B . Scheme showing a sporozoite moving through a polymeric network in 3D . graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel . Box ‐ ‐ whisker plots depict 25 % quantile , median , 75 % quantile , nearest observations within 1 . 5 times interquartile range ( whiskers ) . Significance determined by Kruskal - Wallis with Bonferroni ' s Multiple Comparison .

O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM 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

B. Scheme showing a sporozoite moving through a polymeric network in 3D. The graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


C . Selected trajectories of 20 moving sporozoites observed over a period of 3 min . Scale bar , 10 μm .

O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

C. Selected trajectories of 20 moving sporozoites observed over a period of 3 min. Scale bar, 10 μm.


A Representative pictures of at 18 . 5 days postcoitum ( E18 . 5 ) hematoxylin eosin of whole - mount sections from PR - SET7loxp / Alfp - Cre ( PR - SET7ΔHepE ) mice control ( WT ) .

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

A Representative pictures of embryos at 18.5 days postcoitum (E18.5) and hematoxylin and eosin staining of whole-mount embryo sections from PR-SET7loxp/Alfp-Cre (PR-SET7ΔHepE) mice and control littermates (WT).


O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O

B Hematoxylin and eosin staining of liver sections.


C Immunohistochemical of liver sections with albumin antibody .

O B-EXP_ASSAY O B-TISSUE O O B-GENEPROD O O

C Immunohistochemical staining of liver sections with albumin antibody.


D qPCR of Albumin , Afp HNF4 mRNA . Bars represent mean values of mRNA normalized to glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) mRNA from samples of four mice . * P - value < 0 . 01 .

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

D qPCR analysis of Albumin,Afp and HNF4 mRNA levels. Bars represent mean values of mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and SEM from samples of four individual mice. *P-value < 0.01.


E Immunohistochemical of liver sections with cyclin B1 antibody .

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

E Immunohistochemical staining of liver sections with cyclin B1 antibody.


F Immunohistochemical of liver sections with γH2AX antibody .

F Immunohistochemical staining of liver sections with γH2AX antibody.


A Macroscopic appearance of livers in 120 - day - old ( P120 ) wild - type ( WT ) PR - SET7loxp / Alb - Cre ( KO ) mice . Note , small adenomatous foci in KO livers .

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

A Macroscopic appearance of livers in 120-day-old (P120) wild-type (WT) and PR-SET7loxp/Alb-Cre (KO) mice. Note, small adenomatous foci in KO livers.


B Representative hematoxylin eosin of liver sections from P120 wild - type ( WT ) PR - SET7loxp / Alb - Cre ( PR - SET7ΔHepA ) mice . Arrows show three areas containing morphologically different hepatocytes . Right panels : zoom - in to - A - ' normal zone ' , to - B - ' necrotic zone ' to - C - ' regenerative zone ' .

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

B Representative hematoxylin and eosin staining of liver sections from P120 wild-type (WT) and PR-SET7loxp/Alb-Cre (PR-SET7ΔHepA) mice. Arrows show three areas containing morphologically different hepatocytes. Right panels: zoom-in to Area-A-'normal zone', to Area-B-'necrotic zone' and to Area-C-'regenerative zone'.


C Immunohistological of liver sections from P120 PR - SET7ΔHepA mice control ( WT ) with HNF4 antibody .

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

C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with HNF4 antibody.


D TUNEL of liver sections from P120 PR - SET7ΔHepA mice control ( WT ) . Note that containing enlarged nuclei ( arrows ) are TUNEL negative .

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

D TUNEL staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT). Note that cells containing enlarged nuclei (white arrows) are TUNEL negative.


E Immunohistological with γH2AX albumin ( Alb ) antibodies .

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

E Immunohistological staining with γH2AX and albumin (Alb) antibodies.


A Electronmicroscopic of containing enlarged nuclei in P120 PR - SET7ΔHepA livers normal hepatocytes in control ( WT ) . arrows indicate : ( Nuc ) nuclei ; ( ER ) endoplasmic reticulum ; ( Mit ) mitochondrium ; ( PM ) plasma ; ( a ) swollen mitochondria ; ( b ) swollen endoplasmic reticulum ; ( c ) disorganized endoplasmic reticulum ; ( d ) disrupted plasma ; ( e ) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes .

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

A Electronmicroscopic images of cells containing enlarged nuclei in P120 PR-SET7ΔHepA livers and normal hepatocytes in control littermates (WT). White arrows indicate: (Nuc) nuclei; (ER) endoplasmic reticulum; (Mit) mitochondrium; (PM) plasma membrane; (a) swollen mitochondria; (b) swollen endoplasmic reticulum; (c) disorganized endoplasmic reticulum; (d) disrupted plasma membrane; and (e) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes.


B Immunohistological of liver sections from P120 PR - SET7ΔHepA mice control ( WT ) with H4K20Me1 H4K20Me3 antibodies . Normal ( - A ) , Necrotic ( - B ) Regenerative ( - C ) hepatocyte - containing areas are indicated . Arrows show loss of H4K20Me1 signal from large necrotic nuclei .

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

B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with H4K20Me1 and H4K20Me3 antibodies. Normal (Area-A), Necrotic (Area-B) and Regenerative (Area-C) hepatocyte-containing areas are indicated. Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei.


C - F Partial ( 2 / 3rd ) hepatectomy was performed in 45 - day - old PR - SET7ΔHepA mice control ( WT ) . Two later , at postnatal day 60 ( P60 ) liver sections were stained with hematoxylin eosin ( C ) or γH2AX ( D ) or H4K20Me1 ( E ) or HNF4 F4 / 80 ( F ) antibodies .

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

C-F Partial (2/3rd) hepatectomy was performed in 45-day-old PR-SET7ΔHepA mice and control littermates (WT). Two weeks later, at postnatal day 60 (P60) liver sections were stained with hematoxylin and eosin (C) or γH2AX (D) or H4K20Me1 (E) or HNF4 and F4/80 (F) antibodies.


A , B Immunohistological of liver sections from P120 PR - SET7ΔHepA mice control ( WT ) with Ki67 , albumin ( Alb ) A6 or HNF4 antibodies as indicated . Arrows depict different areas in sections as indicated above ( Fig . 1B ) . Asterisks ( * ) indicate Ki67 + hepatocytes . Small arrows ( > ) indicate Ki67 + non - hepatic . Note lack of co - of with HNF4 A6 antibodies .

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

A, B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with Ki67, albumin (Alb) and A6 or HNF4 antibodies as indicated. Arrows depict the different areas in the sections as indicated above (Fig.1B). Asterisks (*) indicate Ki67+ hepatocytes. Small arrows (>) indicate Ki67+ non-hepatic cells. Note the lack of co-staining of cells with HNF4 and A6 antibodies.


C Immunohistological of liver sections from P120 PR - SET7ΔHepA mice control ( WT ) with FGF7 A6 antibodies .

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

C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with FGF7 and A6 antibodies.


D Relative mRNA of FGF7 . Bars represent mean FGF7 mRNA normalized to GAPDH mRNA from samples of 5 45 days or 120 - day - old mice . data are presented as fold over values obtained with wild - type P45 samples . * P - value < 0 . 01 .

O O O O B-GENEPROD 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-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

D Relative mRNA levels of FGF7. Bars represent mean FGF7 mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 45 days or 120-day-old mice. The data are presented as fold over values obtained with wild-type P45 samples. *P-value < 0.01.


A of reactive oxygen species ( ROS ) accumulation in frozen liver sections from 120 - day - old ( P120 ) 240 - day - old ( P240 ) PR - SET7ΔHepA mice control ( WT ) .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-TISSUE 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-ORGANISM O O O O O

A Analysis of reactive oxygen species (ROS) accumulation in frozen liver sections from 120-day-old (P120) and 240-day-old (P240) PR-SET7ΔHepA mice and control littermates (WT).


B Immunohistological of liver sections from P240 with phospho - STAT3 antibody .

O B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O O

B Immunohistological staining of liver sections from P240 animals with phospho-STAT3 antibody.


C Western blot of liver extracts from P45 , P120 P240 mice with STAT3 phospho - STAT3 antibodies .

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

C Western blot analysis of liver extracts from P45, P120 and P240 mice with STAT3 and phospho-STAT3 antibodies.


D Macroscopic appearance of livers in P240 PR - SET7ΔHepA mice control ( WT ) .

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

D Macroscopic appearance of livers in P240 PR-SET7ΔHepA mice and control littermates (WT).


E Serum alanine aminotransferase ( ALT ) in P45 , P120 P240 mice . Bars represent mean values of ALT from liver extracts of five mice . * P - value < 0 . 01 .

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

E Serum alanine aminotransferase (ALT) levels in P45, P120 and P240 mice. Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice. *P-value < 0.01.


F Representative hematoxylin eosin of liver sections from P240 PR - SET7ΔHepA mice control ( WT ) . Two different magnifications are shown .

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

F Representative hematoxylin and eosin staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT). Two different magnifications are shown.


G Relative mRNA of selected oncofetal genes . Bars represent mean Afp , H19 , GPC - 3 CTGF mRNA normalized to GAPDH mRNA from samples of 5 240 - day - old mice . data are presented as fold over values obtained with wild - type samples . * P - value < 0 . 01 .

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

G Relative mRNA levels of selected oncofetal genes. Bars represent mean Afp, H19, GPC-3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240-day-old mice. The data are presented as fold over values obtained with wild-type samples. *P-value < 0.01.


A , B Immunohistological of liver sections from P240 PR - SET7ΔHepA mice control ( WT ) with Afp HNF4 ( A ) or Ki - 67 albumin ( Alb ) ( B ) antibodies .

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

A, B Immunohistological staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT) with Afp and HNF4 (A) or Ki-67 and albumin (Alb) (B) antibodies.


C - E Double of liver sections with A6 HNF4 ( C ) or Sox9 Alb ( D ) or CD133 HNF4 ( E ) antibodies . Right panels show larger magnifications of stainings performed in PR - SET7ΔHepA liver sections .

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

C-E Double staining of liver sections with A6 and HNF4 (C) or Sox9 and Alb (D) or CD133 and HNF4 (E) antibodies. Right panels show larger magnifications of stainings performed in PR-SET7ΔHepA liver sections.


A P240 PR - SET7ΔHepA liver sections were double - stained with A6 Sox9 ( upper panel ) or CD133 Sox9 ( lower panel ) . Pie charts at right represent average percentages of stained positively with indicated markers after counting DAPI - stained in 5 high - power fields ( HPF ) in liver sections of 3 different mice .

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

A P240 PR-SET7ΔHepA liver sections were double-stained with A6 and Sox9 (upper panel) or CD133 and Sox9 (lower panel). Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI-stained cells in 5 high-power fields (HPF) in liver sections of 3 different mice.


B duplication rate of hepatocytes from P240 PR - SET7ΔHepA livers in culture .

O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE O O O

B Cell duplication rate of primary hepatocytes from P240 PR-SET7ΔHepA livers in culture.


C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with hepatocytes from P240 PR - SET7ΔHepA livers or Hepa 1 - 6 . Right panel shows average volumes ( n = 3 ) dissected at indicated time points following injection .

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

C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers or Hepa 1-6 cells. Right panel shows average tumor volumes (n = 3) dissected at the indicated time points following injection.


D , E Hematoxylin eosin immunohistological with A6 , HNF4 , Sox9 CD133 antibodies of sections from xenografts dissected 20 or 30 days after injection with hepatocytes from P240 PR - SET7ΔHepA livers .

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

D, E Hematoxylin and eosin and immunohistological staining with A6, HNF4, Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers.


B . T6SS secretion of A . tumefaciens strains : wild type C58 , various mutants lacking one , two , or three toxin - immunity gene pairs , a mutant lacking tssL .

O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM 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 O

B. T6SS secretion assay of A. tumefaciens strains: wild type C58, various mutants lacking one, two, or three toxin-immunity gene pairs, and a mutant lacking tssL.


C . T6SS secretion of various A . tumefaciens strains : wild type C58 , ΔtssL , tde double deletion mutant ( Δtdei ) containing pRL662 pTrc200 empty vector ( V ) only or expression of pTdei1 ( tde1 - tdi1 expressed from pTrc200 ) , pTde2 * ( catalytic site mutated tde2 expressed on pRL662 ) , or pTdei1 + pTde2 * .

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

C. T6SS secretion assay of various A. tumefaciens strains: wild type C58, ΔtssL, the tde double deletion mutant (Δtdei) containing pRL662 and pTrc200 empty vector (V) only or expression of pTdei1 (tde1-tdi1 expressed from pTrc200), pTde2* (catalytic site mutated tde2 expressed on pRL662), or pTdei1+ pTde2*.


D . A . tumefaciens antibacterial activity against E . coli . strains of A . tumefaciens were co - cultured at a of 30 : 1 with E . coli DH10B ( + pRL662 ) on LB agar . survival of target E . coli was quantified by counting CFUs on gentamicin - containing LB agar plates . Data represent mean ± of 6 biological replicates from three independent experiments . One - way ANOVA followed by Turkey HSD was used for statistical . Two groups with significant differences ( P = 0 . 0010053 ) are indicated with different letters ( a b ) .

O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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

D. A. tumefaciens antibacterial activity assay against E. coli. The strains of A. tumefaciens were co-cultured at a ratio of 30:1 with E. coli DH10B (+ pRL662) on LB agar. The survival of target E. coli cells was quantified by counting CFUs on gentamicin-containing LB agar plates. Data represent mean ± SEM of 6 biological replicates from three independent experiments. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Two groups with significant differences (P=0.0010053) are indicated with different letters (a and b).


A . T6SS secretion of A . tumefaciens strains : wild type C58 , ΔtssL , Δtap - 1 , Δatu3641 harboring a pTrc200 vector ( V ) or derivatives pTap - 1 ( tap - 1 expressed from pTrc200 ) , or p3641 ( atu3641 expressed from pTrc200 ) .

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

A. T6SS secretion assay of A. tumefaciens strains: wild type C58, ΔtssL, Δtap-1, and Δatu3641 harboring a pTrc200 vector (V) or derivatives pTap-1 (tap-1 expressed from pTrc200), or p3641 (atu3641 expressed from pTrc200).


B . T6SS secretion of A . tumefaciens C58 Δtap - 1Δatu3641 containing an empty pTrc200 vector ( V ) or its derivatives expressing tap - 1 , atu3641 , tde1 - tdi1 , or tde2 - tdi2 .

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

B. T6SS secretion assay of A. tumefaciens C58 Δtap-1Δatu3641 containing an empty pTrc200 vector (V) or its derivatives expressing tap-1, atu3641, tde1-tdi1, or tde2-tdi2.


C . T6SS secretion of A . tumefaciens C58ΔG2op mutant encoding full - or truncated VgrG1 .

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

C. T6SS secretion assay of A. tumefaciens C58ΔG2op mutant encoding full-length or truncated VgrG1 proteins.


A . Representative of of A . tumefaciens C58 strains ΔtssB , ΔtssLΔtssB , ΔtdeiΔtssB each expressing TssB - GFP from pTrc200 . Upper panel : phase contrast ; Lower panel : green . Examples of GFP streaks were indicated by arrowheads . Scale bar : 2 μm .

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

A. Representative images of cells of A. tumefaciens C58 strains ΔtssB, ΔtssLΔtssB, and ΔtdeiΔtssB cells each expressing TssB-GFP from pTrc200. Upper panel: phase contrast images; Lower panel: green fluorescence images. Examples of GFP streaks were indicated by white arrowheads. Scale bar: 2 μm.


B . of TssB - GFP foci in different genetic background . number of foci were counted using " particle " function in Fiji with a manually set threshold . number of were counted manually . For each , a total of three were obtained from one independent experiment each image contained more than 300 for statistical . One - way ANOVA followed by Turkey HSD was used for statistical . Data are mean ± two groups with significant differences ( P = 0 . 000272 ) are indicated with different letters ( a b ) . Similar results are reproduced in two independent experiments .

O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of TssB-GFP foci in different genetic background. The number of the fluorescent foci were counted using the "analysis particle" function in Fiji with a manually set threshold. The number of cells were counted manually. For each strain, a total of three images were obtained from one independent experiment and each image contained more than 300 cells for quantification and statistical analysis. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Data are mean ± SEM and two groups with significant differences (P=0.000272) are indicated with different letters (a and b). Similar results are reproduced in two independent experiments.


C . Western blots of isolated sheaths , which were prepared via ultracentrifugation of lysates from A . tumefaciens C58 wild type , ΔtssL , Δtdei . T : total from lysate . P : pellet samples containing TssBC sheaths after ultracentrifugation . S : supernatant after ultracentrifugation . were analyzed in western blots probed with indicated antibodies . Fha serves as a control . Molecular markers ( in kDa ) are indicated on left . Similar results were obtained from at least two independent experiments . Data are from one independent experiment reproduced in at least two independent experiments .

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

C. Western blots of the isolated sheaths, which were prepared via ultracentrifugation of cell lysates from A. tumefaciens C58 wild type, ΔtssL, and Δtdei. T: total proteins from the cell lysate. P: pellet samples containing TssBC sheaths after ultracentrifugation. S: supernatant after ultracentrifugation. Proteins were analyzed in western blots probed with indicated antibodies. Fha serves as a cytoplasmic control. Molecular weight markers (in kDa) are indicated on the left. Similar results were obtained from at least two independent experiments. Data are from one independent experiment and reproduced in at least two independent experiments.


D . Pellet samples of wild type Δtdei were visualized with transmission electron microscopy ( ) . Sheaths are indicated by red arrowheads flagella , which are distinguishable on basis of smoother , more solid , thinner structures ( ~ 13 nm in width ) , are indicated by green arrowheads [ 33 ] . Data are from one independent experiment reproduced in at least two independent experiments . Scale bar : 0 . 5 μm ( upper panel ) , 100 nm ( upper panel ) .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR 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 O O O O O O O O O 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. Pellet samples of wild type and Δtdei were visualized with transmission electron microscopy (TEM). Sheaths are indicated by red arrowheads and flagella, which are distinguishable on the basis of smoother, more solid, and thinner structures (~13 nm in width), are indicated by green arrowheads [33]. Data are from one independent experiment and reproduced in at least two independent experiments. Scale bar: 0.5 μm (upper panel), 100 nm (upper panel).


B . Secretion of wild type various mutants of 12D1 , 1D1108 15955 .

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

B. Secretion assay of wild type and various mutants of 12D1, 1D1108 and 15955.


C . Antibacterial activity of wild type mutants of 12D1 , 1D1108 15955 was carried out in a of 30 : 1 against E . coli harboring plasmid pRL662 . target E . coli were serially - diluted grown overnight on gentamicin - containing LB agar prior to photographing . Each competition was done at least four times reproduced in three independent experiments .

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

C. Antibacterial activity assay of wild type and mutants of 12D1, 1D1108 and 15955 was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in three independent experiments.


D . Secretion of wild type 12D1 , ΔtssL , Δv1 , Δv3 - 4 containing pRL662 empty vector ( V ) or its derivatives with v1 or v3 - 4 were analyzed for secretion .

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

D. Secretion assay of wild type 12D1, ΔtssL, Δv1, Δv3-4 containing pRL662 empty vector (V) or its derivatives with v1 or v3-4 were analyzed for secretion.


E . Secretion of wild type 1D1108 , ΔtssL , Δv4 , Δv6 containing pRL662 empty vector ( V ) or its derivatives with v4 or v6 .

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

E. Secretion assay of wild type 1D1108, ΔtssL, Δv4, Δv6 containing pRL662 empty vector (V) or its derivatives with v4 or v6.


A . T6SS secretion of A . tumefaciens wild type C58 , ΔtssL , various single , double , triple toxin - immunity deletion strains .

O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O

A. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, and various single, double, and triple toxin-immunity deletion strains.


B . T6SS secretion of A . tumefaciens wild type C58 , ΔtssL , Δtdei , Δ3TIs harboring indicated plasmids .

O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O

B. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, Δtdei, and Δ3TIs harboring the indicated plasmids.


A . T6SS secretion of A . tumefaciens 12D1 wild type , ΔtssL , various toxin - immunity deletion strains .

O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O

A. T6SS secretion assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains.


B . T6SS secretion of A . tumefaciens 12D1 wild type , ΔtssL , various toxin - immunity deletion strains harboring indicated plasmids .

O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O

B. T6SS secretion assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids.


C . Antibacterial activity of A . tumefaciens 12D1 wild type , ΔtssL , various toxin - immunity deletion strains harboring indicated plasmids was carried out in a of 30 : 1 against E . coli harboring plasmid pRL662 . target E . coli were serially - diluted grown overnight on gentamicin - containing LB agar prior to photographing . Each competition was done at least four times reproduced in two independent experiments .

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

C. Antibacterial activity assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in two independent experiments.


D . T6SS secretion of A . tumefaciens C58 wild type , ΔtssL , various mutant strains .

O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O

D. T6SS secretion assay of A. tumefaciens C58 wild type, ΔtssL, and various mutant strains.


( a ) Intracellular of spermidine ( Spd ) in chronologically ageing wild - type yeast . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 * P 0 . 001 ) .

O O O O O B-SMALL_MOLECULE 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

(a) Intracellular levels of spermidine (Spd) in chronologically ageing wild-type yeast. Data represent means ± s.e.m. (n = 3; *P 0.05 and *P 0.001).


( b ) Survival determined by clonogenicity during chronological ageing of wild - type yeast ( BY4741 ) with ( o ) without ( ▪ ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 5 ) .

O O O B-EXP_ASSAY 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 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

(b) Survival determined by clonogenicity during chronological ageing of wild-type yeast (BY4741) cells with (o) and without (▪) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 5).


( c ) Intracellular of spermidine in chronologically ageing wild - type yeast cultured with ( open bar ) or without ( closed bar ) spermidine ( 4 mM ) for 5 days . Data represent means ± s . e . m . ( n = 3 ; * * P 0 . 001 ) .

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

(c) Intracellular levels of spermidine in chronologically ageing wild-type yeast cells cultured with (open bar) or without (closed bar) spermidine (4 mM) for 5 days. Data represent means ± s.e.m. (n = 3; **P 0.001).


( d ) Replicative lifespan of BY4741 wild - type yeast after separation into old ( fraction V ) young ( fraction II ) by elutriation centrifugation . remaining lifespan with or without ( control ) spermidine ( 1 mM , applied after elutriation ) on 2 % glucose synthetic complete medium is shown .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM 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-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

(d) Replicative lifespan analysis of BY4741 wild-type yeast cells after separation into old (fraction V) and young (fraction II) cells by elutriation centrifugation. The remaining lifespan with or without (control) spermidine (1 mM, applied after elutriation) on 2% glucose synthetic complete medium is shown.


( e ) Survival determined by - specific number of dead individuals of Drosophila with without ( control ) supplementation of food with various concentrations of spermidine ( as indicated ) . A representative ageing experiment of at least 50 flies per sample is shown .

O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM 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

(e) Survival determined by the age-specific number of dead individuals of female Drosophila with and without (control) supplementation of food with various concentrations of spermidine (as indicated). A representative ageing experiment of at least 50 flies per sample is shown.


( f ) Endogenous spermidine of Drosophila fed for 48 h with food supplemented by 1 mM spermidine , compared with normal food ( control ) . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 01 ) .

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

(f) Endogenous spermidine of female Drosophila fed for 48 h with food supplemented by 1 mM spermidine, compared with normal food (control). Data represent means ± s.e.m. (n = 3; *P 0.01).


( g ) Survival determined by annexin V / 7 - AAD co - ( unstained were considered as viable ) of human immune ( PBMC ) cultured for 6 12 days in absence ( black bar ) or presence ( bars ) of various spermidine concentrations ( as indicated ) . Data represent means ± s . e . m . of 3 independent experiments ( each performed on PBMC from different donors ) . * P 0 . 05 * * P 0 . 01 .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O B-CELL_TYPE 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 B-CELL_TYPE O O O O O O O O O O O O O O O O O

(g) Survival determined by annexin V/7-AAD co-staining (unstained cells were considered as viable) of human immune cells (PBMC) cultured for 6 and 12 days in the absence (black bar) or presence (white bars) of various spermidine concentrations (as indicated). Data represent means ± s.e.m. of 3 independent experiments (each performed on PBMC from different donors). *P 0.05 and **P 0.01.


( h , i ) Free thiol group ( RSH ) in serum ( h ) intracellular spermidine in hepatocytes ( i ) of ageing mice with ( open bars ) or without ( closed bar ) supplementation of drinking water with spermidine ( 0 . 3 3 mM ) for 200 days . Data represent mean ± s . e . m . ( n = 3 ; * P 0 . 05 * * P 0 . 01 ) .

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

(h, i) Free thiol group (RSH) concentration in serum (h) and intracellular spermidine concentration in hepatocytes (i) of ageing mice with (open bars) or without (closed bar) supplementation of drinking water with spermidine (0.3 and 3 mM) for 200 days. Data represent mean ± s.e.m. (n = 3; *P 0.05 and **P 0.01).


( a ) Intracellular spermidine of five - day - oldΔspe1 , compared with wild - type . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) .

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

(a) Intracellular spermidine of five-day-oldΔspe1 cells, compared with wild-type cells. Data represent means ± s.e.m. (n = 3; *P 0.001).


( b ) Chronological ageing of wild - type ( ▪ , o ) polyamine - depleted Δspe1 ( ▴ , ▿ ) yeast with ( open symbols ) without ( closed symbols ) supplementation of low doses of spermidine . Data represent means ± s . e . m . ( n = 4 ) . were tested for markers at day 3 .

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 B-ORGANISM 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

(b) Chronological ageing of wild-type (▪, o) and polyamine-depleted Δspe1 (▴, ▿) yeast cells with (open symbols) and without (closed symbols) supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4). Cells were tested for cell death markers at day 3.


( c ) microscopy of DHE → Eth conversion in wild - type Δspe1 indicating ROS production . Scale bars , 10 μm .

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

(c) Fluorescence microscopy of DHE→Eth conversion in wild-type and Δspe1 cells indicating ROS production. Scale bars, 10 μm.


( d ) ( FACS ) of ROS accumulation using DHE → Eth conversion of wild - type ( WT ) Δspe1 with without supplementation of low doses of spermidine . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 001 ) .

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

(d) Quantification (FACS analysis) of ROS accumulation using DHE→Eth conversion of wild-type (WT) and Δspe1 cells with and without supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4; *P 0.001).


( e ) ( FACS ) of phosphatidylserine externalization loss of integrity using annexin V / PI co - ( at day 5 ) of fragmentation using TUNEL ( at day 3 ) of chronologically ageing wild - type ( WT ) Δspe1 with or without supplementation of 0 . 1 mM spermidine as indicated . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) .

O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY 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 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

(e) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity using annexin V/PI co-staining (at day 5) and of DNA fragmentation using TUNEL staining (at day 3) of chronologically ageing wild-type (WT) and Δspe1 cells with or without supplementation of 0.1 mM spermidine as indicated. Data represent means ± s.e.m. (n = 3; *P 0.001).


( a ) microscopy of DHE → Eth conversion annexin V ( green ) / PI ( red ) co - of wild - type at day 18 of chronological ageing experiment shown in Fig . 1b . Scale bars , 10 μm . ( b , c ) of DHE → Eth conversion ( b ) annexin V / PI co - ( c ) using FACS performed at indicated time - points of chronological ageing experiment shown in Fig . 1b . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O 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 B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O

(a) Fluorescence microscopy of DHE→Eth conversion and annexin V (green)/PI (red) co-staining of wild-type cells at day 18 of the chronological ageing experiment shown in Fig. 1b. Scale bars, 10 μm. (b, c) Quantification of DHE→Eth conversion (b) and annexin V/PI co-staining (c) using FACS analysis performed at indicated time-points of the chronological ageing experiment shown in Fig. 1b. Data represent means ± s.e.m. (n = 3; *P 0.001).


( d ) Electron microscopy of young log - phase ( day 0 ) of 20 - day - old wild - type aged without ( control ) or with spermidine ( 4 mM ) . Representative are shown ( for an overview see Supplementary Information , Fig . S3c ) .

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 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

(d) Electron microscopy of young log-phase cells (day 0) and of 20-day-old wild-type cells aged without (control) or with spermidine (4 mM). Representative cells are shown (for an overview see Supplementary Information, Fig. S3c).


( e ) microscopy of chronologically aged wild - type ( day 3 14 ) expressing an EGFP - tagged version of yeast HMGB1 homologue ( Nhp6A - EGFP ) with or without ( control ) addition of 4 mM spermidine . Scale bars , 5 μm .

O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

(e) Fluorescence microscopy of chronologically aged wild-type cells (day 3 and 14) expressing an EGFP-tagged version of the yeast HMGB1 homologue (Nhp6A-EGFP) with or without (control) addition of 4 mM spermidine. Scale bars, 5 μm.


( a ) Relative acetylation ( normalized to controls at each day ) of indicated histone H3 Lys residues determined by of immunoblot . A representative blot using site specific antibodies is shown in b . Data represent means ± s . e . m . ( n = 3 ) . Serial dilutions of extracts were applied in western blots to verify linearity before ( examples are shown in Supplementary Information , Fig . S4 ) . For calculation details see section on Methods . P values indicate result of a two - factor ANOVA corrected by Bonferroni post hoc . ( b ) Immunoblot of whole - extracts of wild - type chronologically aged to designated time - points with ( + ) or without ( − ) spermidine application . Blots were probed with antibodies against total histone H3 or H3 acetylation sites at indicated Lys residues . Full scans of blots are available in Supplementary Information , Fig . S5 .

O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O 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 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 B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(a) Relative acetylation (normalized to controls at each day) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis. A representative blot using site specific antibodies is shown in b. Data represent means ± s.e.m. (n = 3). Serial dilutions of protein extracts were applied in western blots to verify linearity before quantification (examples are shown in Supplementary Information, Fig. S4). For calculation details see section on Methods. P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test. (b) Immunoblot of whole-cell extracts of wild-type cells chronologically aged to designated time-points with (+) or without (−) spermidine application. Blots were probed with antibodies against total histone H3 or H3 acetylation sites at the indicated Lys residues. Full scans of blots are available in Supplementary Information, Fig. S5.


( c ) Relative acetylation ( normalized to wild - type controls at each day ) of indicated histone H3 Lys residues of Δspe1 ( open bars ) , compared with wild - type ( closed bars ) during chronological ageing . Data represent means ± s . e . m . ( n = 3 ) . P values indicate result of a two - factor ANOVA corrected by Bonferroni post hoc .

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

(c) Relative acetylation (normalized to wild-type controls at each day) of indicated histone H3 Lys residues of Δspe1 cells (open bars), compared with wild-type cells (closed bars) during chronological ageing. Data represent means ± s.e.m. (n = 3). P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test.


( d ) Relative acetylation ( normalized to day 1 ) of histone H3 Lys 18 residue of chronologically ageing wild - type . Data represent means ± s . e . m . ( n = 3 ) .

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

(d) Relative acetylation (normalized to day 1) of histone H3 Lys 18 residue of chronologically ageing wild-type cells. Data represent means ± s.e.m. (n = 3).


( e ) Relative acetylation ( normalized to controls ) of histone H3 Lys 14 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine . Data represent of two independent experiments performed with obtained from two different donors .

O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE 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

(e) Relative acetylation (normalized to controls) of histone H3 Lys 14 and 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine. Data represent quantification of two independent experiments performed with cells obtained from two different donors.


( f ) Immunoblot of liver extracts ( applied in serial dilutions ) obtained from mice fed with 3 mM spermidine for 200 days respective control mice of same . Blots were probed with antibodies recognizing total histone H3 ( C - terminal epitope ) or specific for acetylated lysine 18 residue ( N - terminal epitope ) . Full scans of blots are available in Supplementary Information , Fig . S5 .

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

(f) Immunoblot analysis of liver cell extracts (applied in serial dilutions) obtained from mice fed with 3 mM spermidine for 200 days and respective control mice of the same age. Blots were probed with antibodies recognizing total histone H3 (C-terminal epitope) or specific for acetylated lysine 18 residue (N-terminal epitope). Full scans of blots are available in Supplementary Information, Fig. S5.


( a ) Relative acetylation of histone H3 Lys 9 14 residues determined by of immunoblot performed at day 20 of ageing experiment shown in panel b . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 ) . ( b ) Chronological ageing of wild - type ( ▪ , o ) Δiki3Δsas3 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 4 ) . ( c ) ( FACS ) of phosphatidylserine externalization loss of integrity ( annexinV / PI costaining ) as well as ROS production ( DHE - > Eth conversion ) performed at day 20 of experiment shown in b . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 001 ) .

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

(a) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b. Data represent means ± s.e.m. (n = 3; *P 0.05). (b) Chronological ageing of wild-type (▪, o) and Δiki3Δsas3 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4). (c) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity (annexinV/PI costaining) as well as ROS production (DHE->Eth conversion) performed at day 20 of the experiment shown in b. Data represent means ± s.e.m. (n = 4; *P 0.001).


( d ) Relative inhibition of histone acetyltransferase activity ( HAT - activity ) by spermidine determined by an in vitro HAT - activity of yeast extracts of wild - type . Data represent means ± s . e . m . of three independent experiments ( * P = 0 . 024 ) .

O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE 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

(d) Relative inhibition of histone acetyltransferase activity (HAT-activity) by spermidine determined by an in vitro HAT-activity assay of yeast nuclear extracts of wild-type cells. Data represent means ± s.e.m. of three independent experiments (*P = 0.024).


( g ) Relative change of ATG7 , ATG11 ATG15 mRNA by spermidine supplementation ( normalised to controls ) after ten days of chronological ageing as determined by reverse transcriptase real - time PCR . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 * * P 0 . 01 ) .

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

(g) Relative change of ATG7, ATG11 and ATG15 mRNA levels by spermidine supplementation (normalised to controls) after ten days of chronological ageing as determined by reverse transcriptase real-time PCR. Data represent means ± s.e.m. (n = 3; *P 0.05 and **P 0.01).


( h ) Relative histone H3 Lys 18 acetylation of indicated promoter regions normalized to acetylation of ATG7 promoter ( pATG7 ) determined by immunoprecipitation with H3 - K18Ac specific antibody subsequent of precipitated promoter using quantitative real time PCR . Signals specific for indicated promoter regions were initially compared with non - immunoprecipitated samples afterwards normalized to pATG7 signals . Data represent means ± s . e . m . ( n = 3 ) . P value was calculated using a two - factor ANOVA with promoter condition ( treated non - treated ) as independent factors .

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

(h) Relative histone H3 Lys 18 acetylation of indicated promoter regions normalized to the acetylation of ATG7 promoter (pATG7) determined by chromatin immunoprecipitation with H3-K18Ac specific antibody and subsequent quantification of precipitated promoter DNA using quantitative real time PCR. Signals specific for indicated promoter regions were initially compared with non-immunoprecipitated samples and afterwards normalized to the pATG7 signals. Data represent means ± s.e.m. (n = 3). The P value was calculated using a two-factor ANOVA with promoter and condition (treated and non-treated) as independent factors.


( a ) microscopy of wild - type yeast expressing an EGFP - Atg8p fusion with or without ( control ) treatment of 4 mM spermidine for 48 h . arrows indicate vacuolar localization of EGFP - Atg8p indicative of autophagy . Scale bars , 5 μm .

O O O B-EXP_ASSAY O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(a) Fluorescence microscopy of wild-type yeast cells expressing an EGFP-Atg8p fusion protein with or without (control) treatment of 4 mM spermidine for 48 h. White arrows indicate vacuolar localization of EGFP-Atg8p indicative of autophagy. Scale bars, 5 μm.


( b ) Relative alkaline phosphatase activity ( ALP activity ) indicative of autophagy during chronological ageing of pho8ΔC60 yeast with ( open bars ) or without ( closed bars ) application of spermidine ( 4 mM ) . Data represent means ± s . e . m . ( n = 3 * ; P 0 . 01 * * P 0 . 001 ) .

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

(b) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of pho8ΔC60 yeast with (open bars) or without (closed bars) application of spermidine (4 mM). Data represent means ± s.e.m. (n = 3 *; P 0.01 and **P 0.001).


( c ) Relative alkaline phosphatase activity ( ALP activity ) indicative of autophagy during chronological ageing of wild - type ( closed bars ) Δiki3Δsas3 ( open bars ) . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 01 * * P 0 . 001 ) .

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

(c) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of wild-type (closed bars) and Δiki3Δsas3 (open bars) cells. Data represent means ± s.e.m. (n = 3; *P 0.01 and **P 0.001).


( d ) Chronological ageing of wild - type ( ▪ , o ) Δatg7 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 4 ) .

O O O O O O O O O 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

(d) Chronological ageing of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4).


( e ) Chronological ageing on water of wild - type ( ▪ , o ) Δatg7 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 8 mM ) at day 0 . Data represent means ± s . e . m . ( n = 4 ) .

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 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

(e) Chronological ageing on water of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (8 mM) at day 0. Data represent means ± s.e.m. (n = 4).


( f ) ( reader ) of ROS production ( DHE → Eth conversion ) by wild - type Δatg7 with without supplementation of spermidine ( 8 mM ) , obtained from ageing experiment shown in panel e . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 07 * * P 0 . 01 ) .

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

(f) Quantification (fluorescence reader) of ROS production (DHE→Eth conversion) by wild-type and Δatg7 cells with and without supplementation of spermidine (8 mM), obtained from the ageing experiment shown in panel e. Data represent means ± s.e.m. (n = 4; *P 0.07 and **P 0.01).


( a ) microscopy of Hoechst - counterstained HeLa transiently transfected with LC3 - GFP subjected to 100 μM spermidine for 6 h . Representative pictures are shown . ( b ) of adherent exhibiting a clear LC3 - GFP relocalization into vacuoles . Numbers were determined using of Hoechst - counterstained HeLa as representatively shown in a . Data represent means ± s . d . ( n = 3 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O

(a) Fluorescence microscopy of Hoechst-counterstained HeLa cells transiently transfected with LC3-GFP subjected to 100 μM spermidine for 6 h. Representative pictures are shown. (b) Percentage of adherent cells exhibiting a clear LC3-GFP relocalization into cytoplasmic vacuoles. Numbers were determined using micrographs of Hoechst-counterstained HeLa cells as representatively shown in a. Data represent means ± s.d. (n = 3).


( c ) LysoTracker Red of vacuoles indicative of autophagy in oesophagus tissue from flies fed with 1 mM spermidine for two days , compared with controls ( without spermidine ) . Nuclei were visualized by Hoechst . Scale bars , 10 μm .

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

(c) LysoTracker Red staining of vacuoles indicative of autophagy in oesophagus tissue from flies fed with 1 mM spermidine for two days, compared with controls (without spermidine). Nuclei were visualized by Hoechst staining. Scale bars, 10 μm.


( d ) of autophagic vesicles per nucleus in LysoTracker Red stained muscle tissue of flies fed with supplementation of 1 mM spermidine or with 10 % glucose ( starved ) for 48 h , compared with normal food ( control ) . Data represent means ± s . e . m . of at least 20 flies for each group ( * P 0 . 01 ) .

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

(d) Quantification of autophagic vesicles per nucleus in LysoTracker Red stained muscle tissue of female flies fed with supplementation of 1 mM spermidine or with 10% glucose (starved) for 48 h, compared with normal food (control). Data represent means ± s.e.m. of at least 20 flies for each group (*P 0.01).


( e , f ) Survival of Drosophila during ageing without ( control ) with supplementation of food at various concentrations of spermidine ( as indicated ) . Autophagy - deficient flies ( f ) homozygous mutant for Atg7 ( Δatg7 ) were compared with flies capable of autophagy ( e ) heterozygous for Atg7 ( control ) . For details of strains additional wild - type controls , see Supplementary Information Methods Fig . S6h , i .

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

(e, f) Survival of Drosophila during ageing without (control) and with supplementation of food at various concentrations of spermidine (as indicated). Autophagy-deficient flies (f) homozygous mutant for Atg7 (Δatg7) were compared with flies capable of autophagy (e) and heterozygous for Atg7 (control). For details of strains and additional wild-type controls, see Supplementary Information Methods and Fig. S6h, i.


( g ) microscopy of C . elegans transgenic expressing a full - plgg - 1DsRED : : LGG - 1 fusion indicative of autophagic activity . Shown are two representative pictures of untreated ( control ) or treated with spermidine ( 0 . 2 mM ) supplementation of food . ( h ) of autophagic activity through measurement of DsRED : : LGG - 1 pixel from of wild - type shown in g , bec - 1 RNAi knockdown ( bec - 1 RNAi ) . Data represent means ± s . e . m . ( n = 3 ) with at least 25 processed for each trial .

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

(g) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRED::LGG-1 fusion protein indicative of autophagic activity. Shown are two representative pictures of embryos untreated (control) or treated with spermidine (0.2 mM) supplementation of food. (h) Quantification of autophagic activity through measurement of DsRED::LGG-1 pixel intensity from images of wild-type animals shown in g, and bec-1 RNAi knockdown animals (bec-1 RNAi). Data represent means ± s.e.m. (n = 3) with at least 25 images processed for each trial.


( i ) Survival of C . elegans during ageing with without ( control ) supplementation of food ( UV - killed E . coli ) with spermidine ( 0 . 2 mM ) . Wild - type ( N2 ) were compared with bec - 1 RNAi deficient in autophagy induction . For mean life spans see Supplementary Information , Table S3 .

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

(i) Survival of C. elegans during ageing with and without (control) supplementation of food (UV-killed E. coli) with spermidine (0.2 mM). Wild-type (N2) animals were compared with bec-1 RNAi animals deficient in autophagy induction. For mean life spans see Supplementary Information, Table S3.


A . Measurement of oxygen consumption rate ( OCR ) , extracellular acidification rate ( ECAR ) oxidative burst of monocytes from COVID - 19 ( COVID ) healthy controls ( CTR ) . OCR was measured in real time , under basal condition in response to mitochondria inhibitors : oligomycin ( Oligo , 2 μM ) , cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 0 . 5 μM ) , antimycin A plus rotenone ( Rot / AA , 0 . 5 μM ) . Oxidative burst was measured in response to PMA / ionomycin ( PMA / Iono , 1 μg / mL ) . Scatter plots show of basal respiration ( indicated as Basal OCR ) , ATP - linked respiration ( ATP - linked ) , maximal respiration ( Max Resp ) , proton - leak , spare respiratory capacity ( Spare ) , oxidative burst immediate response basal ECAR . maximal respiration kinetic range oxidative burst kinetic range are also reported were obtained by analyzing under curve ( AUC ) from sixth to tenth measurement from tenth to thirteenth measurement , respectively . Data represent values , mean standard error of mean ( CTR , n = 12 ; COVID , n = 13 ) . Mann - Whitney was used for statistical . Exact p values are reported in figure . Representative traces of OCR of monocytes from COVID - 19 ( COVID ) healthy controls ( CTR ) are also reported .

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

A. Measurement of oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and oxidative burst of monocytes from COVID-19 patients (COVID) and healthy controls (CTR). OCR was measured in real time, under basal condition and in response to mitochondria inhibitors: oligomycin (Oligo, 2 μM), cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.5 μM), and antimycin A plus rotenone (Rot/AA, 0.5 μM). Oxidative burst was measured in response to PMA/ionomycin (PMA/Iono, 1 μg/mL). Scatter plots show the quantification of basal respiration (indicated as Basal OCR), ATP-linked respiration (ATP-linked), maximal respiration (Max Resp), proton-leak, spare respiratory capacity (Spare), oxidative burst immediate response and basal ECAR. The maximal respiration kinetic range and the oxidative burst kinetic range are also reported and were obtained by analyzing the area under the curve (AUC) from the sixth to the tenth measurement and from the tenth to the thirteenth measurement, respectively. Data represent individual values, mean and standard error of the mean (CTR, n=12; COVID, n=13). Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure. Representative traces of OCR of monocytes from COVID-19 patients (COVID) and healthy controls (CTR) are also reported.


B . Representative electron of mitochondrial ultrastructural changes observed in COVID - 19 monocytes . Panels " a " " c " are representative electron of monocytes from healthy controls , whereas panels " b " , " d " " e " are representative electron of monocytes from COVID - 19 . In panel " f " of mitochondrial , external perimeter , Feret ' s diameter , aspect circularity roundness are reported ( n = 225 mitochondria for each group ) . Black arrows in panel " d " indicate mitochondria ; N refers to nucleus . Exact p values are reported in panel " f " .

O O O B-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE I-DISEASE I-DISEASE B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-DISEASE I-DISEASE I-DISEASE O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O

B. Representative electron micrographs of mitochondrial ultrastructural changes observed in COVID-19 monocytes. Panels "a" and "c" are representative electron micrographs of monocytes from healthy controls, whereas panels "b", "d" and "e" are representative electron micrographs of monocytes from COVID-19 patients. In panel "f" the quantification of mitochondrial area, external perimeter, Feret's diameter, aspect ratio circularity and roundness are reported (n=225 mitochondria for each group). Black arrows in panel "d" indicate mitochondria; N refers to nucleus. Exact p values are reported in panel "f".


B . Heatmaps representing different monocyte clusters identified by FlowSOM , with percentages in healthy donors ( CTR ) COVID - 19 . colors in heat map represent median of arcsinh , 0 - 1 transformed marker expression calculated over from samples , varying from blue for lower expression to red for higher expression . Each cluster has a unique color assigned ( bar on left which colors correspond to those used in panel A ) .

O O O O O B-CELL_TYPE O O O O O O O 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-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O 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. Heatmaps representing different monocyte clusters identified by FlowSOM, with percentages in healthy donors (CTR) and COVID-19 patients. The colors in the heat map represent the median of the arcsinh, 0-1 transformed marker expression calculated over cells from all the samples, varying from blue for lower expression to red for higher expression. Each cluster has a unique color assigned (bar on the left which colors correspond to those used in panel A).


C . Differential between healthy donors ( CTR , n = 8 ) COVID - 19 ( COVID , n = 7 ) . represents arcsine - square - root transformed frequencies that were subsequently normalized per cluster ( rows ) to mean of zero standard deviation of one . color of heat varies from blue , indicating relative under - representation , to red indicating relative over - representation . Bar numbers at right indicate significant differentially abundant clusters ( green ) adjusted p values ( Differential performed by diffcyt package present in Catalyst package ) . statistical analyses was done using GLMM ( according to actual guidelines ) p - values were Bonferroni corrected . Clusters were sorted according to adjusted p values , so that cluster at top shows most significant abundance changes between two conditions . D . Boxes whiskers plots representing maximum , minimum , median value , 25 75 percentile of different subpopulations of monocytes in healthy donors ( CTR , n = 8 ) COVID - 19 ( COVID , n = 7 ) .

O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O

C. Differential analysis between healthy donors (CTR, n=8) and COVID-19 (COVID, n=7). The heatmap represents arcsine-square-root transformed cell frequencies that were subsequently normalized per cluster (rows) to mean of zero and standard deviation of one. The color of the heat varies from blue, indicating relative under-representation, to red indicating relative over-representation. Bar and numbers at the right indicate significant differentially abundant clusters (green) and adjusted p values (Differential analysis performed by diffcyt package present in the Catalyst package). The statistical analyses was done using GLMM (according to the actual guidelines) and all p-values were Bonferroni corrected. Clusters were sorted according to the adjusted p values, so that the cluster at the top shows the most significant abundance changes between the two conditions. D. Boxes and whiskers plots representing maximum, minimum, median value, 25 and 75 percentile of different subpopulations of monocytes in healthy donors (CTR, n=8) and COVID-19 patients (COVID, n=7).


E . Scatter plots showing frequency of PD - 1 + , PD - L1 + among total monocytes in healthy controls ( CTR , n = 8 ) COVID - 19 ( COVID , n = 7 ) . A scatter plot reporting median ( MFI ) of PD - L1 in total monocytes is also shown . Data represent values , mean ± standard error of mean . Mann - Whitney was used for statistical . Exact p values are reported in figure .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD 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

E. Scatter plots showing the frequency of PD-1+ cells, PD-L1+ cells among total monocytes in healthy controls (CTR, n=8) and COVID-19 patients (COVID, n=7). A scatter plot reporting the median fluorescence intensity (MFI) of PD-L1 in total monocytes is also shown. Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


A . of cytokines other mediators in plasma obtained from COVID - 19 ( COVID , n = 15 ) healthy controls ( CTR , n = 15 ) . Data represent values , mean ± standard error of mean . Mann - Whitney was used for statistical . Exact p values are reported in figure .

O O O O O O O B-TISSUE O O B-DISEASE I-DISEASE I-DISEASE O B-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 O O O O O O O O O O O O O O

A. Quantification of cytokines and other mediators in plasma obtained from COVID-19 patients (COVID, n=15) and healthy controls (CTR, n=15). Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


B . Representative dot plots showing percentages of immature mature circulating monocytes in CTR COVID - 19 . Scatter plots showing of immature mature monocytes in healthy controls ( CTR , n = 6 ) COVID - 19 ( COVID , n = 7 ) are reported . Mann - Whitney was used for statistical . Exact p values are reported in figure .

O O O O O O O O O O O B-CELL_TYPE 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 B-DISEASE I-DISEASE I-DISEASE O B-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

B. Representative dot plots showing percentages of immature and mature circulating monocytes in CTR and COVID-19 patients. Scatter plots showing the quantification of immature and mature monocytes in healthy controls (CTR, n=6) and COVID-19 patients (COVID, n=7) are reported. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


( D ) A anisotropy experiment demonstrated direct binding of Pxp3 motif with purified Pex5 ( Kd = 9 . 5 + / - 2 . 9 µM ) , in strength similar to binding of known PTS1 motif from Idp3 ( Kd = 5 . 5 + / - 0 . 5 µM ) . plots represent mean of three independent experiments , error bars represent standard deviation .

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

(D) A fluorescence anisotropy experiment demonstrated direct binding of the Pxp3 motif with purified Pex5 protein (Kd = 9.5 +/-2.9 µM), in strength similar to the binding of the known PTS1 motif from Idp3 (Kd = 5.5 +/- 0.5 µM). The plots represent the mean of three independent experiments, and the error bars represent the standard deviation.


( F ) Subcellular fractionations followed by enzymatic activity assays show that untagged , native , Glr1 is active in peroxisomes , in addition to its known activity in mitochondria . Fum1 Pox2 were used as markers for mitochondrial , peroxisomal fractions , respectively .

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

(F) Subcellular fractionations followed by enzymatic activity assays show that untagged, native, Glr1 is active in peroxisomes, in addition to its known activity in mitochondria. Fum1 and Pox2 were used as markers for mitochondrial, and peroxisomal fractions, respectively.


( H ) Yeast - 2 - hybrid shows Glr1 interacts in vivo with both Pex5 full - Pex5 N ' domain .

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

(H) Yeast-2-hybrid assay shows Glr1 interacts in vivo with both Pex5 full-length and Pex5 N' domain.


( B ) Metabolomic focused on strains with over - expression or deletion of AYT1 gene shows a significant reduction or accumulation , respectively , in acetyl - glutamate compared to matched control of each mutant . Only metabolites changing significantly in at least one of two conditions are presented . D - EIP is D - erythro - 1 - ( Imidazole - 4 - yl ) glycerol 3 - phosphate .

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

(B) Metabolomic analysis focused on strains with over-expression or deletion of the AYT1 gene shows a significant reduction or accumulation, respectively, in acetyl-glutamate compared to the matched control strain of each mutant. Only metabolites changing significantly in at least one of the two conditions are presented. D-EIP is D-erythro-1-(Imidazole-4-yl) glycerol 3-phosphate.


( D ) A growth in a condition that does not induce high of acetyl - CoA in peroxisomes ( glucose ) shows that strains grow similarly to control until stationary phase when peroxisomes become vital . In stationary phase , over - expression ( OE ) of AYT1 grows to a lower density than control , plausibly due to burdening of peroxisomal functions . ( E ) A growth in a condition expected to elevate acetyl - glutamate in peroxisomes ( oleate as a sole carbon source glutamate as a nitrogen source ) demonstrates that AYT1 overexpressing grows faster to a higher density than control . Data information : growth assays in D E were done in three biologically independent replicates error bars were plotted . Note that some error bars are shorter than symbol , hence are not visible in graph .

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

(D) A growth assay in a condition that does not induce high levels of acetyl-CoA in peroxisomes (glucose) shows that all strains grow similarly to the control until stationary phase when peroxisomes become vital. In stationary phase, the over-expression (OE) of AYT1 grows to a lower density than the control strain, plausibly due to burdening of peroxisomal functions. (E) A growth assay in a condition expected to elevate acetyl-glutamate levels in peroxisomes (oleate as a sole carbon source and glutamate as a nitrogen source) demonstrates that the AYT1 overexpressing strain grows faster and to a higher density than the control. Data information: The growth assays in D and E were done in three biologically independent replicates and error bars were plotted. Note that some error bars are shorter than the symbol, hence are not visible in the graph.


( G ) A β - oxidation activity of Δlpx1 , Δfsh3 , Δlpx1Δfsh3 strains supplemented with labeled 8 carbon - or 18 carbon - free fatty in media supplemented with 0 . 5 % glucose shows a significant reduction in β - oxidation activity compared to wild type two single mutants , suggesting an overlapping role for Fsh3 with Lpx1 . This was done in three biologically independent replicates . Bars represent mean error bars represent standard deviation .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O

(G) A β-oxidation activity assay of Δlpx1, Δfsh3, and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon- or 18 carbon-free fatty acids in media supplemented with 0.5% glucose shows a significant reduction in β-oxidation activity compared to the wild type and the two single mutants, suggesting an overlapping role for Fsh3 with Lpx1.This assay was done in three biologically independent replicates. Bars represent the mean and error bars represent the standard deviation.


( E ) Western blot of Fbp1 - HA in mutants Δpex3 ( no peroxisomes ) , Δpex5 ( abolished targeting of GID subunits to peroxisomes ) , Δpex7 ( no β - oxidation due to loss of Pot1 targeting ) demonstrates that two hours after transition from glucose to ethanol ( gluconeogenic conditions ) , control Δpex7 upregulated Fbp1 - HA , while for Δpex3 Δpex5 Fbp1 - HA remain low . Antibodies were used against HA tag ( for Fbp1 ) Actin as a loading control .

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

(E) Western blot analysis of Fbp1-HA levels in mutants Δpex3 (no peroxisomes), Δpex5 (abolished targeting of GID subunits to peroxisomes), and Δpex7 (no β-oxidation due to loss of Pot1 targeting) demonstrates that two hours after the transition from glucose to ethanol (gluconeogenic conditions), control and Δpex7 cells upregulated Fbp1-HA levels, while for Δpex3 and Δpex5 cells Fbp1-HA levels remain low. Antibodies were used against the HA tag (for Fbp1) and Actin as a loading control.


( G ) - exclusion chromatography profiles corresponding SDS - PAGE of indicated samples . Two mutant RZZ complexes containing mutations in ROD L110F - L119F - L120K or I191M are indicated respectively as FFK I / M . Note that in bottom SDS - PAGE first second lane were deliberately inverted contain first eluted fraction molecular marker . For other shown SDS - PAGE gels , marker precedes first fraction .

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

(G) Size-exclusion chromatography profiles and corresponding SDS-PAGE of the indicated samples. Two mutant RZZ complexes containing mutations in ROD L110F-L119F-L120K or I191M are indicated respectively as FFK and I/M. Note that in the bottom SDS-PAGE the first and second lane were deliberately inverted and contain the first eluted fraction and the molecular weight marker. For all other shown SDS-PAGE gels, the marker precedes the first fraction.


( B ) Coomassie ProQ Diamond - stained gels on mChRZZSF ( 4 µM ) treated with indicated kinases ( 1 µM ) for 15 hours at 20ºC . Rev = reversine , used at 10 µM .

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

(B) Coomassie and ProQ Diamond-stained gels on mChRZZSF (4 µM) treated with the indicated kinases (1 µM) for 15 hours at 20ºC. Rev = reversine, used at 10 µM.


( D ) of Zwilch at kinetochores of HeLa that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h then released into mitosis . Subsequently , were immediately treated with 500 nM reversine , 3 . 3 µM nocodazole , 10 µM MG132 for 1 hour imaged while in mitosis . CREST serum was used to visualize kinetochores DAPI to stain . Scale bar : 10 µm . Three biological replicates were performed . ( E ) treated as for panel D were treated for visualization of Spindly . Scale bar : 10 µm .

O O O O B-GENEPROD O B-SUBCELLULAR O B-CELL_LINE 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O B-SUBCELLULAR B-SMALL_MOLECULE O 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 B-GENEPROD O O O O O O O

(D) Levels of Zwilch at kinetochores of HeLa cells that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 500 nM reversine, 3.3 µM nocodazole, and 10 µM MG132 for 1 hour and imaged while in mitosis. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed. (E) Cells treated as for panel D were treated for visualization of Spindly. Scale bar: 10 µm.


( F ) Scatter dot plots representing normalized total of Zwilch Spindly signals , normalized to reversine - untreated control , in indicated number of from experiment shown in panels C - D . Red lines indicate mean standard deviation . Statistical was performed with a nonparametric t - comparing two unpaired groups ( Mann - Whitney ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

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

(F) Scatter dot plots representing normalized total area of the Zwilch and Spindly signals, normalized to the reversine-untreated control, in the indicated number of cells from the experiment shown in panels C-D. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.


( G ) Representative showing effects of a knockdown of endogenous Spindly in HeLa . RNAi treatment was performed for 48 h with 50 nM siRNA . Before fixation , were synchronized in G2 phase with 9 μM RO3306 for 16 h then released into mitosis . Subsequently , were immediately treated with 3 . 3 μM nocodazole for an additional hour . CREST serum was used to visualize kinetochores DAPI to stain . Scale bar : 10 µm . Three biological replicates were performed .

O O O O O O O O O O O B-GENEPROD O B-CELL_LINE 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 B-SMALL_MOLECULE O O O O O O B-TISSUE O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O

(G) Representative images showing the effects of a knockdown of the endogenous Spindly in HeLa cells. RNAi treatment was performed for 48 h with 50 nM siRNA. Before fixation, cells were synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM nocodazole for an additional hour. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed.


( H ) Scatter dot plots representing normalized ratios of Spindly over CREST for kinetochores of from experiment shown in panel G . Red lines indicate median with interquartile range .

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

(H) Scatter dot plots representing normalized intensity ratios of Spindly over CREST for individual kinetochores of cells from the experiment shown in panel G. Red lines indicate median with interquartile range.


( I ) of CENP - E Zwilch were assessed in control in treated as in panel G to knockdown Spindly . Three biological replicates were performed . ( J ) Scatter dot plots representing normalized total of CENP - E Zwilch signals , normalized to RNAi negative control , in indicated number of from experiment shown in panel I . Red lines indicate mean standard deviation . Statistical was performed with a nonparametric t - comparing two unpaired groups ( Mann - Whitney ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

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

(I) Levels of CENP-E and Zwilch were assessed in control cells and in cells treated as in panel G to knockdown Spindly. Three biological replicates were performed. (J) Scatter dot plots representing normalized total area of the CENP-E and Zwilch signals, normalized to the RNAi negative control, in the indicated number of cells from the experiment shown in panel I. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.


( E ) phosphorylation state of RZZ SpindlyF was monitored by ProQ Diamond after SDS - PAGE separation of reactions . Data information : Two technical replicates for panels ,

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

(E) The phosphorylation state of RZZ and SpindlyF was monitored by ProQ Diamond after SDS-PAGE separation of reactions. Data information: Two technical replicates for panels , E


( D ) Heat - induced mChRZZGFPSF MPS1 - induced rings of mChRZZmChSF have similar diameters . As shown in panel B , heat - MPS1 - induced rings of mChRZZmChSF also have very similar dimensions . Data information : Experiments in D were carried out once .

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

(D) Heat-induced mChRZZGFPSF and MPS1-induced rings of mChRZZmChSF have similar diameters. As shown in panel B, heat- and MPS1-induced rings of mChRZZmChSF also have very similar dimensions. Data information: Experiments in D were carried out once.


( A ) - exclusion chromatography SDS - PAGE of elution fractions of indicated samples . Each Spindly construct was incubated with RZZ in absence ( dotted lines ) or in presence ( continuous lines ) of farnesyl transferase farnesyl pyrophosphate . Elution shifts of SpindlyF of resulting RZZSF complexes is indicative of successful interaction . Two technical replicates were performed .

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

(A) Size-exclusion chromatography and SDS-PAGE of elution fractions of the indicated samples. Each Spindly construct was incubated with RZZ in absence (dotted lines) or in presence (continuous lines) of farnesyl transferase and farnesyl pyrophosphate. Elution shifts of SpindlyF and of the resulting RZZSF complexes is indicative of successful interaction. Two technical replicates were performed.


( D ) Representative of fixed HeLa electroporated with full - mChSpindly mChSpindly354 - 605 constructs in depleted of endogenous Spindly by RNAi . Spindly localization was detected with an antibody against C - terminal region of Spindly . Corona expansion or lack thereof were monitored through CENP - E . CREST serum was used to visualize kinetochores , DAPI to stain for . Scale bar : 10 µm . Experiments were replicated three times .

O O O O O O B-CELL_LINE B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O

(D) Representative images of fixed HeLa cells electroporated with full-length mChSpindly and mChSpindly354-605 constructs in cells depleted of endogenous Spindly by RNAi. Spindly localization was detected with an antibody against the C-terminal region of Spindly. Corona expansion or lack thereof were monitored through CENP-E. CREST serum was used to visualize kinetochores, DAPI to stain for DNA. Scale bar: 10 µm. Experiments were replicated three times.


( E ) Scatter dot plots representing normalized ratios of indicated Spindly constructs over CREST for kinetochores of from experiment shown in panel D . Red lines indicate median with interquartile range . Statistical was performed with a nonparametric t - comparing two unpaired groups ( Mann - Whitney ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 . ( G ) Kinetochore intensities were quantified like in panel E . Red lines indicate median with interquartile range . Statistical was performed with a nonparametric t - comparing two unpaired groups ( Mann - Whitney ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

O O O O O O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter dot plots representing normalized intensity ratios of the indicated Spindly constructs over CREST for individual kinetochores of cells from the experiment shown in panel D. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000. (G) Kinetochore intensities were quantified like in panel E. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.


A . Schematic representation of experimental protocol . Abbreviations : LCM , capture microdissection . B , C . RT - PCR performed on extracted from axons collected by LCM ( Ax ) or from eyes . ( B ) β - Actin mRNA is present both in eyes axons , while MAP2 H4 are present only in eye sample , suggesting absence of contamination from bodies or other in LCM axonal samples ( Bellon et al , 2017 ) . Abbreviations : Ax , axonal sample ; Eye , stage 37 / 38 eye ; LCM , capture microdissection ; - , PCR no template control ; NT , RT no template control .

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

A. Schematic representation of the experimental protocol. Abbreviations: LCM, laser capture microdissection. B, C. RT-PCR performed on RNA extracted from axons collected by LCM (Ax) or from eyes. (B) β-Actin mRNA is present both in eyes and axons, while MAP2 and H4 are present only in the eye sample, suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples (Bellon et al, 2017). Abbreviations: Ax, axonal sample; Eye, stage 37/38 eye; LCM, laser capture microdissection; -, PCR no template control; NT, RT no template control.


F . Thermal denaturation profiles of MB , in absence ( solid ) presence ( dashed ) of increasing target . Each melting curve represents average of three separate replicates . Yellow boxes indicate range of working temperatures for ex vivo trafficking experiments . Abbreviations : MB , molecular beacon ; Tm , MB melting .

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 O O O O O 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. Thermal denaturation profiles of MB, in the absence (solid line) and presence (dashed line) of increasing target concentration. Each melting curve represents the average of three separate replicates. Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments. Abbreviations: MB, molecular beacon; Tm, MB melting temperature.


H . of expression of pre - mir - 181a - 1 using 2 - ΔCt method U6 as normalizer from small total fraction ( < ~ 150 nt ) . Each data point corresponds to one independent experiment . n = 3 independent experiments . Values are mean ± . Abbreviations : ns , not significant .

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

H. Quantification of the expression levels of pre-mir-181a-1 using the 2-ΔCt method and U6 as normalizer from small total RNA fraction (<~150 nt). Each data point corresponds to one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.


I . Total number of MB normalized to axon ( µm ) . Each data point corresponds to one axon . Total number of axons analyzed : 928 , 61 axons ( WT ) , 226 , 15 axons ( co - MO ) , 208 , 35 axons ( MO ) . n = 4 independent experiments . Values are mean ± . Abbreviations : ns , not significant ; co - MO , control morpholino ; pri - miR - MO , morpholino blocking pre - miR - 181a - 1 processing by targeting Drosha cleavage site .

O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

I. Total number of MB puncta normalized to axon length (µm). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 928 puncta, 61 axons (WT), 226 puncta, 15 axons (co-MO), 208 puncta, 35 axons (MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site.