyzed by anti-GFP or anti-Flag blotting of whole cell extracts. Five times less extracts were analyzed for ltn1-ΔRing-Flag cells. A nonspecific band served as a loading control. White lanes indicate that intervening lanes have been spliced out. WT, wild type.


B | hFwe - Lose mRNA expression is more abundant in elderly people . hFwe - Lose mRNA expression was analyzed by RT - qPCR in 86 lung tissue biopsies taken from non - COVID patients with age between 20 and 82 years . Older patients show a significant upregulation of hFwe - Lose expression . A log - Linear regression model demonstrates a positive correlation between age and hFwe - Lose expression ( R2 = 0 . 13 ; slope confidence interval of 95 % ( CI ) = [ 2 . 0 - 12 . 2 ] ; p - value of the linear regression model < 6 ⋅ 10 - 4 ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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 | hFwe-Lose mRNA expression is more abundant in elderly people. hFwe-Lose mRNA expression was analyzed by RT-qPCR in 86 lung tissue biopsies taken from non-COVID patients with age between 20 and 82 years. Older patients show a significant upregulation of hFwe-Lose expression. A log-Linear regression model demonstrates a positive correlation between age and hFwe-Lose expression (R2= 0.13; slope confidence interval of 95% (CI) = [2.0-12.2]; p-value of the linear regression model < 6⋅10-4).


C | hFwe - Lose expression is elevated in lung tissue biopsies from patients with comorbidities . Box plot illustrates an increased expression of hFwe - Lose in lungs of patients with hypertension ( HT ; n = 129 ) , obesity ( n = 45 ) , chronic obstructive pulmonary disease ( COPD n = 51 ) , diabetes ( n = 48 ) , cardio - vascular disease ( CVD ; n = 63 ) versus disease - free control lungs ( n = 42 ) . Patient ` s age is depicted in color . Two - sided Student ' s t - test was performed for each comorbidity ( compared to disease - free patients ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-ORGANISM O B-DISEASE O B-DISEASE O O O O O O B-DISEASE O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O B-DISEASE O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 | hFwe-Lose expression is elevated in lung tissue biopsies from patients with comorbidities. Box plot illustrates an increased expression of hFwe-Lose in lungs of patients with hypertension (HT; n = 129), obesity (n = 45), chronic obstructive pulmonary disease (COPD n = 51), diabetes (n = 48), cardio-vascular disease (CVD; n = 63) versus disease-free control lungs (n = 42). Patient`s age is depicted in color. Two-sided Student's t-test was performed for each comorbidity (compared to disease-free patients), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


D | hFwe - Lose expression is upregulated in lung tissue of COVID - 19 patients . Box plot illustrates an increased expression of hFwe - Lose in lung tissue of patients diagnosed with COVID - 19 ( n = 11 ) , individuals affected with host comorbidities ( n = 216 ) versus disease - free control lungs ( n = 42 ) . Patient ' s age is depicted in color . Two - sided Student ' s t - test was performed ( compared to disease - free patients ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O B-DISEASE I-DISEASE I-DISEASE B-ORGANISM O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-ORGANISM O O B-DISEASE I-DISEASE I-DISEASE O O O O 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-ORGANISM O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D | hFwe-Lose expression is upregulated in lung tissue of COVID-19 patients. Box plot illustrates an increased expression of hFwe-Lose in lung tissue of patients diagnosed with COVID-19 (n = 11), individuals affected with host comorbidities (n = 216) versus disease-free control lungs (n = 42). Patient's age is depicted in color. Two-sided Student's t-test was performed (compared to disease-free patients), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


E | SARS - CoV - 2 - infection manifests histological changes in patients ' lungs as demonstrated by H & E staining . 1 ) For comparison , the normal lung of an elderly individual is shown , containing air - filled empty looking alveolar spaces and thin alveolar septae / gas - exchange membranes . Note that there are almost no inflammatory cells and that capillaries are only merely visible since not congested . 2 ) Diffuse ( proliferative ) alveolar damage showing evidence of cell death . An alveolar space filled with desquamated pneumocytes and macrophages , lymphocytes and focal erythrocyte extravasation as well as one multinucleated pneumocyte type II . The still recognizable epithelial lining is detached . Cells and surfactant are lost creating perforations in the alveolar wall , allowing migration of blood cells and fluid to enter inside the alveolar space . The adjacent interstitial space is significantly widened , showing an increase of mononuclear inflammatory cells and extravasation of erythrocytes . On the top and the lower right , dilated and congested capillaries can be seen ( Hematoxylin and Eosin ( H & E ) stain , 200x ) . Abbreviations : a - Alveolar structure destroyed by lymphocytes , desquamated epithelium , as well as extravasated blood ; The alveoli should be empty , but here it is filled with a combination of degenerated cells and fibrin ; b - Next alveoli ; c - Expanded pulmonary interstitium . The interstitium should be as thin as 10um , but here it is 100um ; d - Extravasation of erythrocytes ; e - Lymphocytes ; f - Congested capillaries and arterioles ; g - Multinucleated alveolocyte / pneumocyte type II ; h - Detached epithelium ; i - A histiocyte with a kidney - shaped nucleus at the bottom of the circle has ingested erythrocytes ( left - handed ) , representing the first step of cellular elimination . Round inlet with encircled karyopyknotic , karyorrhectic and " ghost - cell " figures indicative of apoptosis . 3 ) Presence of type II pneumocyte syncytial giant cells in a collapsing alveolar space with detached epithelial lining . The adjacent interstitial space shows analogous changes to 2A1 . At the bottom , there is a prominently dilated and congested capillary ( H & E , 200x ) . Abbreviations : a - Alveoli with detached epithelium ; b - Next alveoli ; c - E Extravasation of erythrocytes ; d - Expanded interstitium ; e - Lymphocytes ; f - Multinucleated pneumocyte type II . 4 ) Diffuse alveolar damage showing massive extravasation of fibrin ( homogeneous eosinophilic material in the center of the alveolar space ) . The lining pneumocytes are almost all apoptotic / necrotic . The fibrin exudate is intermingled with mononuclear inflammatory cells and cellular debris ( H & E , 200x ) . Abbreviations : a - Alveola ; b - Congested capillaries ; c - Fibrin ; d - Remnants of degenerated / dying epithelium ; e - Expanded interstitium ; f - Cellular debris consisting of macrophages , detached epithelial cells and lymphocytes ; g - Fully degenerated / lacking epithelial coverage within the alveoli . Round inlet with encircled karyopyknotic , karyorrhectic and " ghost - cell " figures indicative of apoptosis . 5 ) Immunohistochemical stain ( IHC ) for fibrin showing microthrombi caused by dysfunction of endothelial cells in capillaries of the alveolar membranes / lung interstitium leading to obstruction of the microcirculation ( IHC for fibrin , 200x ) . Abbreviations : a - All small alveolar septal capillaries are filled out with worm - like fibrin thrombi hampering / obstructing circulation ; b - Normal interstitium .

O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-ORGANISM O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-TISSUE O O O B-CELL_TYPE O O B-TISSUE O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O B-TISSUE O B-TISSUE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE O O O O B-CELL_TYPE O O B-TISSUE O O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-TISSUE O O O B-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O O O B-TISSUE O O O O O O B-CELL_TYPE O O O O B-TISSUE O O O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O B-TISSUE O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O

E | SARS-CoV-2-infection manifests histological changes in patients' lungs as demonstrated by H&E staining. 1)For comparison, the normal lung of an elderly individual is shown, containing air-filled empty looking alveolar spaces and thin alveolar septae/gas-exchange membranes. Note that there are almost no inflammatory cells and that capillaries are only merely visible since not congested. 2) Diffuse (proliferative) alveolar damage showing evidence of cell death. An alveolar space filled with desquamated pneumocytes and macrophages, lymphocytes and focal erythrocyte extravasation as well as one multinucleated pneumocyte type II. The still recognizable epithelial lining is detached. Cells and surfactant are lost creating perforations in the alveolar wall, allowing migration of blood cells and fluid to enter inside the alveolar space. The adjacent interstitial space is significantly widened, showing an increase of mononuclear inflammatory cells and extravasation of erythrocytes. On the top and the lower right, dilated and congested capillaries can be seen (Hematoxylin and Eosin (H&E) stain, 200x). Abbreviations: a- Alveolar structure destroyed by lymphocytes, desquamated epithelium, as well as extravasated blood; The alveoli should be empty, but here it is filled with a combination of degenerated cells and fibrin; b- Next alveoli; c- Expanded pulmonary interstitium. The interstitium should be as thin as 10um, but here it is 100um; d- Extravasation of erythrocytes; e- Lymphocytes; f- Congested capillaries and arterioles; g- Multinucleated alveolocyte/pneumocyte type II; h- Detached epithelium; i- A histiocyte with a kidney-shaped nucleus at the bottom of the circle has ingested erythrocytes (left-handed), representing the first step of cellular elimination. Round inlet with encircled karyopyknotic, karyorrhectic and "ghost-cell" figures indicative of apoptosis. 3) Presence of type II pneumocyte syncytial giant cells in a collapsing alveolar space with detached epithelial lining. The adjacent interstitial space shows analogous changes to 2A1. At the bottom, there is a prominently dilated and congested capillary (H&E, 200x). Abbreviations: a- Alveoli with detached epithelium; b- Next alveoli; c- E Extravasation of erythrocytes; d- Expanded interstitium; e- Lymphocytes; f- Multinucleated pneumocyte type II. 4) Diffuse alveolar damage showing massive extravasation of fibrin (homogeneous eosinophilic material in the center of the alveolar space). The lining pneumocytes are almost all apoptotic/necrotic. The fibrin exudate is intermingled with mononuclear inflammatory cells and cellular debris (H&E, 200x). Abbreviations: a- Alveola; b- Congested capillaries; c- Fibrin; d- Remnants of degenerated / dying epithelium; e- Expanded interstitium; f- Cellular debris consisting of macrophages, detached epithelial cells and lymphocytes; g- Fully degenerated / lacking epithelial coverage within the alveoli. Round inlet with encircled karyopyknotic, karyorrhectic and "ghost-cell" figures indicative of apoptosis. 5) Immunohistochemical stain (IHC) for fibrin showing microthrombi caused by dysfunction of endothelial cells in capillaries of the alveolar membranes/lung interstitium leading to obstruction of the microcirculation (IHC for fibrin, 200x). Abbreviations: a- All small alveolar septal capillaries are filled out with worm-like fibrin thrombi hampering / obstructing circulation; b- Normal interstitium.


F | Healthy lungs show very low cleaved caspase - 3 positive cells . IHC staining for cleaved caspase - 3 ( 20x ) . G | Expression of cleaved ( active ) caspase - 3 yielding apoptotic and pre - apoptotic cells : IHC for cleaved ( active ) caspase - 3 showing a brown nuclear staining signal in respective cells . 31 . 5 % cells were found to be caspase positive , 11 . 7 % with high caspase - 3 positivity and another 19 . 8 % with low . These cells are mainly located in the interstitium , but also in some alveolar epithelial and endothelial compartments . In the lower right , the edge of an alveolar space containing several ( pre - ) apoptotic pneumocytes is seen , while in the upper left a completely denuded / deepithelialized alveolus with two apoptotic remnants is observable ( IHC for cleaved caspase - 3 , 20x ) . Abbreviations : a - Apoptotic large mononuclear cells ; b - Apoptotic alveolocyte / pneumocyte ; i - Amplification of apoptotic alveolocyte / pneumocyte . As shown in the round inlets of 1E2 and 1E4 , homogeneous dark nuclear condensation up to a size of 2μm ( karyopyknosis ) , homogeneous pinkish - greyish nuclear condensation , large nuclear inclusion with marginalization of the condensed chromatin , coarse nuclear angulation , nose - like / polar body - like nuclear protrusions , the latter two being karyorrhectic debris , were all morphologically considered evidence of apoptosis . This has been correlated with and was reflected by the results of the cleaved caspase - 3 staining on step sections . All cells were counted on the 20X fields for n = 42 disease - free and n = 3 COVID - 19 autopsy patients . The expression of hFwe - Lose in COVID - 19 patients is significantly higher than in disease - free individuals , regardless of cleaved caspase - 3 staining ( Cas POS : p < 0 . 004 ; Cas NEG : p < 0 . 006 ) . In COVID - 19 patients we observed a significantly higher expression of hFwe - Lose in sections with positive cleaved caspase - 3 staining ( p < 0 . 004 ) . Two - sided Student ' s t - test was performed ( compared to disease - free samples and NEG samples , respectively ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE I-DISEASE I-DISEASE B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 | Healthy lungs show very low cleaved caspase-3 positive cells. IHC staining for cleaved caspase-3 (20x). G | Expression of cleaved (active) caspase-3 yielding apoptotic and pre-apoptotic cells: IHC for cleaved (active) caspase-3 showing a brown nuclear staining signal in respective cells. 31.5% cells were found to be caspase positive, 11.7% with high caspase-3 positivity and another 19.8% with low. These cells are mainly located in the interstitium, but also in some alveolar epithelial and endothelial compartments. In the lower right, the edge of an alveolar space containing several (pre-)apoptotic pneumocytes is seen, while in the upper left a completely denuded/deepithelialized alveolus with two apoptotic remnants is observable (IHC for cleaved caspase-3, 20x). Abbreviations: a- Apoptotic large mononuclear cells; b-Apoptotic alveolocyte / pneumocyte; i- Amplification of apoptotic alveolocyte / pneumocyte. As shown in the round inlets of 1E2 and 1E4, homogeneous dark nuclear condensation up to a size of 2μm (karyopyknosis), homogeneous pinkish-greyish nuclear condensation, large nuclear inclusion with marginalization of the condensed chromatin, coarse nuclear angulation, nose-like/polar body-like nuclear protrusions, the latter two being karyorrhectic debris, were all morphologically considered evidence of apoptosis. This has been correlated with and was reflected by the results of the cleaved caspase-3 staining on step sections. All cells were counted on the 20X fields for n = 42 disease-free and n = 3 COVID-19 autopsy patients. The expression of hFwe-Lose in COVID-19 patients is significantly higher than in disease-free individuals, regardless of cleaved caspase-3 staining (Cas POS: p < 0.004; Cas NEG: p < 0.006). In COVID-19 patients we observed a significantly higher expression of hFwe-Lose in sections with positive cleaved caspase-3 staining (p < 0.004). Two-sided Student's t-test was performed (compared to disease-free samples and NEG samples, respectively), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


A | hFwe - Lose biomarker expression is more abundant in nasopharyngeal swab probes from older adults . hFwe - Lose expression was analyzed by RT - qPCR in 283 nasopharyngeal swab samples taken from patients with age between 1 and 96 years , taken at the very beginning of the disease ( the earliest contact with physician , before the disease progression ) . The vertical axis represents relative hFwe - Lose expression normalized to the mean of non - hospitalized patients . Colors depict the outcome groups : non - hospitalized ( gray , n = 85 ) , hospitalized ( blue , n = 177 ) and deceased ( red , n = 21 ) . The shape of data points reflects the cohorts : circles for the training cohort ( n = 203 ) and triangles for the validation cohort ( n = 80 ) . The lines show the fitted curves of an asymptotic model with the same asymptotic value but different rate constants per group ( see Materials and Methods ) . Due to the comparatively low number of deceased patients in the dataset ( n = 21 ) , the curve for this group reflects the asymptotic value . Hospitalized and deceased patients show a positive correlation of hFwe - Lose expression and age with a larger rate constant for the hospitalized patients . ( R2 = 0 . 65 ) The p - value ( < 0 . 001 ) indicates that the blue curve ( for hospitalized patients ) grows faster with age , compared to the gray curve for non - hospitalized patients ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O 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-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O

A | hFwe-Lose biomarker expression is more abundant in nasopharyngeal swab probes from older adults. hFwe-Lose expression was analyzed by RT-qPCR in 283 nasopharyngeal swab samples taken from patients with age between 1 and 96 years, taken at the very beginning of the disease (the earliest contact with physician, before the disease progression). The vertical axis represents relative hFwe-Lose expression normalized to the mean of non-hospitalized patients. Colors depict the outcome groups: non-hospitalized (gray, n = 85), hospitalized (blue, n = 177) and deceased (red, n = 21). The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The lines show the fitted curves of an asymptotic model with the same asymptotic value but different rate constants per group (see Materials and Methods). Due to the comparatively low number of deceased patients in the dataset (n = 21), the curve for this group reflects the asymptotic value. Hospitalized and deceased patients show a positive correlation of hFwe-Lose expression and age with a larger rate constant for the hospitalized patients. (R2= 0.65) The p-value (< 0.001) indicates that the blue curve (for hospitalized patients) grows faster with age, compared to the gray curve for non-hospitalized patients).


B | hFwe - Lose expression is elevated in nasopharyngeal swab probes from patients with comorbidities . Box plots illustrate an increased relative expression of hFwe - Lose in nasopharyngeal swabs of patients with diabetes ( n = 129 ) , COPD ( n = 20 ) , obesity ( BMI > 30 ; n = 152 ) , cardiomyopathy ( " CM " , n = 19 ) , heart failure ( " HF " , n = 35 ) , hypertension ( " HT " , n = 121 ) , chronic kidney disease ( " CKD " , n = 60 ) versus disease - free patients ( n = 96 ) . Two - sided Student ' s t - tests were performed ( compared to disease - free patients ) , and p - values are presented on the plot . The vertical axis represents relative hFwe - Lose expression normalized to the mean of non - hospitalized patients . The color refers to the COVID - 19 disease outcome : gray for not hospitalised , blue for hospitalised and red for deceased patients . The shape of data points reflects the cohorts : circles for the training cohort ( n = 203 ) and triangles for the validation cohort ( n = 80 ) . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-ORGANISM O B-DISEASE O O O O O O B-DISEASE O O O O O O B-DISEASE O O O O O O O O O O B-DISEASE O O B-DISEASE O O O O O O O B-DISEASE I-DISEASE O O B-DISEASE O O O O O O O B-DISEASE O O B-DISEASE O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O B-DISEASE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 | hFwe-Lose expression is elevated in nasopharyngeal swab probes from patients with comorbidities. Box plots illustrate an increased relative expression of hFwe-Lose in nasopharyngeal swabs of patients with diabetes (n = 129), COPD (n = 20), obesity (BMI > 30; n = 152), cardiomyopathy ("CM", n = 19), heart failure ("HF", n = 35), hypertension ("HT", n = 121), chronic kidney disease ("CKD", n = 60) versus disease-free patients (n = 96). Two-sided Student's t-tests were performed (compared to disease-free patients), and p-values are presented on the plot. The vertical axis represents relative hFwe-Lose expression normalized to the mean of non-hospitalized patients. The color refers to the COVID-19 disease outcome: gray for not hospitalised, blue for hospitalised and red for deceased patients. The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


D | Elevated hFwe - Lose expression in the nasal swab samples associates with patients ' condition severity and respective medical treatment . Box plots illustrate an increased expression of hFwe - Lose in nasal swabs of patients , who were hospitalized within 14 days of disease progression ( n = 177 ) , admitted to intensive care unit ( ICU ) ( n = 34 ) , underwent intubation ( n = 58 ) , had respiratory rate greater than 30 ( GT30 ) ( n = 76 ) , had blood oxygenation level ( SpO2 ) less than 94 % ( n = 147 ) , and who died within 30 days of disease progression ( n = 21 ) versus patients without respective conditions . Pairwise two - sided Student ' s t - tests were performed ( compared to patients without respective conditions ) , and p - values are presented on the plot . The vertical axis represents hFwe - Lose expression normalized to the mean of non - hospitalized patients . The color refers to the COVID - 19 disease outcome : gray for not hospitalised , blue for hospitalised and red for deceased patients . The shape of data points reflects the cohorts : circles for the training cohort ( n = 203 ) and triangles for the validation cohort ( n = 80 ) . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D | Elevated hFwe-Lose expression in the nasal swab samples associates with patients' condition severity and respective medical treatment. Box plots illustrate an increased expression of hFwe-Lose in nasal swabs of patients, who were hospitalized within 14 days of disease progression (n = 177), admitted to intensive care unit (ICU) (n = 34), underwent intubation (n = 58), had respiratory rate greater than 30 (GT30) (n = 76), had blood oxygenation level (SpO2) less than 94% (n = 147), and who died within 30 days of disease progression (n = 21) versus patients without respective conditions. Pairwise two-sided Student's t-tests were performed (compared to patients without respective conditions), and p-values are presented on the plot. The vertical axis represents hFwe-Lose expression normalized to the mean of non-hospitalized patients. The color refers to the COVID-19 disease outcome: gray for not hospitalised, blue for hospitalised and red for deceased patients. The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


E | Elevated hFwe - Lose expression in nasal swab associates with patients ' disease outcome . Box plot emphasizes an increased expression of hFwe - Lose in nasal swabs of patients , who were hospitalized within 14 days of disease progression ( n = 177 ) , and who died within 30 days of disease progression ( n = 21 ) versus patients without respective conditions . Two - sided Student ' s t - tests were performed ( compared to non - hospitalised patients ) , and p - values are presented on the plot . The vertical axis represents hFwe - Lose expression normalized to the mean of non - hospitalized patients . The color refers to the COVID - 19 disease outcome : gray for not hospitalised , blue for hospitalised and red for deceased patients . The shape of data points reflects the cohorts : circles for the training cohort ( n = 203 ) and triangles for the validation cohort ( n = 80 ) . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-TISSUE O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-ORGANISM O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 |Elevated hFwe-Lose expression in nasal swab associates with patients' disease outcome. Box plot emphasizes an increased expression of hFwe-Lose in nasal swabs of patients, who were hospitalized within 14 days of disease progression (n = 177), and who died within 30 days of disease progression (n = 21) versus patients without respective conditions. Two-sided Student's t-tests were performed (compared to non-hospitalised patients), and p-values are presented on the plot. The vertical axis represents hFwe-Lose expression normalized to the mean of non-hospitalized patients. The color refers to the COVID-19 disease outcome: gray for not hospitalised, blue for hospitalised and red for deceased patients. The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


( a ) Miz1ΔPOZNes mice move slowly and lack motor coordination . The panel shows the average speed of movement and time on a rotarod ( maximum : 300 s ) maintained at constant speed of control and age - matched Miz1ΔPOZNes mice . The average age of mice tested was 16 months . P - values were calculated using an unpaired two - tailed Student ' s t - test ( the number of animals tested is shown below each graph ) .

O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O 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) Miz1ΔPOZNes mice move slowly and lack motor coordination. The panel shows the average speed of movement and time on a rotarod (maximum: 300 s) maintained at constant speed of control and age-matched Miz1ΔPOZNes mice. The average age of mice tested was 16 months. P-values were calculated using an unpaired two-tailed Student's t-test (the number of animals tested is shown below each graph).


( b ) Defect of cerebellar growth in Miz1ΔPOZNes mice . The panels show sagittal sections of brains of control and Miz1ΔPOZNes mice at the indicated age . Dotted lines show the circumference of the cerebellum . For each mouse , the cerebellar surface area was determined ; numbers show the ratio between control and Miz1ΔPOZNes mice . The number of mice analysed for each genotype is indicated on the right . Scale bar , 1 mm .

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(b) Defect of cerebellar growth in Miz1ΔPOZNes mice. The panels show sagittal sections of brains of control and Miz1ΔPOZNes mice at the indicated age. Dotted lines show the circumference of the cerebellum. For each mouse, the cerebellar surface area was determined; numbers show the ratio between control and Miz1ΔPOZNes mice. The number of mice analysed for each genotype is indicated on the right. Scale bar, 1 mm.


( c , d ) Histology of the cerebellum . Sections of the cerebellum of 11 - month - old control ( c ) and Miz1ΔPOZNes ( d , please note different scale ) mice were stained with antibodies directed against calbindin , a marker for PCs , and glial fibrillary acid protein ( GFAP ) , a marker for Bergman glial cells . Boxes indicate the area of a detail picture in the overviews . GFAP staining is shown for adjacent section ( arrowheads : regions of missing PCs ) . Scale bar , 500 μm ( upper panels ) and 100 μm ( lower panels ) .

O O O O O B-EXP_ASSAY O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

(c,d) Histology of the cerebellum. Sections of the cerebellum of 11-month-old control (c) and Miz1ΔPOZNes (d, please note different scale) mice were stained with antibodies directed against calbindin, a marker for PCs, and glial fibrillary acid protein (GFAP), a marker for Bergman glial cells. Boxes indicate the area of a detail picture in the overviews. GFAP staining is shown for adjacent section (arrowheads: regions of missing PCs). Scale bar, 500 μm (upper panels) and 100 μm (lower panels).


( e ) Enhanced apoptosis in the granular layer of cerebella in Miz1ΔPOZNes mice . The upper panel shows representative TUNEL ( terminal deoxynucleotidyl transferase dUTP nick end labelling ) staining of cerebella of age - matched control and Miz1ΔPOZNes mice . The lower panel shows a quantification of staining observed in four control and five Miz1ΔPOZNes mice . Error bars show s . e . m . P - value was calculated using an unpaired two - tailed Student ' s t - test .

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(e) Enhanced apoptosis in the granular layer of cerebella in Miz1ΔPOZNes mice. The upper panel shows representative TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining of cerebella of age-matched control and Miz1ΔPOZNes mice. The lower panel shows a quantification of staining observed in four control and five Miz1ΔPOZNes mice. Error bars show s.e.m. P-value was calculated using an unpaired two-tailed Student's t-test.


( a ) ISH documenting the expression of Miz1 mRNA in PCs of the cerebellum in wild - type mice . Left panel shows ISH , right panel shows haematoxylin and eosin staining . Scale bar , 50 μm . ( b ) IHC documenting the presence of Miz1 in the nuclei of PCs . The panel on the left shows staining with α Miz1 ( H190 ) antibody , staining on the right is a control staining lacking primary antibody . Scale bar , 50 μm .

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(a) ISH documenting the expression of Miz1 mRNA in PCs of the cerebellum in wild-type mice. Left panel shows ISH, right panel shows haematoxylin and eosin staining. Scale bar, 50 μm. (b) IHC documenting the presence of Miz1 in the nuclei of PCs. The panel on the left shows staining with α Miz1 (H190) antibody, staining on the right is a control staining lacking primary antibody. Scale bar, 50 μm.


( a ) Representative examples for ChIP - sequencing traces obtained from NPCs using an α - Miz1 antibody . Input traces are shown as negative control . For each gene , the intron / exon structure is shown below the traces with exons indicated as vertical bars and the transcriptional start site ( TSS ) at the left . An enlarged picture of the peak region is shown on the right .

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(a) Representative examples for ChIP-sequencing traces obtained from NPCs using an α-Miz1 antibody. Input traces are shown as negative control. For each gene, the intron/exon structure is shown below the traces with exons indicated as vertical bars and the transcriptional start site (TSS) at the left. An enlarged picture of the peak region is shown on the right.


( b ) Miz1 binds predominantly to core promoters . The panel shows a histogram of the percentage of promoters with the indicated distances between the peak of the Miz1 - binding site and the next TSS . Of all the peaks , 140 / 261 ( 54 % ) are located in core promoters ( ± 1 . 5 kb ) .

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(b) Miz1 binds predominantly to core promoters. The panel shows a histogram of the percentage of promoters with the indicated distances between the peak of the Miz1-binding site and the next TSS. Of all the peaks, 140/261 (54%) are located in core promoters (±1.5 kb).


( c ) Miz1 binding is conserved across different cell types and species . The plot shows a Venn diagram documenting the overlap in Miz1 - bound promoters between murine NPCs and human MDA - MB231 cells ( a mammary epithelial tumour cell line ) . The P - value was calculated using a hypergeometric test . Note that different α - Miz1 antibodies ( H190 and G18 ) were used for the analysis of mouse and human Miz1 - binding sites .

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(c) Miz1 binding is conserved across different cell types and species. The plot shows a Venn diagram documenting the overlap in Miz1-bound promoters between murine NPCs and human MDA-MB231 cells (a mammary epithelial tumour cell line). The P-value was calculated using a hypergeometric test. Note that different α-Miz1 antibodies (H190 and G18) were used for the analysis of mouse and human Miz1-binding sites.


( e ) Electrophoretic mobility shift assay documenting binding of Miz1 to the conserved sequence element ( ' Miz1 - binding motif ' ) . Nuclear extracts from control HeLa cells ( ' Mock ' ) or cells ectopically expressing Miz1 were incubated with radioactive oligonucleotides spanning the Miz1 - binding motif of the Ambra1 and Vps28 promoters . Where indicated , binding reactions included a 25 - fold excess of non - labelled oligonucleotides ( ' Competitor ' ) , the indicated mutants thereof or of an oligonucleotide containing an E - box sequence from the Ubap2 promoter . Supershift assays were performed using α - Miz1 ( H190 , G18 and C19 ) , α - Myc ( N262 ) or control antibodies . * Non - specific band . The sequences of the oligonucleotides used are shown above the panel ; mutated nucleotides are shown in bold .

O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O 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) Electrophoretic mobility shift assay documenting binding of Miz1 to the conserved sequence element ('Miz1-binding motif'). Nuclear extracts from control HeLa cells ('Mock') or cells ectopically expressing Miz1 were incubated with radioactive oligonucleotides spanning the Miz1-binding motif of the Ambra1 and Vps28 promoters. Where indicated, binding reactions included a 25-fold excess of non-labelled oligonucleotides ('Competitor'), the indicated mutants thereof or of an oligonucleotide containing an E-box sequence from the Ubap2 promoter. Supershift assays were performed using α-Miz1 (H190, G18 and C19), α-Myc (N262) or control antibodies. *Non-specific band. The sequences of the oligonucleotides used are shown above the panel; mutated nucleotides are shown in bold.


( a ) Miz1 activates the Rorc and Ambra1 promoters , and the POZ domain is required for activation . HEK293 cells were transiently transfected with plasmids containing promoter sequences of Ambra1 or Rorc in front of luciferase and with expression vectors encoding Miz1 or MizΔPOZ . Empty vectors were used as control . Values were normalized with CMV - βGal . Error bars are s . d . of biological triplicates .

O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

(a) Miz1 activates the Rorc and Ambra1 promoters, and the POZ domain is required for activation. HEK293 cells were transiently transfected with plasmids containing promoter sequences of Ambra1 or Rorc in front of luciferase and with expression vectors encoding Miz1 or MizΔPOZ. Empty vectors were used as control. Values were normalized with CMV-βGal. Error bars are s.d. of biological triplicates.


( b ) Direct target genes of Miz1 are downregulated in cerebella of Miz1ΔPOZNes mice . Microarray analysis was performed from cerebella of mice at the indicated ages . The panels show box plots ( line : median , box : 50 % of data points , whisker : 1 . 5 interquantile range , outliers are not shown ) of relative gene expression ( plotted as log2 - fold change ) of the 140 Miz1 direct target genes comparing control and Miz1ΔPOZNes mice . The P - value was calculated using a Mann - Whitney - Wilcoxon test . Microarrays were performed as technical duplicates from RNA pooled from two animals for each genotype .

O O O O O O O B-GENEPROD O O O B-TISSUE O B-GENEPROD B-ORGANISM O B-EXP_ASSAY O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O 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) Direct target genes of Miz1 are downregulated in cerebella of Miz1ΔPOZNes mice. Microarray analysis was performed from cerebella of mice at the indicated ages. The panels show box plots (line: median, box: 50% of data points, whisker: 1.5 interquantile range, outliers are not shown) of relative gene expression (plotted as log2-fold change) of the 140 Miz1 direct target genes comparing control and Miz1ΔPOZNes mice. The P-value was calculated using a Mann-Whitney-Wilcoxon test. Microarrays were performed as technical duplicates from RNA pooled from two animals for each genotype.


( d ) qRT - PCR documenting the expression of selected Miz1 target genes in cerebella of control and of Miz1ΔPOZNes mice at the indicated ages . Data represent the mean of two independent biological experiments each with three technical replicates . Error bars are s . e . m .

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(d) qRT-PCR documenting the expression of selected Miz1 target genes in cerebella of control and of Miz1ΔPOZNes mice at the indicated ages. Data represent the mean of two independent biological experiments each with three technical replicates. Error bars are s.e.m.


( a , b ) Immunostaining using an α - ubiquitin antibody of ( a ) PCs and ( b ) the molecular layer of Miz1ΔPOZNes and age - matched control mice ( dotted area : higher magnification ) . Arrowheads designate ubiquitin - positive structures . Scale bar , ( a ) 25 μm ; ( b ) 50 μm .

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(a,b) Immunostaining using an α-ubiquitin antibody of (a) PCs and (b) the molecular layer of Miz1ΔPOZNes and age-matched control mice (dotted area: higher magnification). Arrowheads designate ubiquitin-positive structures. Scale bar, (a) 25 μm; (b) 50 μm.


( c ) Immunofluorescence staining using an α - p62 and an α - ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age - matched control mice . Nuclei were counterstained with DAPI ( 4 ' , 6 - diamidino - 2 - phenylindole ) and overviews of the cerebella clarifying the selected areas are shown . Scale bar , 25 μm .

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(c) Immunofluorescence staining using an α-p62 and an α-ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age-matched control mice. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) and overviews of the cerebella clarifying the selected areas are shown. Scale bar, 25 μm.


( a , b ) Immunostaining using an α - ubiquitin antibody of ( a ) PCs and ( b ) the molecular layer of Miz1ΔPOZNes and age - matched control mice ( dotted area : higher magnification ) . Arrowheads designate ubiquitin - positive structures . Scale bar , ( a ) 25 μm ; ( b ) 50 μm . ( c ) Immunofluorescence staining using an α - p62 and an α - ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age - matched control mice . Nuclei were counterstained with DAPI ( 4 ' , 6 - diamidino - 2 - phenylindole ) and overviews of the cerebella clarifying the selected areas are shown . Scale bar , 25 μm . ( d ) Quantification of a , b and c . Error bars represent s . e . m . derived from the indicated numbers of animals . P - values were calculated using an unpaired two - tailed Student ' s t - test .

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(a,b) Immunostaining using an α-ubiquitin antibody of (a) PCs and (b) the molecular layer of Miz1ΔPOZNes and age-matched control mice (dotted area: higher magnification). Arrowheads designate ubiquitin-positive structures. Scale bar, (a) 25 μm; (b) 50 μm. (c) Immunofluorescence staining using an α-p62 and an α-ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age-matched control mice. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) and overviews of the cerebella clarifying the selected areas are shown. Scale bar, 25 μm. (d) Quantification of a, b and c. Error bars represent s.e.m. derived from the indicated numbers of animals. P-values were calculated using an unpaired two-tailed Student's t-test.


( e ) Immunoblots of Triton X - 100 soluble ( ' TX100 ' ) and insoluble ( ' 1 % SDS ' ) fractions of cerebella obtained from control and Miz1ΔPOZNes mice of the indicated ages . Blots were probed with antibodies recognizing either mono - or polyubiquitinated proteins . Staining of the membrane with amido black was used as loading control .

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(e) Immunoblots of Triton X-100 soluble ('TX100') and insoluble ('1% SDS') fractions of cerebella obtained from control and Miz1ΔPOZNes mice of the indicated ages. Blots were probed with antibodies recognizing either mono- or polyubiquitinated proteins. Staining of the membrane with amido black was used as loading control.


( f ) Transmission electron microscopy documenting the accumulation of large multilamellar bodies in PC bodies ( ' PC ' ) and Purkinje dendrites ( ' D ' ) of Miz1ΔPOZNes mice ( N , nucleus ) . Such structures were not observed in age - matched control mice ( as quantified in Supplementary Fig . S7b ) . Mean age of mice for all electron microscopy pictures is 13 monthsfor Miz1ΔPOZNes and 12 . 5 months for control mice . Morphologically similar structures accumulate in neurons of mice deficient in late steps of autophagy . Scale bars , 2 , 0 . 2 , 0 . 5 and 1 μm , respectively .

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(f) Transmission electron microscopy documenting the accumulation of large multilamellar bodies in PC bodies ('PC') and Purkinje dendrites ('D') of Miz1ΔPOZNes mice (N, nucleus). Such structures were not observed in age-matched control mice (as quantified in Supplementary Fig. S7b). Mean age of mice for all electron microscopy pictures is 13 monthsfor Miz1ΔPOZNes and 12.5 months for control mice. Morphologically similar structures accumulate in neurons of mice deficient in late steps of autophagy. Scale bars, 2, 0.2, 0.5 and 1 μm, respectively.


( a ) Genomic PCR documenting recombination of the floxed Miz1 alleles in Cre - ER - positive and - negative MEFs . 4 - OHT was added to the media for 4 days .

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(a) Genomic PCR documenting recombination of the floxed Miz1 alleles in Cre-ER-positive and -negative MEFs. 4-OHT was added to the media for 4 days.


( b ) qRT - PCR assays of Miz1 target genes in Miz1flox / floxCre - ER − and Miz1flox / floxCre - ER + MEFs . The experiment was performed independently from four embryos with identical results . Error bars depict s . d . of technical triplicates from one experiment .

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(b) qRT-PCR assays of Miz1 target genes in Miz1flox/floxCre-ER− and Miz1flox/floxCre-ER+ MEFs. The experiment was performed independently from four embryos with identical results. Error bars depict s.d. of technical triplicates from one experiment.


( c ) Immunoblots using an α - Lc3 antibody detecting Lc3 - I and conjugated Lc3 - II . 4 - OHT - treated and control - treated MEFs of the indicated genotype were incubated with leupeptin and ammonium chloride ( lys PI ) for 2 h in DMEM ( + amino acids ) or in EBSS ( − amino acids ) medium . The expression of α - tubulin ( upper panels ) or α - vinculin ( lower panels ) was used as loading control .

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(c) Immunoblots using an α-Lc3 antibody detecting Lc3-I and conjugated Lc3-II. 4-OHT-treated and control-treated MEFs of the indicated genotype were incubated with leupeptin and ammonium chloride (lys PI) for 2 h in DMEM (+amino acids) or in EBSS (−amino acids) medium. The expression of α-tubulin (upper panels) or α-vinculin (lower panels) was used as loading control.


( d ) qRT - PCR assays of Miz1 target genes in control and Miz1ΔPOZ MEFs after incubation in DMEM or in EBSS . Error bars depict s . d . of technical triplicates from one experiment .

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(d) qRT-PCR assays of Miz1 target genes in control and Miz1ΔPOZ MEFs after incubation in DMEM or in EBSS. Error bars depict s.d. of technical triplicates from one experiment.


( e ) qRT - PCR assays documenting expression of the indicated Miz1 target genes in control and Miz1ΔPOZ neurospheres ; assays were performed at passage three . s . e . m . values are derived from six biological replicates , each with technical triplicates .

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(e) qRT-PCR assays documenting expression of the indicated Miz1 target genes in control and Miz1ΔPOZ neurospheres; assays were performed at passage three. s.e.m. values are derived from six biological replicates, each with technical triplicates.


( f ) Recombination of Miz1flox / floxCre - ER + neurospheres was tested by genomic PCR . 4 - OHT was added to the media for 3 days .

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(f) Recombination of Miz1flox/floxCre-ER+ neurospheres was tested by genomic PCR. 4-OHT was added to the media for 3 days.


( g ) Immunoblots using an α - Lc3 antibody detecting Lc3 - I and Lc3 - II . 4 - OHT - treated and non - treated Miz1flox / floxCre - ER + neurospheres were incubated with leupeptin and ammonium chloride ( lys PI ) for 30 min . The expression of α - tubulin was used as loading control .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(g) Immunoblots using an α-Lc3 antibody detecting Lc3-I and Lc3-II. 4-OHT-treated and non-treated Miz1flox/floxCre-ER+ neurospheres were incubated with leupeptin and ammonium chloride (lys PI) for 30 min. The expression of α-tubulin was used as loading control.


( h ) Fluorescent microscopy of SHEP cells stably infected with lentiviruses expressing Lc3 - GFP and Lamp1 - RFP ( to mark lysosomes ) . Vps28 was depleted with siRNAs and a non - targeting pool ( siNTP ) was used as a control . Note the accumulation of Lc3 in vesicles that do not overlap with lysosomes upon depletion of Vps28 . Scale bar , 25 μm .

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(h) Fluorescent microscopy of SHEP cells stably infected with lentiviruses expressing Lc3-GFP and Lamp1-RFP (to mark lysosomes). Vps28 was depleted with siRNAs and a non-targeting pool (siNTP) was used as a control. Note the accumulation of Lc3 in vesicles that do not overlap with lysosomes upon depletion of Vps28. Scale bar, 25 μm.


A : Brightfield and fluorescence images of a NTH / GFP zygote at 4h post - fertilization , showing dispersed extranuclear fluorescence . Hoechst DNA staining ( blue ) marks nucleus . Scale bar = 5μm . B : Image of an NTH / GFP ookinete , showing fluorescent spots co - localizing with pigment clusters ( arrows ) . Hoechst DNA staining ( blue ) marks nucleus . Scale bar = 5μm .

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O

A: Brightfield and fluorescence images of a NTH/GFP zygote at 4h post-fertilization, showing dispersed extranuclear fluorescence. Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. B: Image of an NTH/GFP ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows). Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm.


D : Brightfield and fluorescence images of a zygote at 4h post - fertilization simultaneously expressing NTH : : GFP ( green ) and LAP3 : : mCherry ( red ) . Scale bar = 5μm . E : An ookinete simultaneously expressing NTH : : GFP ( green ) and LAP3 : : mCherry ( red ) , showing co - localization in the crystalloid . Scale bar = 5μm .

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O

D: Brightfield and fluorescence images of a zygote at 4h post-fertilization simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red). Scale bar = 5μm. E: An ookinete simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red), showing co-localization in the crystalloid. Scale bar = 5μm.


F : Western blot of purified NTH / GFP ookinete samples with ( + ) and without ( - ) prior in vivo crosslinking . Mr markers ( kDa ) are indicated on the right - hand side .

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F: Western blot of purified NTH/GFP ookinete samples with (+) and without (-) prior in vivo crosslinking. Mr markers (kDa) are indicated on the right-hand side.


A : Brightfield images of a NTH - KO and NTH / GFP ookinete , showing normal ookinete morphology , but lack of pigment clusters associated with crystalloids ( arrows ) in the NTH null mutant . Scale bar = 5μm .

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A: Brightfield images of a NTH-KO and NTH/GFP ookinete, showing normal ookinete morphology, but lack of pigment clusters associated with crystalloids (arrows) in the NTH null mutant. Scale bar = 5μm.


B : Aligned dot plot of oocyst numbers in Anopheles stephensi infected with parasite lines NTH / GFP , NTH - KO and NTH ∆ PP . Horizontal lines mark mean values ( n = 20 ) ; ns , not significantly different ( Mann - Whitney ) .

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B: Aligned dot plot of oocyst numbers in Anopheles stephensi infected with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20); ns, not significantly different (Mann-Whitney).


C : Brightfield and fluorescence images of oocysts of parasite lines NTH / GFP and NTH - KO at 15 days post - infection , showing lack of sporulation in the NTH null mutant . Hoechst DNA staining ( artificially depicted red ) labels parasite nuclei . The large nuclei of the epithelial midgut cells also show . Scale bar = 20μm .

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C: Brightfield and fluorescence images of oocysts of parasite lines NTH/GFP and NTH-KO at 15 days post-infection, showing lack of sporulation in the NTH null mutant. Hoechst DNA staining (artificially depicted red) labels parasite nuclei. The large nuclei of the epithelial midgut cells also show. Scale bar = 20μm.


D : Aligned dot plot of oocyst diameters in Anopheles stephensi at 15 days post - infection with parasite lines NTH / GFP , NTH - KO and NTH ∆ PP . Horizontal lines mark mean values ( n = 20 ) . * * mark statistically significant differences ( P < 0 . 0001 , Mann - Whitney ) ; ns , not significant .

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D: Aligned dot plot of oocyst diameters in Anopheles stephensi at 15 days post-infection with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20). ** mark statistically significant differences (P<0.0001, Mann-Whitney); ns, not significant.


E : Images of ookinetes of parasite lines NTH ∆ PP showing dispersed NTH ∆ PP : : GFP fluorescence ; LAP3 / mCherry showing focal LAP3 : : mCherry fluorescence in crystalloids ; Cross ( carrying modified alleles encoding NTH ∆ PP : : GFP and LAP3 : : mCherry ) , showing dispersed LAP3 : : mCherry fluorescence . Arrow marks pigment cluster associated with crystalloids . Scale bar = 5μm .

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E: Images of ookinetes of parasite lines NTH∆PP showing dispersed NTH∆PP::GFP fluorescence; LAP3/mCherry showing focal LAP3::mCherry fluorescence in crystalloids; Cross (carrying modified alleles encoding NTH∆PP::GFP and LAP3::mCherry), showing dispersed LAP3::mCherry fluorescence. Arrow marks pigment cluster associated with crystalloids. Scale bar = 5μm.


G : PCR diagnostic for the presence of modified nth alleles in blood stage infections obtained after sporozoite transmission of a NTH ∆ PP x LAP3 / mCherry genetic cross ( lane NTH ∆ PP ) , amplifying an approximately 4 . 4kb fragment . NTH / GFP parasites are used as a PCR control , amplifying approximately 4 . 6kb . Parental parasites provide a negative control .

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G: PCR diagnostic for the presence of modified nth alleles in blood stage infections obtained after sporozoite transmission of a NTH∆PP x LAP3/mCherry genetic cross (lane NTH∆PP), amplifying an approximately 4.4kb fragment. NTH/GFP parasites are used as a PCR control, amplifying approximately 4.6kb. Parental parasites provide a negative control.


H : Image of a NTH / ND500LK ookinete , showing fluorescent spots co - localizing with pigment clusters ( arrows ) indicative of normal crystalloid formation . Scale bar = 5μm .

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H: Image of a NTH/ND500LK ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows) indicative of normal crystalloid formation. Scale bar = 5μm.


A : Brightfield and fluorescence image of an unsporulated NTH / GFP oocyst at 15 days post - infection . Scale bar = 20μm . B : Image of a sporulated NTH / GFP oocyst at 15 days post - infection . Scale bar = 20μm .

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A: Brightfield and fluorescence image of an unsporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm. B: Image of a sporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm.


C : Image of a NTH / GFP sporozoite , showing NTH : : GFP localisation in a tubular structure . Scale bar = 10μm .

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C: Image of a NTH/GFP sporozoite, showing NTH::GFP localisation in a tubular structure. Scale bar = 10μm.


D : Image of a sporulated oocyst simultaneously expressing NTH : : GFP ( green ) and ACP : : mCherry ( red ) , showing co - localization in the apicoplast . Scale bar = 20μm and 10μm ( inset ) .

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D: Image of a sporulated oocyst simultaneously expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast. Scale bar = 20μm and 10μm (inset).


E : Image of sporozoites expressing NTH : : GFP ( green ) and ACP : : mCherry ( red ) , showing co - localization in the apicoplast ( arrows ) . Sporozoites expressing only ACP : : mCherry or NTH : : GFP are also present . Scale bar = 10μm .

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E: Image of sporozoites expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast (arrows). Sporozoites expressing only ACP::mCherry or NTH::GFP are also present. Scale bar = 10μm.


F : Image of group of NTH / GFP sporozoites stained with MitoTracker Red CMXRos , showing distinct subcellular positions of apicoplasts ( green ) and mitochondria ( red ) . Scale bar = 10μm .

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F: Image of group of NTH/GFP sporozoites stained with MitoTracker Red CMXRos, showing distinct subcellular positions of apicoplasts (green) and mitochondria (red). Scale bar = 10μm.


( A ) Immunostaining of ES cells ( Oct4 ) , NPCs ( day4 , Nestin ) , and neurons ( day 8 , Tuj1 ) . Scale bar : 200µm .

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(A) Immunostaining of ES cells (Oct4), NPCs (day4, Nestin), and neurons (day 8, Tuj1). Scale bar: 200µm.


( B ) Expression levels of candidate lncRNAs during three stages of differentiation , measured by RNA - seq . Gene models of lncRNAs appear in green , adjacent protein - coding genes appear in grey . Blue depicts transcripts from the ' + ' strand , orange represents transcripts from the ' - ' strand . All three RNA - seq tracks were scaled separately to the maximum coverage in each locus .

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(B) Expression levels of candidate lncRNAs during three stages of differentiation, measured by RNA-seq. Gene models of lncRNAs appear in green, adjacent protein-coding genes appear in grey. Blue depicts transcripts from the '+' strand, orange represents transcripts from the '-' strand. All three RNA-seq tracks were scaled separately to the maximum coverage in each locus.


( C ) qRT - PCR of Reno1 , Cox10as1 , and lnc - Nr2f1 in ES cells ( top ) and following eight days of neuronal differentiation ( bottom ) , following knockdown using two different shRNAs , normalized to sh - NT . Mean ± SEM is shown for 3 - 6 independent experiments , * P < 0 . 05 , * * P < 0 . 01 ( unpaired two sample t - test ) .

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(C) qRT-PCR of Reno1, Cox10as1, and lnc-Nr2f1 in ES cells (top) and following eight days of neuronal differentiation (bottom), following knockdown using two different shRNAs, normalized to sh-NT. Mean ± SEM is shown for 3-6 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test).


( A ) Immunostaining using anti - TUJ1 antibody of ES - cell - derived neurons following infection with either non - targeting shRNA ( sh - NT ) , or two different shRNAs targeting Reno1 . Scale bar : 200µm . ( B ) Quantification of cell numbers and neurite lengths of ten images of non - overlapping fields for each shRNA . Mean ± SEM is shown , * * P < 0 . 01 ( unpaired two sample t - test ) .

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(A) Immunostaining using anti-TUJ1 antibody of ES-cell-derived neurons following infection with either non-targeting shRNA (sh-NT), or two different shRNAs targeting Reno1. Scale bar: 200µm. (B) Quantification of cell numbers and neurite lengths of ten images of non-overlapping fields for each shRNA. Mean ± SEM is shown, ** P<0.01 (unpaired two sample t-test).


( C ) Mean expression levels of genes which were down - or upregulated following knockdown ( KD ) of Reno1 or lnc - Nr2f1 during an eight time point neuronal differentiation and maturation of mouse ES cells : Neurons1 corresponds to day in vitro ( DIV ) 1 ; Neurons2 - DIV7 ; Neurons3 - DIV16 ; Neurons4 - DIV21 ; and Neurons5 - DIV28 ( Hubbard et al . , 2013 ) .

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(C) Mean expression levels of genes which were down- or upregulated following knockdown (KD) of Reno1 or lnc-Nr2f1 during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV)1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013).


( D ) Transcriptional response of cell cycle and neuron - related genes . Heatmaps show log2FC of cells infected with shRNAs targeting Reno1 or lnc - Nr2f1 compared to cells infected with sh - NT , following eight days of neuronal differentiation , and of day eight of differentiation compared to ES cells during differentiation of WT cells .

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(D) Transcriptional response of cell cycle and neuron-related genes. Heatmaps show log2FC of cells infected with shRNAs targeting Reno1 or lnc-Nr2f1 compared to cells infected with sh-NT, following eight days of neuronal differentiation, and of day eight of differentiation compared to ES cells during differentiation of WT cells.


( A ) Reno1 locus in six vertebrate species . Shaded region indicates the Reno1 locus . Blue gene models are from RefSeq or Ensembl , red gene models are based on PLAR transcript reconstructions ( Hezroni et al . , 2015 ) . RNA - seq datasets from indicated tissues were taken from publicly - available datasets : BodyMap ( Li et al , 2017a ) ( mouse ) , Reference Epigenome ( human ) , FAANG ( sheep ) , SRP011985 ( opossum ) , SRP016501 ( chicken ) , DRP000627 ( coelacanth ) . RNA - seq data was scaled separately in each species to the highest coverage in the window , except for opossum and chicken , where a custom upper threshold was set as indicated .

O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O

(A) Reno1 locus in six vertebrate species. Shaded region indicates the Reno1 locus. Blue gene models are from RefSeq or Ensembl, red gene models are based on PLAR transcript reconstructions (Hezroni et al., 2015). RNA-seq datasets from indicated tissues were taken from publicly-available datasets: BodyMap (Li et al, 2017a) (mouse), Reference Epigenome (human), FAANG (sheep), SRP011985 (opossum), SRP016501 (chicken), DRP000627 (coelacanth). RNA-seq data was scaled separately in each species to the highest coverage in the window, except for opossum and chicken, where a custom upper threshold was set as indicated.