A , B Deacetylase activity on H3K9 ( A ) H3K18 ( B ) residues shows reduced activity in centSIRT6 allele . Designer histones were incubated with purified SIRT6 5 mM NAD + , then resolved by SDS - PAGE analyzed by immunoblotting with acetyl - specific histone antibodies . n = 3 TR ; error bars = SD . Students t - , two . Asterisks indicate p < 0 . 05 compared to WT , color corresponds to allele . H3K9ac , H3K18ac H3 are blots , SIRT6 loading Coomassie stain HeLa histone preps were also

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

A, B Deacetylase activity on H3K9 (A) and H3K18 (B) residues shows reduced activity in centSIRT6 allele. Designer histones were incubated with purified SIRT6 and 5 mM NAD+, then resolved by SDS-PAGE and analyzed by immunoblotting with acetyl-specific histone antibodies. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisks indicate p<0.05 compared to WT, color corresponds to allele. H3K9ac, H3K18ac and H3 are blots, SIRT6 loading Coomassie stain HeLa histone preps were also probed


C Quantitative mass spec of histone H3 peptide purified from human expressing different SIRT6 alleles did not reveal a difference in acetylation . Relative fraction represents portion of peptide encompassing H3K9 - 17 compared to summed total of of peptide quantitation values for same region . n = 3 biological replicates ( BR ) ; error bars = SD . Students t - , two

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

C Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels. Relative fraction represents the portion of the peptide encompassing H3K9-17 compared to the summed total of all of the peptide quantitation values for the same region. n=3 biological replicates (BR); error bars=SD. Students t-test, two tailed


D Whole histone H3 acetylation in cumate - inducible SIRT6 human fibroblasts , assessed by Western blot . Cu , cumate ; PQ , paraquat . Cumate dosage required for equivalent SIRT6 abundance was determined by Western blot , administered accordingly to respective lines were incubated with appropriate cumate dose for 48hrs prior to harvest , with PQ - treated receiving PQ 24hrs after initial cumate induction .

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

D Whole cell histone H3 acetylation levels in cumate-inducible SIRT6 human fibroblasts, assessed by Western blot. Cu, cumate; PQ, paraquat. Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot, and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest, with PQ-treated cells receiving PQ 24hrs after initial cumate induction.


E - H Relative abundance of H3K9ac at SIRT6 - dependent NF - κB target genes . Anti - H3K9ac antibodies were used to perform ChIP in absence ( - Cu ) or presence of ( + Cu ) cumate - induced SIRT6 expression . Samples were normalized using 10 % input . n = 3 BR ; error bars = SD . Students t - , two . Black asterisk indicate p < 0 . 05 compared to - Cu condition . Red asterisk indicated p < 0 . 05 compared to + Cu WT .

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

E-H Relative abundance of H3K9ac at SIRT6-dependent NF-κB target genes. Anti-H3K9ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 BR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition. Red asterisk indicated p<0.05 compared to +Cu WT.


I , J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity . Anti - H3K18ac antibodies were used to perform ChIP in absence ( - Cu ) or presence of ( + Cu ) cumate - induced SIRT6 expression . Samples were normalized using 10 % input . n = 3 TR ; error bars = SD . Students t - , two . Black asterisk indicate p < 0 . 05 compared to - Cu condition .

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

I, J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity. Anti-H3K18ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 TR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition.


A Self - ribosylation of SIRT6 using biotin - labeled NAD + . Recombinant SIRT6 was incubated with NAD + conjugated with a biotin residue then run on an SDS - PAGE . Each allele was assessed relative to its 0 hr time point normalized to SIRT6 total loading controls . n = 3 TR ; error bars = SD . Students t - , two . Asterisk indicate p < 0 . 05 . Black asterisks indicate significance over 0 hr control . Red asterisks indicated significance over time - matched WT SIRT6 .

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

A Self-ribosylation of SIRT6 using biotin-labeled NAD+. Recombinant SIRT6 was incubated with NAD+ conjugated with a biotin residue and then run on an SDS-PAGE. Each allele was assessed relative to its 0 hr time point and normalized to SIRT6 total protein loading controls. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05. Black asterisks indicate significance over 0 hr control. Red asterisks indicated significance over time-matched WT SIRT6.


B Self - ribosylation of SIRT6 with titration of NAD + . mADPr - specific antibody was used to detect ribosylated wild type SIRT6 centSIRT6 . Plot was fit Michaelis - Menten best - fit with GraphPad software . Km NAD was 142 + 29 for centSIRT6 106 + 45 for wild type SIRT6 maximal signal was ~ 2x greater for centSIRT6 compared to wild type SIRT6 . Two separate batches of recombinant of each SIRT6 allele were prepared assayed . n = 3 TR ; error bars = SD .

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

B Self-ribosylation of SIRT6 with titration of NAD+. mADPr-specific antibody was used to detect ribosylated wild type SIRT6 and centSIRT6 proteins. Plot was fit the Michaelis-Menten best-fit with GraphPad software. Km NAD was 142+29 for centSIRT6 and 106+45 for wild type SIRT6 and maximal signal was ~2x greater for centSIRT6 compared to the wild type SIRT6. Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed. n=3 TR; error bars=SD.


C Activation of PARP1 by SIRT6 variants . SIRT6 was incubated with human PARP1 then analyzed by immunoblotting with poly - ADPr antibody . Poly - ADPr activity of PARP1 results in a wide range of product . Activity was assessed by quantifying poly - ADPr signal in whole lanes for each sample . n = 3 TR ; error bars = SD . Two - way ANOVA . Asterisk indicate p < 0 . 05 . Black asterisks indicate significance over HPRT control . Red asterisks indicated significance over wild type SIRT6 .

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

C Activation of PARP1 by SIRT6 variants. SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly-ADPr antibody. Poly-ADPr activity of PARP1 results in a wide range of product size. Activity was assessed by quantifying poly-ADPr signal in whole lanes for each sample. n=3 TR; error bars=SD. Two-way ANOVA. Asterisk indicate p<0.05. Black asterisks indicate significance over HPRT control. Red asterisks indicated significance over wild type SIRT6.


A qRT - PCR of LINE1 expression in cumate - inducible SIRT6 fibroblasts . Primers assessed both 5 ' ( ORF1 ) 3 ' ( ORF2 ) LINE1 sequences from L1HS family of evolutionarily active LINE1 retrotransposons . Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes . Asterisk indicate p < 0 . 05 . n = 3 TR ; error bars = SD . Students t - , two .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of LINE1 expression in cumate-inducible SIRT6 fibroblasts. Primers assessed both 5' (ORF1) and 3' (ORF2) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons. Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes. Asterisk indicate p<0.05. n=3 TR; error bars=SD. Students t-test, two tailed.


B LINE1 EGFP retrotransposition . Three independent transfections with LINE1 EGFP reporter plasmid were conducted quantified by flow cytometry . Asterisk indicate p < 0 . 05 . Red asterisk indicated significance over SIRT6 KO control . Black asterisk indicate significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVA .

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

B LINE1 EGFP retrotransposition assay. Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicated significance over SIRT6 KO control. Black asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.


C In vitro KAP1 ribosylation by SIRT6 . Asterisk indicate p < 0 . 05 . Red asterisk indicate significance over 0 hr , black asterisk indicate significance over compared to WT SIRT6 . n = 3 BR ; error bars = SD . Students t - , two .

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

C In vitro KAP1 ribosylation by SIRT6. Asterisk indicate p<0.05. Red asterisk indicate significance over 0 hr, black asterisk indicate significance over compared to WT SIRT6. n=3 BR; error bars=SD. Students t-test, two tailed.


D , E Stimulation of NHEJ HR by SIRT6 variants . Reporter lines were co - transfected with SIRT6 - expressing plasmid , I - Sce1 plasmid , DsRed transfection control . After 72 hr recovery , reactivation of GFP reporter was measured by flow cytometry . Stimulation of NHEJ or HR was calculated as radio of GFP + / DsRed + positive . Blots demonstrate equivalent SIRT6 abundance in transfected . Asterisk indicate p < 0 . 05 Black asterisk indicate significance over control , red asterisk indicate significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way

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

D, E Stimulation of NHEJ and HR by SIRT6 variants. Reporter cell lines were co-transfected with SIRT6-expressing plasmid, I-Sce1 plasmid, and DsRed transfection control. After 72 hr recovery, reactivation of the GFP reporter was measured by flow cytometry. Stimulation of NHEJ or HR was calculated as radio of GFP+/DsRed+ positive cells. Blots demonstrate equivalent SIRT6 abundance in transfected cells. Asterisk indicate p<0.05 Black asterisk indicate significance over control, red asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA


F Basal γH2AX foci in cumate - inducible SIRT6 fibroblasts . Foci were scored in at least 80 per condition . Asterisk indicate p < 0 . 05 . n = 3 BR ; error bars = SD . Students t - , two .

O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Basal γH2AX foci in cumate-inducible SIRT6 fibroblasts. Foci were scored in at least 80 cells per condition. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.


G repair kinetics in cumate - inducible SIRT6 fibroblasts . were grown on slides irradiated with 2 Gy gamma radiation , followed by immunostaining for γH2AX . Irradiation was conducted when were at 75 % confluency on slides . were fixed foci scored at t = 0 . 5 hr , 2 hr , 4 hr , 6 hr , 24 hr post - irradiation . Foci were scored in at least 80 per genotype per time point . Asterisk indicate p < 0 . 05 . Asterisks indicate significance over WT SIRT6 . Color of asterisk corresponds to SIRT6 allele . n = 3 BR ; error bars = SD . Students t - , two .

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

G DNA repair kinetics in cumate-inducible SIRT6 fibroblasts. Cells were grown on slides and irradiated with 2 Gy gamma radiation, followed by immunostaining for γH2AX. Irradiation was conducted when the cells were at 75% confluency on slides. Cells were fixed and foci scored at t=0.5 hr, 2 hr, 4 hr, 6 hr, and 24 hr post-irradiation. Foci were scored in at least 80 cells per genotype per time point. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.


H , I Oxidative stress resistance . Cumate - inducible SIRT6 fibroblasts were induced for SIRT6 expression exposed to paraquat for 24 hours . Resistance was determined by apoptosis 48 hours after exposure . Asterisk indicate p < 0 . 05 . Asterisks indicate significance over WT SIRT6 . Color of asterisk corresponds to SIRT6 allele . n = 3 BR ; error bars = SD . Students t - , two .

O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD B-CELL_TYPE O O O B-GENEPROD O O O B-SMALL_MOLECULE 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 B-GENEPROD 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

H, I Oxidative stress resistance. Cumate-inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours. Resistance was determined by apoptosis staining 48 hours after exposure. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.


A Number of adherent after transfection with SIRT6 variants . were transfected with SIRT6 plasmids encoding different SIRT6 alleles numbers were counted after 72 hours . HCA2 are normal human foreskin fibroblasts . Asterisk indicate p < 0 . 05 . n = 3 BR ; error bars = SD . Students t - , two .

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

A Number of adherent cells after transfection with SIRT6 variants. Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours. HCA2 are normal human foreskin fibroblasts. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.


B , C Apoptosis of cancer lines 48 hours after transfection . were stained with Annexin V / PI analyzed by flow cytometry . Asterisk indicate p < 0 . 05 . Red asterisk indicates significance over control , black asterisk indicates significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVA .

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

B, C Apoptosis staining of cancer cell lines 48 hours after transfection. Cells were stained with Annexin V/PI and analyzed by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicates significance over control, black asterisk indicates significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.


B IP experiments on lysates from cumate - induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6 , LMNA , mADPr . SIRT6 expression was induced 48 hours prior to IP . n = 3 BR . One representative set of IPs is shown .

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

B IP experiments on lysates from cumate-induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6, LMNA, and mADPr. SIRT6 expression was induced 48 hours prior to IP. n=3 BR. One representative set of IPs is shown.


C CentSIRT6 shows stronger interaction with LMNA compared to wild type SIRT6 . of IP experiment shown in ( B ) . SIRT6 IP followed by Western blot with antibodies to LMNA . D LMNA shows enhanced interaction with centSIRT6 compared to wild type . of IP experiment shown in ( B ) . LMNA IP from cumate - inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6 . Data information : n = 3 BR ; error bars = SD . Students t - , two . Asterisk indicate p < 0 .

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

C CentSIRT6 shows stronger interaction with LMNA compared to the wild type SIRT6. Quantification of the IP experiment shown in (B). SIRT6 IP followed by Western blot with antibodies to LMNA. D LMNA shows enhanced interaction with centSIRT6 compared to the wild type. Quantification of the IP experiment shown in (B). LMNA IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05


E centSIRT6 shows enhanced mADPr . of IP experiment shown in ( B ) . SIRT6 IP from cumate - inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues F LMNA shows enhanced mADPr signal in expressing centSIRT6 . of IP experiment shown in ( B ) . IP with mADPr antibody using extract from cumate - induced SIRT6 fibroblasts , followed by Western blot with antibodies to LMNA . Data information : n = 3 BR ; error bars = SD . Students t - , two . Asterisk indicate p < 0 .

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

E centSIRT6 shows enhanced mADPr. Quantification of the IP experiment shown in (B). SIRT6 IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues F LMNA shows enhanced mADPr signal in cells expressing centSIRT6. Quantification of the IP experiment shown in (B). IP with mADPr antibody using extract from cumate-induced SIRT6 fibroblasts, followed by Western blot with antibodies to LMNA. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05


( H ) - ( J ) mRNA expression of ( H ) U2AF1 , ( I ) SMN2 - FL , or ( J ) SMN2 ∆ 7 in SMA - derived fibroblasts ( red bars ) 72 hours post - transfection with SMN2 SSO , scrambled siRNA , or U2AF1 siRNA at indicated dose . Unaffected , SMA carrier fibroblasts ( blue bars ) treated with scrambled siRNA . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; empirical p - values are determined by a simulation sampling method error bars show Standard Error of Mean ( ) . Data represent 3 biological replicates . Three technical replicates were performed during qPCR for each biological replicate averaged . Scrambled siRNA condition for each experiment was set to 1 .

O 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 B-GENEPROD O O O B-DISEASE O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-DISEASE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(H)-(J) mRNA expression of (H) U2AF1, (I) SMN2-FL, or (J) SMN2∆7 in SMA patient-derived fibroblasts (red bars) 72 hours post-transfection with SMN2 SSO, scrambled siRNA, or U2AF1 siRNA at the indicated dose. Unaffected, SMA carrier fibroblasts (blue bars) treated with scrambled siRNA. Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates. Three technical replicates were performed during qPCR for each biological replicate and averaged. Scrambled siRNA condition for each experiment was set to 1.


( K ) Representative Western blot . ( L ) - ( M ) of expression of ( L ) U2AF1 or ( M ) SMN normalized to GAPDH ( n = 3 ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; empirical p - values are determined by a simulation sampling method error bars show Standard Error of Mean ( ) . Data represent 3 biological replicates .

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

(K) Representative Western blot. (L)-(M) Quantification of protein expression of (L) U2AF1 or (M) SMN normalized to GAPDH (n=3). Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates.


a , Mice were infected for 21 days liver granulomas analysed by transmission electron microscopy ( ) . Shown are spacious vacuoles ( SLAPs ) containing multiple . Magnification , × 5 , 200 . Region 1 2 ( boxes ) are enlarged in lower panels .

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a, Mice were infected for 21 days and liver granulomas analysed by transmission electron microscopy (TEM). Shown are spacious vacuoles (SLAPs) containing multiple bacteria. Magnification, ×5,200. Region 1 and 2 (white boxes) are enlarged in the lower panels.


b , SCID mice were infected as in a liver sections stained for LAMP1 ( green ) , ( red ) ( blue ) . Shown is a LAMP1 + SLAP .

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b, SCID mice were infected as in a and liver sections stained for LAMP1 (green), bacteria (red) and DNA (blue). Shown is a LAMP1+ SLAP.


c , of in LAMP1 + compartments was quantified characterized as either spacious or tight - fitting . Mean ± s . e . m . for three mice examined . image in a ( from ref . 2 ) tissue sections were provided by E . Unanue .

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c, The percentage of bacteria in LAMP1+ compartments was quantified and characterized as either spacious or tight-fitting. Mean ± s.e.m. for three mice examined. The image in a (from ref. 2) and the tissue sections were provided by E. Unanue.


a , RAW 264 . 7macrophages infected for 4 or 8 h were analysed by . Shown are spacious vacuoles ( SLAPs ) containing multiple . arrow indicates septum of dividing . Scale bars , 0 . 5 µm .

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a, RAW 264.7macrophages infected for 4 or 8 h were analysed by TEM. Shown are spacious vacuoles (SLAPs) containing multiple bacteria. The arrow indicates septum of dividing bacteria. Scale bars, 0.5 µm.


b , arrow indicates a LAMP1 + SLAP colocalizing with green ( GFP ) - LC3 in infected for 4 h . of GFP - LC3 + ( c )

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b, The arrow indicates a LAMP1+ SLAP colocalizing with green fluorescent protein (GFP)-LC3 in cells infected for 4 h.The percentage of GFP-LC3+ (c)


d , arrowhead indicates a LAMP1 + SLAP devoid of cathepsin D in infected for 4 h . Scale bars , 5 µm . of GFP - LC3 + ( c ) or cathepsin D + ( e )

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d, The arrowhead indicates a LAMP1+ SLAP devoid of cathepsin D in cells infected for 4 h. Scale bars, 5 µm.The percentage of GFP-LC3+ (c) or cathepsin D+ (e)


f , GFP - LC3 - transfected macrophages were infected with wild - type or D actA . Where indicated , chloramphenicol ( CM ) was added to media at 3 h post infection . number of per SLAP was quantified . Brackets indicate significant differences , corresponding P values are shown .

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f, GFP-LC3-transfected macrophages were infected with wild-type or D actA bacteria. Where indicated, chloramphenicol (CM) was added to the media at 3 h post infection. The number of bacteria per SLAP was quantified. Brackets indicate significant differences, and corresponding P values are shown.


g , Macrophages were infected with wild - type for 7 h , pulsed with BrdU for 1 h , stained for LAMP1 ( red ) , ( blue ) BrdU ( green ) . Magnified arrow indicate SLAPs containing actively replicating ( BrdU + ) . h , of BrdU + in SLAPs , compared to PFA - killed , was quantified as in c .

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g, Macrophages were infected with wild-type bacteria for 7 h, pulsed with BrdU for 1 h, and stained for LAMP1 (red), bacteria (blue) and BrdU (green). Magnified images and the arrow indicate SLAPs containing actively replicating bacteria (BrdU+). h, The percentage of BrdU+ bacteria in SLAPs, compared to bacteria and PFA-killed bacteria, was quantified as in c.


a , GFP - LC3 - transfected macrophages were infected for 4 h , of infected exhibiting SLAPs was quantified . Mean ± s . e . m . for three independent experiments . P values for strains with significant differences from wild - type are shown .

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a, GFP-LC3-transfected macrophages were infected for 4 h, and the percentage of infected cells exhibiting SLAPs was quantified. Mean ± s.e.m. for three independent experiments. P values for strains with significant differences from wild-type levels are shown.


b , arrow indicates a GFP - LC3 + SLAP with internal LLO expression in infected for 4 h . Scale bar , 5 µm .

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b, The arrow indicates a GFP-LC3+ SLAP with internal LLO expression in cells infected for 4 h. Scale bar, 5 µm.


c , Arrowheads indicate GFP - LC3 + SLAPs devoid of Lysotracker Red in infected for 4 h .

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c, Arrowheads indicate GFP-LC3+ SLAPs devoid of Lysotracker Red in cells infected for 4 h.


e , arrow indicates a LAMP1 + SLAP colocalizing with v - ATPase in infected for 4 h . Scale bar , 5 µm . f , of v - ATPase + SLAPs was quantified as in Fig . 2c . Mean ± s . e . m . for three independent experiments . P values for conditions significantly different from PFA - killed are shown .

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e, The arrow indicates a LAMP1+ SLAP colocalizing with v-ATPase staining in cells infected for 4 h. Scale bar, 5 µm. f, The percentage of v-ATPase+ SLAPs was quantified as in Fig. 2c. Mean ± s.e.m. for three independent experiments. P values for conditions significantly different from PFA-killed bacteria are shown.


a , Arrows indicate LAMP1 - wild - type with actin ' comet tails ' in infected for 6 h . boxed region is magnified in bottom left panels . merged image for this region is shown at bottom right . Scale bar , 5 µm .

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a, Arrows indicate LAMP1- wild-type bacteria with actin 'comet tails' in cells infected for 6 h. The boxed region is magnified in the bottom left panels. The merged image for this region is shown at the bottom right. Scale bar, 5 µm.


b , Macrophages were infected with IPTG - induced iLLO . Arrowheads indicate actin - iLLO within LAMP1 + vacuoles .

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b, Macrophages were infected with IPTG-induced iLLO bacteria. Arrowheads indicate actin- iLLO bacteria within LAMP1+ vacuoles.


c , Macrophages were infected with wild - type ( triangles ) or IPTG - induced iLLO ( circles ) , of LAMP1 + was quantified . Mean ± s . e . m . for three independent experiments .

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c, Macrophages were infected with wild-type (triangles) or IPTG-induced iLLO (circles) bacteria, and the percentage of LAMP1+ bacteria was quantified. Mean ± s.e.m. for three independent experiments.


d , Macrophages were infected as in c with or without IPTG induction . Intracellular replication was determined using a gentamicin - protection . Shown is fold replication compared to 2 h post infection . Clear triangles , wild type ; filled triangles , wild type + IPTG ; clear circles , iLLO ; filled circles , iLLO + IPTG . Mean ± s . e . m . or range for three ( wild type , wild type + IPTG , iLLO + IPTG ) or two ( iLLO - IPTG ) independent experiments , respectively .

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d, Macrophages were infected as in c with or without IPTG induction. Intracellular bacterial replication was determined using a gentamicin-protection assay. Shown is fold replication compared to 2 h post infection. Clear triangles, wild type; filled triangles, wild type + IPTG; clear circles, iLLO; filled circles, iLLO + IPTG. Mean ± s.e.m. or range for three (wild type, wild type + IPTG, iLLO + IPTG) or two (iLLO-IPTG) independent experiments, respectively.


e , Wild - type or Atg5 - / - MEFs were infected with iLLO , intracellular replication was determined as in d . Mean ± s . e . m . for three independent experiments . Clear circles , wild - type MEFs ; filled circles , wild - type MEFs + IPTG ; clear squares , Atg5 - / - MEFs ; filled squares , Atg5 - / - MEFs + IPTG .

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e, Wild-type or Atg5-/- MEFs were infected with iLLO bacteria, and intracellular bacterial replication was determined as in d. Mean ± s.e.m. for three independent experiments. Clear circles, wild-type MEFs; filled circles, wild-type MEFs + IPTG; clear squares, Atg5-/- MEFs; filled squares, Atg5-/- MEFs + IPTG.


f , Wild - type ( circles ) or Atg5 - / - ( squares ) MEFs were infected as in e with IPTG induction . of LAMP1 + was quantified as in c . Mean ± s . e . m . for three independent experiments .

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f, Wild-type (circles) or Atg5-/- (squares) MEFs were infected as in e with IPTG induction. The percentage of LAMP1+ bacteria was quantified as in c. Mean ± s.e.m. for three independent experiments. g


( A ) Isolation of FTCD from rat liver extract . p47 ( 1 - 170 ) fragment , which does not contain p97 - binding site , was immobilized on beads incubated with rat liver extract . Bound were fractionated by SDS - PAGE , stained with CBB .

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(A) Isolation of FTCD from rat liver extract. The p47(1-170) fragment, which does not contain the p97-binding site, was immobilized on beads and incubated with rat liver extract. Bound proteins were fractionated by SDS-PAGE, and stained with CBB.


( B ) FTCD binds directly to p47 . GST - tagged p47 ( 0 . 54 μg ) or GST - tagged p37 ( 0 . 45 μg ) was incubated with His - tagged FTCD ( 0 . 80 μg ) , then isolated on glutathione beads . blots were probed with antibodies to FTCD GST .

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(B) FTCD binds directly to p47. GST-tagged p47 (0.54 μg) or GST-tagged p37 (0.45 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.


( C ) FTCD p47 form a in . HepG2 were solubilized incubated with anti - p47 antibodies . immunoprecipitates were fractionated by SDS - PAGE , followed by Western blotting with antibodies to p47 FTCD .

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(C) FTCD and p47 form a complex in cells. HepG2 cells were solubilized and incubated with anti-p47 antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to p47 and FTCD.


( D ) FTCD , p47 p97 form a in . HepG2 were solubilized incubated with anti - FTCD antibodies . immunoprecipitates were fractionated by SDS - PAGE , followed by Western blotting with antibodies to FTCD , VCIP135 , p97 , p47 syntaxin5 .

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(D) FTCD, p47 and p97 form a complex in cells. HepG2 cells were solubilized and incubated with anti-FTCD antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to FTCD, VCIP135, p97, p47 and syntaxin5.


( E ) p47 binds to CD domain of FTCD . GST - tagged p47 ( 0 . 50 μg ) was incubated with either His - tagged FTCD ( 1 - 326 ) ( FT , 0 . 88 μg ) or His - tagged FTCD ( 327 - 541 ) ( CD , 0 . 63 μg ) . blots were probed with antibodies to His - tag GST .

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(E) p47 binds to the CD domain of FTCD. GST-tagged p47 (0.50 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.63 μg). The blots were probed with antibodies to His-tag and GST.


( F ) p47 ( E86K , E88K ) does not bind to FTCD . GST - tagged p47wt / mutant ( 0 . 50 μg ) was incubated with indicated His - tagged ( p97 , 1 . 0 μg ; FTCD , 0 . 75 μg ) . blots were probed with antibodies to p97 , FTCD GST .

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(F) p47(E86K, E88K) does not bind to FTCD. GST-tagged p47wt/mutant (0.50 μg) was incubated with the indicated His-tagged proteins (p97, 1.0 μg; FTCD, 0.75 μg). The blots were probed with antibodies to p97, FTCD and GST.


( G ) FTCD ( R382A ) , which lacks binding ability to polyglutamates , does not bind to p47 . GST - tagged p47 ( 0 . 35 μg ) was incubated with His - tagged FTCDwt / mutant ( 0 . 53 μg ) . blots were probed with antibodies to His - tag GST .

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(G) FTCD(R382A), which lacks binding ability to polyglutamates, does not bind to p47. GST-tagged p47 (0.35 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.


( H ) p47 binds to FTCD in Golgi membranes . Salt - washed Golgi membranes were first incubated with either GST - tagged p47wt , p47 ( E86K , E88K ) or p47 ( F253S ) . isolated membranes were solubilized , then GST - tagged p47wt / mutants their binding were recovered with glutathione - beads . bound were assayed by Western blotting with antibodies to FTCD GST .

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(H) p47 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were first incubated with either GST-tagged p47wt, p47(E86K, E88K) or p47(F253S). The isolated membranes were solubilized, and then GST-tagged p47wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.


( A ) FTCD directly binds to p97 . GST - tagged p97 ( 2 . 1 μg ) was incubated with His - tagged FTCD ( 0 . 80 μg ) , then isolated on glutathione beads . blots were probed with antibodies to FTCD GST .

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(A) FTCD directly binds to p97. GST-tagged p97 (2.1 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.


( B ) Nucleotide dependency of binding of FTCD with p97 . FTCD ( 0 . 80 μg ) GST - tagged p97 ( 2 . 1 μg ) were incubated in presence of indicated nucleotide ( 1 mM ) . blots were probed with antibodies to FTCD GST .

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(B) Nucleotide dependency of the binding of FTCD with p97. FTCD (0.80 μg) and GST-tagged p97 (2.1 μg) were incubated in the presence of the indicated nucleotide (1 mM). The blots were probed with antibodies to FTCD and GST.


( C ) FTCD binds to C - terminal region of p97 . GST - tagged p97 fragments were incubated with either FTCD or p47 . blots were probed with antibodies to FTCD p47 .

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(C) FTCD binds to the C-terminal region of p97. GST-tagged p97 fragments were incubated with either FTCD or p47. The blots were probed with antibodies to FTCD and p47.


( D ) p97 also binds to CD domain of FTCD . GST - tagged p97 ( 2 μg ) was incubated with either His - tagged FTCD ( 1 - 326 ) ( FT , 0 . 88 μg ) or His - tagged FTCD ( 327 - 541 ) ( CD , 0 . 62 μg ) . blots were probed with antibodies to His - tag GST .

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(D) p97 also binds to the CD domain of FTCD. GST-tagged p97 (2 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.62 μg). The blots were probed with antibodies to His-tag and GST.


( E ) p97 ( V722K , E724K ) shows very low binding affinity to FTCD . GST - tagged p97wt / mutant ( 2 . 0 μg ) was incubated with indicated His - tagged ( FTCD , 2 . 0 μg ; p47 , 0 . 5 μg ) . blots were probed with antibodies to p47 , FTCD GST .

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(E) p97(V722K, E724K) shows very low binding affinity to FTCD. GST-tagged p97wt/mutant ( 2.0 μg) was incubated with the indicated His-tagged proteins (FTCD, 2.0 μg; p47, 0.5 μg). The blots were probed with antibodies to p47, FTCD and GST.


( F ) FTCD ( R382A ) , which lacks binding ability to polyglutamates , shows a very low binding affinity to p97 . GST - tagged p97 ( 1 . 4 μg ) was incubated with His - tagged FTCDwt / mutant ( 0 . 53 μg ) . blots were probed with antibodies to His - tag GST .

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(F) FTCD(R382A), which lacks binding ability to polyglutamates, shows a very low binding affinity to p97. GST-tagged p97 (1.4 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.


( G ) p97 binds to FTCD in Golgi membranes . Salt - washed Golgi membranes were incubated with either GST - tagged p97wt or p97 ( V722k , E724K ) . isolated membranes were solubilized , then GST - tagged p97wt / mutants their binding were recovered with glutathione - beads . bound were assayed by Western blotting with antibodies to FTCD GST .

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(G) p97 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were incubated with either GST-tagged p97wt or p97(V722k, E724K). The isolated membranes were solubilized, and then GST-tagged p97wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.


( A ) Immunofluorescence of FTCD GM130 , a Golgi marker . HepG2 were fixed , permeabilized , stained with a monoclonal antibody to FTCD , a polyclonal antibody to GM130 DAPI , observed by confocal microscopy . Scale bar = 10 μm .

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(A) Immunofluorescence staining of FTCD and GM130, a Golgi marker. HepG2 cells were fixed, permeabilized, stained with a monoclonal antibody to FTCD, a polyclonal antibody to GM130 and DAPI, and observed by confocal microscopy. Scale bar = 10 μm.


( B ) Depletion of FTCD by FTCD siRNA duplexes . HepG2 were either mock transfected with water or transfected with two distinct siRNA duplexes specific to FTCD . After incubation for 48 hrs , were analyzed by Western blotting with antibodies to FTCD α - tubulin .

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(B) Depletion of FTCD by FTCD siRNA duplexes. HepG2 cells were either mock transfected with water or transfected with two distinct siRNA duplexes specific to FTCD. After incubation for 48 hrs, the cells were analyzed by Western blotting with antibodies to FTCD and α-tubulin.


( C ) Golgi in which were depleted of either FTCD or p47 . HepG2 , which were treated with either FTCD siRNAs , p47 siRNA or mock for 48 hrs , were fixed stained with a monoclonal antibody to GM130 . Scale bar = 10 μm . ( D ) results of of ( C ) . values are from five sets of independent experiments . Results are expressed as mean ± SD ( n = 5 ) , with 100 counted in each group of each set . Asterisks indicate a significant difference at P < 0 . 01 compared with mock - treated ( Bonferroni method ) .

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(C) The Golgi in cells which were depleted of either FTCD or p47. HepG2 cells, which were treated with either FTCD siRNAs, p47 siRNA or mock for 48 hrs, were fixed and stained with a monoclonal antibody to GM130. Scale bar = 10 μm. (D) The results of quantification of (C). The values are from five sets of independent experiments. Results are expressed as the mean±SD (n=5), with 100 cells counted in each group of each set. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).


( E ) Representative EM of Golgi in HepG2 treated with either mock or FTCD siRNAs for 42 hrs . Scale bar = 0 . 5 μm . ( F ) results of of ( E ) . Golgi membranes in HepG2 were classified into cisternae , vesicles tubules , counted as previously described ( Shorter & Warren , 1999 ) . results are shown as mean ± SD of five sets of independent experiments , with 15 - 20 counted in each group in each independent experiment . An asterisk indicates a significant difference ( P < 0 . 05 ) compared with mock - treated in each category ( Bonferroni method ) .

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(E) Representative EM images of the Golgi in HepG2 cells treated with either mock or FTCD siRNAs for 42 hrs. Scale bar = 0.5 μm. (F) The results of quantification of (E). Golgi membranes in HepG2 cells were classified into cisternae, vesicles and tubules, and counted as previously described (Shorter & Warren, 1999). The results are shown as the mean±SD of five sets of independent experiments, with 15-20 cells counted in each group in each independent experiment. An asterisk indicates a significant difference (P<0.05) compared with the mock-treated cells in each category (Bonferroni method).


( A ) localization of FTCD in mitotic . Mitotic HepG2 were collected by flushing cultured on coated coverslips for 10 - 40 mins . were fixed , stained with a monoclonal antibody to FTCD polyclonal antibodies to GM130 α - tubulin , observed by confocal microscopy . Panels d , h , l indicate of α - tubulin other panels show distribution of FTCD or / GM130 . Scale bar = 10 μm . Arrowheads indicate colocalization of FTCD GM130 .

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(A) The localization of FTCD in mitotic cells. Mitotic HepG2 cells were collected by flushing and cultured on coated coverslips for 10-40 mins. The cells were fixed, stained with a monoclonal antibody to FTCD and polyclonal antibodies to GM130 and α-tubulin, and observed by confocal microscopy. Panels d, h, and l indicate the images of α-tubulin and the other panels show the distribution of FTCD or/and GM130. Scale bar = 10 μm. Arrowheads indicate the colocalization of FTCD and GM130.


( B ) effects of FTCD siRNA treatment on Golgi reassembly at end of mitosis . HepG2 were treated with either FTCD siRNA ( FTCD siRNA 2 duplex ) or mock for 48 hrs before collection of mitotic . collected mitotic were cultured for 35 mins then fixed . were stained with a polyclonal antibody to GM130 a monoclonal antibody to α - tubulin , observed by confocal microscopy . Panels a - p display representative . Scale bar = 10 μm .

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(B) The effects of FTCD siRNA treatment on Golgi reassembly at the end of mitosis. HepG2 cells were treated with either FTCD siRNA (FTCD siRNA 2 duplex) or mock for 48 hrs before the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and then fixed. The cells were stained with a polyclonal antibody to GM130 and a monoclonal antibody to α-tubulin, and observed by confocal microscopy. Panels a-p display representative images. Scale bar = 10 μm.


( C ) Rescue experiments in FTCD siRNA - treated HepG2 . indicated HA - tagged FTCDs , which were insensitive to FTCD siRNA , were expressed 25 hrs after treatment of with FTCD siRNA2 . were further cultured for 23 hrs then used for collection of mitotic . collected mitotic were cultured for 35 mins fixed . were stained with a monoclonal antibody to α - tubulin ( panels g h ) polyclonal antibodies to GM130 HA ( panels a - f ) . Scale bar = 10 μm .

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(C) Rescue experiments in FTCD siRNA-treated HepG2 cells. The indicated HA-tagged FTCDs, which were insensitive to FTCD siRNA, were expressed 25 hrs after the treatment of cells with FTCD siRNA2. The cells were further cultured for 23 hrs and then used for the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and fixed. The cells were stained with a monoclonal antibody to α-tubulin (panels g and h) and polyclonal antibodies to GM130 and HA (panels a-f). Scale bar = 10 μm.


( D ) at cytokinesis in ( B ) ( C ) were analyzed . results are shown as mean ± SD of five sets of independent experiments , with 100 counted in each group in each independent experiment . Asterisks indicate a significant difference ( P < 0 . 01 ) compared with mock - treated ( Bonferroni method ) .

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(D) The cells at cytokinesis in (B) and (C) were analyzed. The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference (P<0.01) compared with the mock-treated cells (Bonferroni method).


( A ) p47 requires its association with FTCD for its localization to Golgi at end of mitosis . HepG2 were treated with FTCD siRNA then indicated HA - tagged FTCDs were expressed as described in Figure 4C . mitotic were collected , cultured for 35 mins , then fixed . were stained with polyclonal antibodies to p47 HA a monoclonal antibody to GM130 , observed by confocal microscopy . Panels a - l show distribution of p47 or / GM130 panels m - o show HA - FTCD expression . Scale bar = 10 μm . ( B ) Insets in ( A ) . p47 ( red ) GM130 ( a Golgi marker , green ) were visualized . Scale bar = 5 μm . ( C ) results of of ( A ) . results are shown as mean ± SD of five sets of independent experiments , with 100 counted in each group in each independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with mock - treated ( Bonferroni method ) .

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(A) p47 requires its association with FTCD for its localization to the Golgi at the end of mitosis. HepG2 cells were treated with FTCD siRNA and then the indicated HA-tagged FTCDs were expressed as described in Figure 4C. The mitotic cells were collected, cultured for 35 mins, and then fixed. The cells were stained with polyclonal antibodies to p47 and HA and a monoclonal antibody to GM130, and observed by confocal microscopy. Panels a-l show the distribution of p47 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm. (B) Insets in (A). p47 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm. (C) The results of quantification of (A). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).


( D ) p97 requires its association with FTCD for its localization to Golgi at end of mitosis . were treated in same way as ( A ) stained with polyclonal antibodies to p97 HA a monoclonal antibody to GM130 . Panels a - l show distribution of p97 or / GM130 panels m - o show HA - FTCD expression . Scale bar = 10 μm . ( E ) Insets in ( D ) . p97 ( red ) GM130 ( a Golgi marker , green ) were visualized . Scale bar = 5 μm . ( F ) results of of ( D ) . results are shown as mean ± SD of five sets of independent experiments , with 100 counted in each group in each independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with mock - treated ( Bonferroni method ) .

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(D) p97 requires its association with FTCD for its localization to the Golgi at the end of mitosis. The cells were treated in the same way as (A) and stained with polyclonal antibodies to p97 and HA and a monoclonal antibody to GM130. Panels a-l show the distribution of p97 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm. (E) Insets in (D). p97 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm. (F) The results of quantification of (D). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).


( A ) Anti - FTCD antibodies inhibit p97 / p47 - mediated Golgi fusion . Golgi fragments were mixed with indicated components ( p97 , 60 μg / ml ; p47 , 30 μg / ml ; p37 , 24 μg / ml ; p115 , 40 μg / ml ) together with either anti - FTCD or anti - p115 antibodies . Results are expressed as mean + SD of five sets of independent experiments ; 0 % 100 % represent buffer only ( 25 . 9 % in cisternal membranes ) p97 / p47 with no antibodies ( ' + none ' , 48 . 8 % ) , respectively . Asterisks indicate a significant difference at P < 0 . 01 compared with no antibody groups ( ' + none ' ) ( Bonferroni method ) .

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(A) Anti-FTCD antibodies inhibit p97/p47-mediated Golgi membrane fusion. Golgi fragments were mixed with the indicated components (p97, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml) together with either anti-FTCD or anti-p115 antibodies. Results are expressed as mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (25.9% in cisternal membranes) and p97/p47 with no antibodies ('+ none', 48.8%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the no antibody groups ('+none') (Bonferroni method).


( B ) p47 ( E86K , E88K ) , which lacks binding affinity to FTCD , does not function in p97 / p47 - mediated Golgi fusion . Golgi fragments were incubated with p97 ( 60 μg / ml ) p47wt / mutant ( 30 μg / ml ) . Data are shown as mean + SD of five sets of independent experiments ; 0 % 100 % represent buffer only ( 27 . 3 % in cisternal membranes ) p97 + p47wt ( ' + p47wt ' , 50 . 9 % ) , respectively . Asterisks indicate a significant difference at P < 0 . 01 compared with group in which p47wt as well as p97 were added ( ' + p47wt ' ) ( Bonferroni method ) .

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(B) p47(E86K, E88K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with p97 (60 μg/ml) and p47wt/mutant (30 μg/ml). Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (27.3% in cisternal membranes) and p97+p47wt ('+p47wt', 50.9%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the group in which p47wt as well as p97 were added ('+p47wt') (Bonferroni method).


( C ) p97 ( V722K , E724K ) , which lacks binding affinity to FTCD , does not function in p97 / p47 - mediated Golgi fusion . Golgi fragments were incubated with following components : p97wt / mutant , 60 μg / ml ; p47 , 30 μg / ml ; p37 , 24 μg / ml ; p115 , 40 μg / ml . Data are shown as mean + SD of five sets of independent experiments ; 0 % 100 % represent buffer only ( 29 . 5 % in cisternal membranes ) , p47 + p97wt ( ' + p97wt ' . 57 . 5 % ) , respectively . An asterisk indicates a significant difference at P < 0 . 01 compared with group in which p97wt as well as p47 were added ( ' + p97wt ' ) ( Bonferroni method ) .

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(C) p97(V722K,E724K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with the following components: p97wt/mutant, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml. Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (29.5% in cisternal membranes), and p47+p97wt ('+p97wt'. 57.5%), respectively. An asterisk indicates a significant difference at P<0.01 compared with the group in which p97wt as well as p47 were added ('+p97wt') (Bonferroni method).


( D ) Panels a - e show representative EM . Scale bar = 0 . 5 μm .

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(D) Panels a-e show representative EM images. Scale bar = 0.5 μm.


( A ) Two sets of FTCD molecules a p97 / p47 form a quaternary . His - tagged FTCD was biotinylated immobilized on streptavidin beads ( His - FTCD - beads ) . beads ( 1 . 0 μg ) were incubated with Flag - tagged FTCD ( 2 . 0 μg ) in absence or presence of p47 ( 1 . 0 μg ) p97 ( 2 . 0 μg ) . blots were probed with HRP - conjugated avidin antibodies to Flag - tag , p97 p47 .

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(A) Two sets of FTCD molecules and a p97/p47 complex form a quaternary complex. His-tagged FTCD was biotinylated and immobilized on streptavidin beads (His-FTCD-beads). The beads (1.0 μg) were incubated with Flag-tagged FTCD (2.0 μg) in the absence or presence of p47 (1.0 μg) and p97 (2.0 μg). The blots were probed with HRP-conjugated avidin and antibodies to Flag-tag, p97 and p47.


( B ) Formation of quaternary in presence of nucleotides . His - FTCD - beads ( 1 . 0 μg ) were incubated with Flag - tagged FTCD ( 2 . 0 μg ) , p97 ( 2 . 0 μg ) p47 ( 1 . 0 μg ) in presence of nucleotides ( 1 mM ) . blots were probed as described in ( A ) .

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(B) Formation of the quaternary complex in the presence of nucleotides. His-FTCD-beads (1.0 μg) were incubated with Flag-tagged FTCD (2.0 μg), p97 (2.0 μg) and p47 (1.0 μg) in the presence of nucleotides (1 mM). The blots were probed as described in (A).


( C ) Mutants that lack binding affinity disrupt formation of quaternary . His - FTCD - beads ( 1 . 0 μg ) Flag - tagged FTCD ( 2 . 0 μg ) were incubated in presence of AMP - PNP together with p97wt / mutant ( 2 . 0 μg ) p47wt / mutant ( 1 . 0 μg ) . blots were probed as described in ( A ) .

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(C) Mutants that lack binding affinity disrupt the formation of the quaternary complex. His-FTCD-beads (1.0 μg) and Flag-tagged FTCD (2.0 μg) were incubated in the presence of AMP-PNP together with p97wt/mutant (2.0 μg) and p47wt/mutant (1.0 μg). The blots were probed as described in (A).


( D ) Negative stain electron microscopy of quaternary . panels show class averages of centered aligned negative stain particles for different populations present within dataset , i . e . , full quaternary FTCD - p97 / p47 - FTCD ( left panel ) , its partial complexes ( two variations , middle two panels ) . Class averages contain approximately 20 per class . A class average showing a p97 / p47 side view from cryo - EM data a projection generated from FTCD structure ( PDB code 1TT9 ) have been included for comparisons . Brackets arrow heads indicate p97 hexamer FTCD octamer , respectively . Scale bar = 15 nm .

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(D) Negative stain electron microscopy of the quaternary complex. The panels show class averages of centered and aligned negative stain particles for the different populations present within the dataset, i.e., the full quaternary complex FTCD-p97/p47-FTCD (left panel), and its partial complexes (two variations, middle two panels). Class averages contain approximately 20 images per class. A class average showing a p97/p47 side view from cryo-EM data and a projection generated from FTCD structure (PDB code 1TT9) have been included for comparisons. Brackets and arrow heads indicate the p97 hexamer and the FTCD octamer, respectively. Scale bar = 15 nm.


( E ) FTCDwt - beads are aggregated in presence of both p97 p47 . Biotinylated FTCDwt FTCD ( R382A ) were immobilized on streptavidin magnetic beads to form FTCDwt - beads FTCD ( R382A ) - beads , respectively . beads were incubated with indicated ( p97 , 0 . 1 μg / μl ; p47 , 0 . 1 μg / μl ; p37 , 0 . 1 μg / μl ) in presence of AMP - PNP ( 1 mM ) , observed under a bright - field microscope . Scale bar = 50 μm . ( F ) results of of ( E ) . percentages of beads in were calculated . Results are expressed as mean + SD of ten sets of independent experiments . An asterisk indicates a significant difference at P < 0 . 01 compared with others ( Bonferroni method ) .

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(E) FTCDwt-beads are aggregated in the presence of both p97 and p47. Biotinylated FTCDwt and FTCD(R382A) were immobilized on streptavidin magnetic beads to form FTCDwt-beads and FTCD(R382A)-beads, respectively. The beads were incubated with the indicated proteins (p97, 0.1 μg/μl; p47, 0.1 μg/μl; p37, 0.1 μg/μl) in the presence of AMP-PNP (1 mM), and observed under a bright-field microscope. Scale bar = 50 μm. (F) The results of quantification of (E). The percentages of beads in the aggregates were calculated. Results are expressed as the mean+SD of ten sets of independent experiments. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method).


( G ) p97 / p47 - mediated aggregation of FTCDwt - beads is nucleotide - dependent . FTCDwt - beads were incubated together with p97 ( 0 . 1 μg / μl ) p47 ( 0 . 1 μg / μl ) in presence of indicated nucleotides ( 1mM ) . representative are presented in Figure EV1E . Data are shown as mean + SD of nine sets of independent experiments . An asterisk hash tags indicate significant differences at P < 0 . 01 compared with others negative control ( ' none ' ) , respectively ( Bonferroni method ) .

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(G) p97/p47-mediated aggregation of FTCDwt-beads is nucleotide-dependent. FTCDwt-beads were incubated together with p97 (0.1 μg/μl) and p47 (0.1 μg/μl) in the presence of the indicated nucleotides (1mM). The representative images are presented in Figure EV1E. Data are shown as the mean+SD of nine sets of independent experiments. An asterisk and hash tags indicate significant differences at P<0.01 compared with the others and the negative control ('none'), respectively (Bonferroni method).


( H ) p47 p97 mutants that lack binding affinity inhibit p97 / p47 - mediated aggregation of FTCDwt - beads . FTCDwt - beads were incubated with p97wt / mutant ( 0 . 1 μg / μl ) p47wt / mutants ( 0 . 1 μg / μl ) in presence of AMP - PNP . representative are presented in Figure EV1F . Data are shown as mean + SD of nine sets of independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with positive control ( ' p97wt + p47wt ' ) ( Bonferroni method ) .

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

(H) p47 and p97 mutants that lack binding affinity inhibit p97/p47-mediated aggregation of FTCDwt-beads. FTCDwt-beads were incubated with p97wt/mutant (0.1 μg/μl) and p47wt/mutants (0.1 μg/μl) in the presence of AMP-PNP. The representative images are presented in Figure EV1F. Data are shown as the mean+SD of nine sets of independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the positive control ('p97wt+p47wt') (Bonferroni method).


( A ) expression of FTCDwt - HA - MAO causes aggregation of mitochondria . HeLa Tet - off inducibly expressing either FTCDwt - HA - MAO or FTCD ( R382A ) - HA - MAO were cultured in DOX - free medium for 48 hrs . which were cultured in DOX - containing medium were used as a control . were fixed stained with a monoclonal antibody to mitochondria a polyclonal antibody to HA , followed by observation by confocal microscopy . Panels a - j display representative . Scale bar = 10 μm . ( B ) results of of ( A ) . Results are shown as mean ± SD of five sets of independent experiments , with 100 counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with others ( Bonferroni method ) .

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

(A) The expression of FTCDwt-HA-MAO causes the aggregation of mitochondria. HeLa Tet-off cells inducibly expressing either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO were cultured in DOX-free medium for 48 hrs. Cells which were cultured in the DOX-containing medium were used as a control. The cells were fixed and stained with a monoclonal antibody to mitochondria and a polyclonal antibody to HA, followed by observation by confocal microscopy. Panels a-j display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method).


( C ) Representative EM of a expressing FTCDwt - HA - MAO . HeLa Tet - off inducibly expressing FTCDwt - HA - MAO were cultured for 48 hrs in absence of DOX , used for EM observation . Panel b displays inset in panel a . Arrowheads show narrow gaps between mitochondria . Scale bar = 0 . 2 μm . low magnification image is presented in Figure EV2C .

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

(C) Representative EM images of a cell expressing FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were cultured for 48 hrs in the absence of DOX, and used for EM observation. Panel b displays the inset in panel a. Arrowheads show narrow gaps between mitochondria. Scale bar = 0.2 μm. The low magnification image is presented in Figure EV2C.


( D ) Endogenous p47 p97 are necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . HeLa Tet - off inducibly expressing FTCDwt - HA - MAO were transfected with either mock , p47 siRNA or p97 siRNA duplexes cultured for 24 hrs . were further cultured in DOX - free medium for 48 hrs for induction of FTCDwt - HA - MAO , then analyzed as in ( A ) . Panels a - l display representative . Scale bar = 10 μm . ( E ) results of of ( D ) . Results are shown as mean ± SD of five sets of independent experiments , with 100 counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with others ( Bonferroni method ) .

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

(D) Endogenous p47 and p97 are necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with either mock, p47 siRNA or p97 siRNA duplexes and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCDwt-HA-MAO, and then analyzed as in (A). Panels a-l display representative images. Scale bar = 10 μm. (E) The results of quantification of (D). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method).


( F ) Binding between FTCD p47 between p47 p97 is necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . HeLa Tet - off inducibly expressing FTCDwt - HA - MAO were transfected with mammalian expression constructs of siRNA - insensitive Flag - tagged p47wt / mutants at same time as treatment of p47 siRNA , cultured for 24 hrs . were further cultured in DOX - free medium for 48 hrs for induction of FTCD - HA - MAO . After fixation , were visualized with a monoclonal antibody to mitochondria polyclonal antibodies to HA Flag . Panels a - l display representative . Scale bar = 10 μm . ( G ) Binding between FTCD p97 is necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . HeLa Tet - off inducibly expressing FTCDwt - HA - MAO were transfected with mammalian expression construct of siRNA - insensitive Flag - tagged p97wt / mutant at same time as treatment with p97 siRNA . following procedures were same as in ( F ) . Panels a - i display representative . Scale bar = 10 μm . ( H ) results of of ( F ) ( G ) . Results are shown as mean ± SD of five sets of independent experiments , with 100 counted in each group in each independent experiment . Asterisks indicate a significant difference at P < 0 . 01 compared with siRNA treatment alone ( ' none ' ) compared with mutant expression ( Bonferroni method ) .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binding between FTCD and p47 and between p47 and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with mammalian expression constructs of siRNA-insensitive Flag-tagged p47wt/mutants at the same time as the treatment of p47 siRNA, and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCD-HA-MAO. After fixation, the cells were visualized with a monoclonal antibody to mitochondria and polyclonal antibodies to HA and Flag. Panels a-l display representative images. Scale bar = 10 μm. (G) Binding between FTCD and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with the mammalian expression construct of siRNA-insensitive Flag-tagged p97wt/mutant at the same time as treatment with p97 siRNA. The following procedures were the same as in (F). Panels a-i display representative images. Scale bar = 10 μm. (H) The results of quantification of (F) and (G). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with siRNA treatment alone ('none') and compared with mutant expression (Bonferroni method).


( A ) FTCD located in mitochondria causes mitochondria aggregation during mitosis . Mitotic were collected by flushing from HeLa Tet - off in which either FTCDwt - HA - MAO or FTCD ( R382A ) - HA - MAO had been induced for 24 hrs . mitotic were further cultured for 4 hrs to enable complete cytokinesis . in which FTCDwt - HA - MAO was induced for 28 hrs without synchronization were used as asynchronous control group . were fixed visualized with a monoclonal antibody to mitochondria a polyclonal antibody to HA . Panels a - f display representative . Scale bar = 10 μm . ( B ) results of of ( A ) . Results are shown as mean ± SD of seven sets of independent experiments , with 100 counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with others ( Bonferroni method ) .

O O O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-CELL_LINE B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR 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 O O O O 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) FTCD located in mitochondria causes mitochondria aggregation during mitosis. Mitotic cells were collected by flushing from HeLa Tet-off cells in which either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO had been induced for 24 hrs. The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis. Cells in which FTCDwt-HA-MAO was induced for 28 hrs without synchronization were used as the asynchronous control group. Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA. Panels a-f display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of seven sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method).


( C ) A single living expressing FTCDwt - HA - MAO was tracked during mitosis . Mitotic were collected by flushing from HeLa Tet - off inducibly expressing FTCDwt - HA - MAO , cultured in absence or presence of DOX . were stained with MitoTracker at 22 hr time point confocal of 30 - 40 were randomly taken at 23 hr time point . From 23 hr to 27 hr time point , were tracked in bright field every 30 mins . At 27 hr time point , confocal were taken of 21 - 38 that were successfully tracked . Panels a - j display representative . Asterisks indicate tracked . Scale bar = 10 μm . ( D ) results of of ( C ) . experiment was repeated five times each shows average of an independent experiment . percentages of that underwent mitosis are as follows : 41 . 5 % in DOX ( - ) group 54 . 7 % in DOX ( + ) group .

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

(C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm. (D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group.


( E ) FTCD located in mitochondria causes mitochondria aggregation at end of mitosis . Mitotic were collected from HeLa Tet - off inducibly expressing FTCDwt - HA - MAO cultured in absence or presence of DOX as in ( C ) . were stained with MitoTracker at 23hr time point observed by confocal microscopy from 24 hr to 26 hr time point . Panels a - t display representative . Scale bar = 10 μm .

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

(E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm.


( F ) results of of ( E ) . were classified by their cycle phases into following three categories : prometaphase / metaphase , anaphase / telophase cytokinesis . Results are shown as mean ± SD of three sets of independent experiments , with 10 - 14 counted in each group in each independent experiment . Asterisks indicate a significant difference at P < 0 . 01 compared with DOX ( + ) group with prometaphase / metaphase category ( Bonferroni method ) .

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

(F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).


A , Heat map of reported cancer blood metabolites ( each blue mark denotes that metabolite was reported to be increased or decreased ) in various cancer types , illustrating that most studies report only a small subset of previously measured metabolites . It is typically not described whether metabolites that were not reported ( " empty " space ) were not measured , or measured but not reported . Labelings : X - axis ( top ) : ribbon colour code , denoting cancer type ( right ; indicated by red arrow ) ; X - axis ( bottom ) : cohorts , arranged from 1 to 71 ; Y - axis ( right ) : 1 , 206 metabolites reported in at least one of studies ; Y - axis ( left ) : ribbon colour code , denoting metabolite class ( , carbohydrates , etc ; " other " refers to other metabolites than listed classes ) .

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

A, Heat map of all reported cancer blood metabolites (each dark blue mark denotes that the metabolite was reported to be increased or decreased) in various cancer types, illustrating that most individual studies report only a small subset of all previously measured metabolites. It is typically not described whether the metabolites that were not reported (white "empty" space) were not measured, or measured but not reported. Labelings: X-axis (top): ribbon colour code, denoting the cancer type (right; indicated by red arrow); X-axis (bottom): cohorts, arranged from 1 to 71; Y-axis (right): all 1,206 metabolites reported in at least one of the studies; Y-axis (left): ribbon colour code, denoting the metabolite class (amino acids, carbohydrates, etc; "other" refers to all other metabolites than the listed classes).


B , Vote counting of cancer blood metabolites ( reported in at least six cohorts ) showed consistently deregulated metabolites . Blue bars : decreased metabolites ; red bars : increased metabolites . * ( asterisk ) in front of name of metabolite indicates a statistical trend ( p < 0 . 1 ) ; red arrowheads denote metabolites mentioned in main text .

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

B, Vote counting of cancer blood metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrowheads denote metabolites mentioned in the main text.


C , D , Volcano plot of bloodmetabolites ( C ) tissuemetabolites ( D ) , reported in at least six cancer studies , with vote counting score on X - axis - log10 adjusted p - value on Y - axis . Cyan indicates deregulated metabolites that show a trend ( p < 0 . 1 ; for blood , corresponding to metabolites marked with * in panel 2B ; for tissue , corresponding to metabolites marked with * in Figure 3 ) or statistical significance ( p < 0 . 05 ; above black dashed horizontal ) ; red indicates metabolites with a p - value > 0 . 1 . A subset of metabolites is annotated ( see Table EV4 ( blood ) EV5 ( tissue ) for full annotation ) .

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

C,D, Volcano plot of bloodmetabolites (C) and tissuemetabolites (D), reported in at least six cancer studies, with the vote counting score on the X-axis and the -log10 adjusted p-value on the Y-axis. Cyan indicates deregulated metabolites that show a trend (p < 0.1; for blood, corresponding to metabolites marked with * in panel 2B; for tissue, corresponding to metabolites marked with * in Figure 3) or statistical significance (p < 0.05; above black dashed horizontal line); red indicates metabolites with a p-value > 0.1. A subset of metabolites is annotated (see Table EV4 (blood) and EV5 (tissue) for full annotation).


Vote counting of cancer tissue metabolites ( reported in at least six cohorts ) showed consistently deregulated metabolites . Blue bars : decreased metabolites ; red bars : increased metabolites . * ( asterisk ) in front of name of metabolite indicates a statistical trend ( p < 0 . 1 ) ; red arrows denote key metabolites mentioned in main text ( see Table EV5 for full annotation ) .

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

Vote counting of cancer tissue metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrows denote key metabolites mentioned in the main text (see Table EV5 for full annotation).


Representative ( top ) ( bottom ) of TRF1 of H676 treated with indicated compounds for 24h at 1 μM . Scale bars , 5μm . Data are representative of n = 3 biological replicates . ( C ) Western blot ( top ) TRF1 ( bottom ) of h676 treated with indicated compounds for 24h at 1 μM . Data are representative of n = 3 ( PLKi , HSP90i RTKi ) n = 4 ( AuroraI , mTOR , CDKi , Docetaxel , Gemcitabine , ERKi , MEKi ) biological replicates .

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

Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates.


Representative ( left ) ( right ) of presenting 1 or more γH2AX RAP1 colocalizing foci ( TIFs ) upon treatment of CHA9 - 3 lung cancer with indicated compounds . arrowheads point to colocalization of γH2AX RAP1 . Scale bars , 5μm . Data are representative of n = 6 ( DMSO ) n = 3 ( mTORi , PI3Ki , RTKi , MEKi , ERKi , HSPO90i , CDKi , Docetaxel ) biological

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

Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5μm. Data are representative of n=6 (DMSO) and n=3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, Docetaxel) biological replicates


Representative ( left ) ( right ) of γH2AX - positive per field in DMSO or compound treated - derived h676 GSCs . Scale bars , 50μm . Data are representative of 6 ( DMSO ) 3 ( mTORi , Docetaxel , ERKi , MEKi , RTKi , HSP90i , Gemcitabine , CDKi ) biological

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

Representative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound treated patient-derived h676 GSCs. Scale bars, 50μm. Data are representative of 6 (DMSO) and 3 (mTORi, Docetaxel, ERKi, MEKi, RTKi, HSP90i, Gemcitabine, CDKi) biological replicates


of multitelomeric signals ( MTS ) in h676 GSC metaphases upon treatment with indicated compounds . Representative of qFISH in metaphases ( left ) . Multitelomeric signals are indicated by arrowheads . Scale bars , 1μm . Data are representative of n = 31 ( DMSO ) , n = 18 ( mTORi ) , n = 11 ( MEKi ) n = 24 ( RTKi ) biological replicates . Data are represented as mean ± . Significant differences using unpaired t ‐ are indicated by * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Quantification of multitelomeric signals (MTS) in h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1μm. Data are representative of n=31 (DMSO), n=18 (mTORi), n=11 (MEKi) and n=24 (RTKi) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.


Dose response curves of h543 h676 GSCs treated with indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± normalized to DMSO .

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

Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.