LAMP . ( a ) Clinical samples tested by qRT - PCR ( Positive , red or Negative , grey ) were sequenced run through LAMP . These results were compared to buffer blanks ( Negative Control ) , grey , Synthetic RNAs or Vero 6 extracts with SARS - CoV - 2 infection ( Positive Controls , red ) , Subway Samples ( Environmental , blue ) . Read proportions are shown on y - axis .

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LAMP.(a) Clinical samples tested by qRT-PCR (Positive, dark red or Negative, light grey) were sequenced and run through the LAMP assay. These results were compared to the buffer blanks (Negative Control), dark grey, Synthetic RNAs or Vero 6 cell extracts with SARS-CoV-2 infection (Positive Controls, light red), and Subway Samples (Environmental, blue). Read proportions are shown on the y-axis.


( b ) SARS - CoV - 2 abundance , as measured with NGS of reads ( y - axis ) is compared to Ct Threshold for qRT - PCR ( x - axis ) , with lower Ct values representing higher viral abundance , LAMP reaction output ( Fluorimeter values , black to yellow scale ) .

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(b) SARS-CoV-2 abundance, as measured with NGS and percentage of reads (y-axis) is compared to the Ct Threshold for qRT-PCR (x-axis), with lower Ct values representing higher viral abundance, and the LAMP reaction output (Fluorimeter values, black to yellow scale).


( c , d ) Read mapping to archaea ( red ) , ( green ) , fungi ( yellow ) , human ( blue ) , SARS - CoV - 2 ( orange ) , other ( grey ) , across clinical controls ( CN , CP ) , environmental samples , qRT - PCR negative , qRT - PCR positive samples .

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(c,d) Read mapping to archaea (red), bacteria (green), fungi (yellow), human (blue), and SARS-CoV-2 (orange), and other viruses (grey), across the clinical controls (CN, CP), environmental samples, qRT-PCR negative, and qRT-PCR positive samples.


coverage ( a ) coverage plot across SARS - CoV - 2 genomes ( viral coordinate on bottom , colored by sample ) from a representative set of clinical positive samples . Sample names with suffixes CN N are clinical negative ( buffer ) , P are qRT - PCR positive , CP are Vero 6 with .

coverage(a) The coverage plot across the SARS-CoV-2 genomes (viral coordinate on bottom, colored by sample) from a representative set of clinical positive samples. Sample names with the suffixes CN and N are clinical negative (buffer), P are qRT-PCR positive, and CP are Vero 6 cells with virus.


phylogenetic placement of these COVID samples is shown on tree ( left ) global map of known COVID genomes ( right ) . Genetic variants called from - seq data show a range of variants that are distinct from Wuhan reference , samples from this study , highlighted in blue , show enrichment for European Asian alleles .

The phylogenetic placement of these COVID samples is shown on the tree (left) and the global map of known COVID genomes (right). Genetic variants called from the RNA-seq data show a range of variants that are distinct from the Wuhan reference strain, and the samples from this study, highlighted in blue, show enrichment for European and Asian alleles.


( b ) Proportion of L ( green ) S ( yellow ) are shown for NYC . Phylogeny of samples from this study on left total GISAID samples on right , with a map of variants in this study ' s samples in middle , colored by event type sized by number of nucleotides impacted . Annotation track on bottom shows frequency of alternate alleles in this study in GISAID database .

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(b) Proportion of the L (green) and S strain (yellow) are shown for the NYC viruses. Phylogeny of samples from this study on the left and total GISAID samples on the right, with a map of variants in this study's samples in the middle, colored by event type and sized by number of nucleotides impacted. Annotation track on the bottom shows frequency of alternate alleles in this study and in the GISAID database.


( c ) 9 - bp deletion in ORRF1b ( NSP1 ) that was detected in samples from two was confirmed in GISAID database ( below ) .

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(c) The 9-bp deletion in ORRF1b (NSP1) that was detected in samples from two patients was confirmed in the GISAID database (below).


( d ) Comparison of ORF1b of SARS - CoV - 2 SARS - CoV genomes with 9 - bp deletion region shown enlarged .

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(d) Comparison of the ORF1b of the SARS-CoV-2 and SARS-CoV genomes with the 9-bp deletion region shown enlarged.


( a ) Six variant alleles were found to be significantly enriched by population allelic fraction within this set of 93 WCM / NYP cases as compared with non - NYC strains of Nextstrain clade A2a ( 1059 : C > T P = 6 . 26e - 22 , 11916 : C > T P = 3 . 09e - 9 , 18998 : C > T P = 2 . 51e - 7 , 20755 : A > C P = 1 . 32e - 4 , 25563 : G > T P = 9 . 6e - 23 , 29540 : G > A P = 2 . 51e - 7 ) . These variants demonstrated similar PAF enrichment within other NYC strains as compared to non - NYC clade A2a ( 1059 : C > T P = 4 . 35e - 84 , 11916 : C > T P = 2 . 16e - 43 , 18998 : C > T P = 1 . 34e - 25 , 20755 : A > C P = 1 . 67e - 4 , 25563 : G > T P = 2 . 60e - 83 , 29540 : G > A P = 1 . 34e - 46 ) .

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(a) Six variant alleles were found to be significantly enriched by population allelic fraction within this set of 93 WCM/NYP cases as compared with non-NYC strains of Nextstrain clade A2a (1059:C>T P = 6.26e-22, 11916:C>T P = 3.09e-9, 18998:C>T P= 2.51e-7, 20755:A>C P = 1.32e-4, 25563:G>T P = 9.6e-23, 29540:G>A P = 2.51e-7). These variants demonstrated similar PAF enrichment within all other NYC strains as compared to non-NYC clade A2a (1059:C>T P = 4.35e-84, 11916:C>T P = 2.16e-43, 18998:C>T P= 1.34e-25, 20755:A>C P = 1.67e-4, 25563:G>T P = 2.60e-83, 29540:G>A P = 1.34e-46).


( b ) ( left ) Phylogenetic tree produced by Nextstrain with clade affiliations nodes corresponding to WCM / NYP in red other NYC cases in green . ( right ) occurrence of six NYC - enriched alleles 9 nucleotide deletion across genomes .

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(b) (left) Phylogenetic tree produced by the Nextstrain analysis with clade affiliations and nodes corresponding to WCM/NYP in red and other NYC cases in green. (right) occurrence of the six NYC-enriched alleles and the 9 nucleotide deletion across genomes.


Samples were quantified by a range of viral detection methods , including LAMP ( Quantifluor ) , - seq ( log10 SARS - CoV - 2 % of reads ) , qRT - PCR ( Ct values ) to create a three - tier range of viral load for positive samples ( right ) compared to clinically - annotated negative samples ( class , red or grey ) . ( bottom ) differentially expressed genes of COVID + compared to COVID - showed up - regulated ( orange ) genes as well as down - regulated ( purple ) genes .

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Samples were quantified by a range of viral detection methods, including LAMP (Quantifluor), RNA-seq (log10 SARS-CoV-2 % of reads), and qRT-PCR (Ct values) to create a three-tier range of viral load for the positive samples (right) compared to the clinically-annotated negative samples (class, red or grey). (bottom) The differentially expressed genes of COVID+ patients compared to COVID-patients showed up-regulated (orange) genes as well as down-regulated (purple) genes.


( b ) Up - regulated genes , with boxplots across samples , include IFI6 , ACE2 , SHFL , HERC6 , IFI27 , IFIT1 , on data from ( c ) , which is total set of DEGs . full set is shown in an intersecting heat map , with a set of up - regulated genes ( orange ) distinct form set of down - regulated genes ( purple ) , compared to genes that are not significantly differently expressed ( grey ) in any comparison ( DESeq2 , q - value < 0 . 01 , | logFC | > 0 . 58 ) .

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(b) Up-regulated genes, with boxplots across all samples, include IFI6, ACE2, SHFL, HERC6, IFI27, and IFIT1, based on data from (c), which is the total set of DEGs. The full set is shown in an intersecting heat map, with a core set of up-regulated genes (orange) distinct form the set of down-regulated genes (purple), compared to genes that are not significantly differently expressed (grey) in any comparison (DESeq2, q-value <0.01, |logFC| >0.58).


( e ) Survival curves for requiring mechanical respiration ( identified by intubation procedure notes ) ( e ) mortality ( f ) . with a history of ACE inhibitor exposure were more likely to require intubation ( HR = 2 . 63 95 % CI 2 . 01 - 3 . 43 , p = 1 . 22E - 12 ; ( e ) less likely to survive ( HR = 1 . 68 95 % CI : 1 . 22 - 2 . 31 , p = 1 . 42E - 03 ; ( f ) . Because several individuals were intubated shortly before they first tested positive for SARS - CoV - 2 infection , each first positive was set back by seven days to account for testing delays .

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(e)Survival curves for requiring mechanical respiration (identified by intubation procedure notes) (e) and mortality (f). Patients with a history of ACE inhibitor exposure were more likely to require intubation (HR=2.63 95%CI 2.01-3.43, p=1.22E-12; (e) and less likely to survive (HR=1.68 95%CI: 1.22-2.31, p=1.42E-03; (f). Because several individuals were intubated shortly before they first tested positive for SARS-CoV-2 infection, each first positive test was set back by seven days to account for testing delays.


A - C . Enzyme - linked immunosorbent ( ELISA ) of IL - 6 , TNF - α IFN - β real - time PCR of IL - 6 , TNF - α IFN - β mRNA in peritoneal macrophage isolated from wild type RKIP - deficient mice treated with VSV ( MOI , 0 . 1 ) ( A ) .

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A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice treated with VSV (MOI,0.1) (A).


A - C . Enzyme - linked immunosorbent ( ELISA ) of IL - 6 , TNF - α IFN - β real - time PCR of IL - 6 , TNF - α IFN - β mRNA in peritoneal macrophage isolated from wild type RKIP - deficient mice treated with SEV ( MOI , 10 ) ( B ) .

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A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice treated with SEV (MOI,10) (B).


A - C . Enzyme - linked immunosorbent ( ELISA ) of IL - 6 , TNF - α IFN - β real - time PCR of IL - 6 , TNF - α IFN - β mRNA in peritoneal macrophage isolated from wild type RKIP - deficient mice transfected with poly ( I : C ) ( 1 μg / ml ) ( C ) .

A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice transfected with poly(I:C) ( 1 μg/ml) (C).


D . - activated sorting ( FACS ) TCID50 ( 50 % tissue culture infective dose ) assessing proliferation level of VSV . RAW264 . 7 stably overexpressed flag - RKIP was infected with VSV - eGFP at MOI of 1 for indicated time . fusion fluorophore were analyzed by FACS . Then fresh medium with VSV - eGFP infection was serially diluted on monolayer of HEK293 for 3 - 7 days TCID50 was measured .

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D. Fluorescence-activated cell sorting (FACS) and TCID50 (50% tissue culture infective dose) assessing the proliferation level of VSV. RAW264.7 cells line stably overexpressed flag-RKIP was infected with VSV-eGFP at MOI of 1 for the indicated time. The fusion protein fluorophore were analyzed by FACS. Then fresh medium with the VSV-eGFP infection was serially diluted on the monolayer of HEK293 cells for 3-7 days and TCID50 was measured.


E . FACS infection of wild type RKIP - deficient peritoneal macrophage infected with VSV - eGFP at MOI of 1 . fresh medium with VSV - eGFP infection 9 hr were collected for TCID50 assessing .

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E. FACS analysis the infection of wild type and RKIP-deficient peritoneal macrophage infected with VSV-eGFP at MOI of 1. The fresh medium with the VSV-eGFP infection 9 hr were collected for TCID50 assessing.


A . Wide - type ( RKIP + / + ) RKIP deficiency ( RKIP - / - ) chimera were intraperitoneally injected with VSV ( 1x107 pfu / g ) , 12 hours later , ELISA of IFN - β IL - 6 in serum ( n = 6 per group ) .

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A. Wide-type (RKIP+/+) and RKIP deficiency (RKIP-/-) chimera were intraperitoneally injected with VSV (1x107 pfu/g), 12 hours later, ELISA of IFN-β and IL-6 in serum (n=6 per group).


B . Determination of VSV loads in organs by TCID50 12 hours later after RKIP + / + RKIP - / - chimera mice ( n = 6 per group ) intraperitoneally injected with VSV ( 1x107 pfu / g ) .

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B. Determination of VSV loads in organs by TCID50 assay 12 hours later after RKIP+/+ and RKIP-/- chimera mice (n = 6 per group) intraperitoneally injected with VSV (1x107 pfu/g).


C . Real - time PCR of IFN - β mRNA VSV expression in liver from RKIP + / + RKIP - / - chimera mice 12 hours later intraperitoneally injected with VSV ( 1x107 pfu / g ) ( n = 6 per group ) .

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C. Real-time PCR analysis of IFN-β mRNA and VSV expression in liver from RKIP+/+ and RKIP-/- chimera mice 12 hours later intraperitoneally injected with VSV (1x107 pfu/g) (n = 6 per group).


D . Pathology of wild type RKIP deficient chimeric mice in response to VSV infection . Hematoxylin eosin of liver lung sections from mice in ( A ) . Scale bar , 100 μm ( left ) or 50 μm ( right ) .

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D. Pathology of wild type and RKIP deficient chimeric mice in response to VSV infection. Hematoxylin and eosin staining of liver and lung sections from mice in (A). Scale bar, 100 μm (left) or 50 μm (right).


E . Real - time PCR of IFN - β mRNA expression in peritoneal macrophages from RKIP + / + RKIP - / - chimera mice 12 hours after intraperitoneally injected with VSV ( 1x107 pfu / g ) ( n = 6 per group ) .

E. Real-time PCR analysis of IFN-β mRNA expression in peritoneal macrophages from RKIP+/+ and RKIP-/- chimera mice 12 hours after intraperitoneally injected with VSV (1x107 pfu/g) (n = 6 per group).


F . Survival curve for 8 - old wild type RKIP deficient chimeras challenged with VSV ( 1x108 pfu / g , i . v . ) ( n = 10 per group ) . * p < 0 . 05 Wilcoxon .

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F. Survival curve for 8 weeks-old wild type and RKIP deficient chimeras challenged with VSV (1x108 pfu/g, i.v.) (n=10 per group). *p<0.05 Wilcoxon Test .


A . Immunoblot of p - TBK1 , - IKKi , - IRF3 , - P38 , - JNK1 / 2 , - ERK1 / 2 - IκBα in lysates of RKIP + / + , RKIP + / - RKIP - / - peritoneal macrophages stimulated with VSV ( MOI , 0 . 1 ) for indicated time .

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A. Immunoblot analysis of p-TBK1, -IKKi, -IRF3, -P38, -JNK1/2, -ERK1/2 and -IκBα in lysates of RKIP+/+, RKIP+/- and RKIP-/- peritoneal macrophages stimulated with VSV (MOI, 0.1) for the indicated time.


B . Immunoblot of RKIP + / + , RKIP + / - RKIP - / - peritoneal macrophage treated with intracellular poly ( I : C ) ( 1 μg / ml ) .

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B. Immunoblot assay of RKIP+/+, RKIP+/- and RKIP-/- peritoneal macrophage cells treated with intracellular poly(I:C) (1 μg/ml).


C . Immunoblot of phosphorylated ( p - ) TBK1 , IKKi , IRF3 , P38 , JNK1 / 2 , ERK1 / 2 in peritoneal macrophage transfected with RKIP - specific or scrambled siRNA , followed by infection with VSV ( MOI , 0 . 1 ) . β - actin was used as a loading control .

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C. Immunoblot analysis of phosphorylated (p-) TBK1, IKKi, IRF3, P38, JNK1/2, ERK1/2 in peritoneal macrophage transfected with RKIP-specific or scrambled siRNA, followed by infection with VSV (MOI, 0.1). β-actin was used as a loading control.


D . Immunoblot of p - TBK1 , - IKKi , - IRF3 , - P38 , - JNK1 / 2 , - ERK1 / 2 - IκBα in lysates of VSV - treated RAW264 . 7 stably over - expressing RKIP .

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D. Immunoblot analysis of p- TBK1, -IKKi, -IRF3, -P38, -JNK1/2, -ERK1/2 and -IκBα in lysates of VSV-treated RAW264.7 cells stably over-expressing RKIP.


E . Immunoblot in lysates of RAW264 . 7 stably overexpressing RKIP treated with intracellular poly ( I : C ) ( 1 μg / ml ) .

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E. Immunoblot analysis in lysates of RAW264.7 cells stably overexpressing RKIP treated with intracellular poly(I:C) (1 μg/ml).


F . Luciferase activity in 293T transfected with plasmid encoding a luciferase reporter for IFN - β ( IFN - β - luc ) or ISRE ( ISRE - luc ) , an expression vector for RKIP ( 0 , 50 , 100 , 200 ng each ) , together with plasmids encoding RIG - I CARD , TBK1 , IKKi IRF3 5D .

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F. Luciferase activity in 293T cells transfected with plasmid encoding a luciferase reporter for IFN-β (IFN-β-luc) or ISRE (ISRE-luc), and an expression vector for RKIP (0, 50, 100, 200 ng each), together with plasmids encoding RIG-I CARD, TBK1, IKKi and IRF3 5D.


A . Immunoblot of extracts of RAW264 . 7 stably overexpressing flag - RKIP infected with VSV ( MOI , 0 . 1 ) for indicated times followed by immunoprecipitation ( IP ) . WCL , whole lysates .

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A. Immunoblot of extracts of RAW264.7 cells stably overexpressing flag-RKIP infected with VSV (MOI, 0.1) for the indicated times followed by immunoprecipitation (IP). WCL, whole cell lysates.


B . Coimmunoprecipitation immunoblot of 293T transfected with vector for myc - RKIP alone or with deletion mutants of TBK1 . Numbers in parentheses indicate included in construct . FL , full ; KD , kinase domain ; ULD , ubiquitin - like domain ; SDD , α - helical scaffold domain ; NBD , C - terminal domain . * indicates targeted band .

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B. Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with vector for myc-RKIP alone or with the deletion mutants of TBK1. Numbers in parentheses indicate amino acids included in construct. FL, full length; KD, kinase domain; ULD, ubiquitin-like domain; SDD, α-helical scaffold domain; NBD, C-terminal domain.* indicates targeted band.


C . Immunoblot of 293T transfected with vector for myc - TBK1 together with plasmid for HA - GSK3β or HA - RKIP , followed by immunoprecipitation . * indicates targeted band .

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C. Immunoblot of 293T cells transfected with vector for myc-TBK1 together with plasmid for HA-GSK3β or HA-RKIP, followed by immunoprecipitation. * indicates targeted band.


D . In vitro kinase was carried out in reaction mixture containing 2 mM ATP , 100 μM GST - RKIP or 100 μM GST - GFP 5 μM flag - TBK1 at 30 ° C for 30 min then followed by immunoblotting with indicated Antibody .

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D. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP and 5 μM flag-TBK1 at 30 °C for 30 min and then followed by immunoblotting with the indicated Antibody.


A . Real - time PCR of IL - 6 , TNF - α IFN - β mRNA in RAW264 . 7 stably overexpressing flag - RKIP , flag - S109A flag - S109D , followed by infection with VSV ( MOI , 0 . 1 ) .

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A. Real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, followed by infection with VSV (MOI, 0.1).


B . Immunoblot of p - TBK1 , - IRF3 , - P38 of as in ( A ) .

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B. Immunoblot analysis of p- TBK1, -IRF3, -P38 of cells as in (A).


C . FACS of RAW264 . 7 stably overexpressing flag - RKIP , flag - S109A flag - S109D infected with VSV - eGFP at MOI of 1 .

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C. FACS analysis of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D infected with VSV-eGFP at MOI of 1.


D . Immunoblot of phosphorylated - TBK1 , - IRF3 , - P38 in lysates of RKIP + / + or RKIP - / - bone marrow derived macrophages ( BMDM ) transfected with flag - RKIP , flag - S109A flag - S109D , then stimulated with VSV ( MOI , 0 . 1 ) .

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D. Immunoblot analysis of phosphorylated-TBK1, -IRF3, -P38 in lysates of RKIP+/+ or RKIP-/- bone marrow derived macrophages (BMDM) transfected with flag-RKIP, flag-S109A and flag-S109D, and then stimulated with VSV (MOI, 0.1).


E . ELISA of IFN - β TNF - α of in ( D ) .

E. ELISA of IFN-β and TNF-α of cells in (D).


A . BMDM were infected with VSV ( MOI , 0 . 1 ) or HSV ( MOI , 5 ) for indicated times . lysates were analyzed by immunoblotting with indicated antibodies .

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A. BMDM cells were infected with VSV (MOI, 0.1) or HSV (MOI, 5) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies.


B . BMDM were transfected with control or TBK1 siRNA . 72 hours after transfection , were analyzed by immunoblotting with antibody for phospho - RKIP or TBK1 .

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B. BMDM cells were transfected with control or TBK1 siRNA. 72 hours after transfection, cells were analyzed by immunoblotting with antibody for phospho-RKIP or TBK1.


C . Immunoblot of RKIP phosphorylation in control or TBK1 - / - mouse embryonic fibroblasts after VSV ( MOI , 0 . 1 ) infection for indicated times .

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C. Immunoblot analysis of RKIP phosphorylation in control or TBK1-/- mouse embryonic fibroblasts after VSV (MOI, 0.1) infection for the indicated times.


D . Recombinant flag - tagged TBK1 was immunoprecipitated with anti - flag M2 beads in flag - TBK1 transfected 293T . Followed by in vitro kinase , which was carried out in reaction mixture containing 2 mM ATP , 100 μM GST - RKIP or 100 μM GST - GFP together with 5 μM flag - TBK1 at 30 ° C for 30 min . mixtures were immunoblotted with indicated antibody for phospho - RKIP , flag , GST or his .

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D. Recombinant flag-tagged TBK1 was immunoprecipitated with anti-flag M2 beads in flag-TBK1 transfected 293T cells. Followed by in vitro kinase assay, which was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP together with 5 μM flag-TBK1 at 30 °C for 30 min. Assay mixtures were immunoblotted with the indicated antibody for phospho-RKIP, flag, GST or his.


E . Immunoblot of extracts of RAW264 . 7 stably overexpressing flag - RKIP , flag - S109A flag - S109D , infected with VSV ( MOI , 0 . 1 ) for indicated times , followed by immunoprecipitation with anti - flag beads .

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E. Immunoblot analysis of extracts of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, infected with VSV (MOI, 0.1) for the indicated times, followed by immunoprecipitation with anti-flag beads.


F . BMDM were infected with VSV ( MOI , 0 . 1 ) for indicated times . lysates were immunoprecipitated with anti - TBK1 ( left ) or anti - RKIP ( right ) . immunoprecipitates were analyzed by immunoblot with anti - RKIP or anti - TBK1 antibody . of endogenous RKIP , TBK1 , p - RKIP were detected by immunoblot .

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F. BMDM cells were infected with VSV (MOI, 0.1) for the indicated times. The cell lysates were immunoprecipitated with anti-TBK1 (left) or anti-RKIP (right). The immunoprecipitates were analyzed by immunoblot with anti-RKIP or anti-TBK1 antibody. The levels of the endogenous RKIP, TBK1, and p-RKIP were detected by immunoblot analysis.


G . Co - immunoprecipitation immunoblot of 293T transfected with myc - TBK1 alone or with flag - RKIP , flag - S109A or flag - S109D .

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G. Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with myc-TBK1 alone or with flag-RKIP, flag-S109A or flag-S109D.


H . GST - pull down experiment was performed with 1 μg fusion GST - GFP , GST - his - RKIP or GST - his - S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium , followed by adding 1 μg flag - TBK1 incubating at 4 ° C for 3 hours with gentle rotation . Pull down ( 1st - 3th lane ) input samples ( 4th lane ) were separated by SDS - PAGE followed by immunoblotting with anti - GST or anti - flag antibody .

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H. GST-pull down experiment was performed with 1 μg fusion protein GST-GFP, GST-his-RKIP or GST-his-S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium, followed by adding 1 μg flag-TBK1 and incubating at 4°C for 3 hours with gentle rotation. Pull down (1st-3th lane) and input samples (4th lane) were separated by SDS-PAGE followed by immunoblotting with anti-GST or anti-flag antibody.


I . Immunoprecipitation immunoblot of 293T transfected with indicated combinations of vector for myc - TBK1 , flag - RKIP , flag - S109A , flag - S109D HA - GSK3β or flag - GFP .

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I. Immunoprecipitation and immunoblot analysis of 293T cells transfected with indicated combinations of vector for myc-TBK1, flag-RKIP, flag-S109A, flag-S109D and HA-GSK3β or flag-GFP.


J . In vitro kinase was carried out in reaction mixture containing 2 mM ATP , 50 μM GST - TBK1 together with 100 μM GST - his - RKIP , 100 μM GST - his - S109D , 100 μM GST - his - S109A or 100 μM GST , respectively . React at 30 ° C for 30 min . Then followed by immunoblotting with anti - p - TBK1or anti - GST antibody .

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J. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 50 μM GST-TBK1 together with 100 μM GST-his-RKIP, 100 μM GST-his-S109D, 100 μM GST-his-S109A or 100 μM GST, respectively. React at 30 °C for 30 min. Then followed by immunoblotting with anti-p-TBK1or anti-GST antibody.


( D ) Immunostaining of PGCLCs d5 cryosections for SOX2 ( magenta ) TFAP2C ( cyan ) . Barplot indicates of SOX2 + , TFAP2C + , SOX2 + / TFAP2C + . n = 1150 , from n = 3 separate inductions , using two biological clones . squares represent position of magnified region at bottom . Scale bar , 50 µm 10 µm for magnified region .

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(D) Immunostaining of PGCLCs d5 cryosections for SOX2 (magenta) and TFAP2C (cyan). Barplot indicates the quantification of SOX2+ cells, TFAP2C+ cells, and SOX2+/TFAP2C+. n=1150 cells, from n=3 separate inductions, using two biological clones. The white squares represent the position of the magnified region at the bottom. Scale bar, 50 µm and 10 µm for the magnified region.


( C ) MA plot of differential gene expression changes between XGFP - XGFP + PGCLCs as determined by - seq . Log2 - mean expression ( log2 - normalised from DESeq2 ) on x - axis log2 - fold change on y - axis are shown . Significantly upregulated downregulated genes are highlighted in red green respectively . False Discovery Rate ( FDR ) < 0 . 001 . Non - significant genes with log2 - mean expression between 0 0 . 2 were removed for easier plot visualisation .

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(C) MA plot of differential gene expression changes between XGFP- and XGFP+ PGCLCs as determined by RNA-seq. Log2-mean expression (log2-normalised counts from DESeq2) on the x-axis and the log2-fold change on the y-axis are shown. Significantly upregulated and downregulated genes are highlighted in red and green respectively. False Discovery Rate (FDR) < 0.001. Non-significant genes with log2-mean expression between 0 and 0.2 were removed for easier plot visualisation.


( D ) Expression ( normalised DEseq2 ) of selected differentially expressed genes between XGFP - XGFP + PGCLCs during differentiation time course . Genes with FDR < 0 . 001 were considered significantly differentially expressed . Points indicate expression of biological replicates .

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(D) Expression levels (normalised DEseq2 counts) of selected differentially expressed genes between XGFP- and XGFP+ PGCLCs during the differentiation time course. Genes with FDR < 0.001 were considered significantly differentially expressed. Points indicate expression of individual biological replicates.


( E ) of - seq normalised showing Zscore across PGCLC induction timepoints of 31 manually selected manually ordered marker genes belonging to categories reported on side .

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(E) Heatmap of RNA-seq normalised counts showing the Zscore across PGCLC induction timepoints of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side.


( G ) FACS of cycle using DAPI . Numbers indicate of in G1 , S G2 / M respectively .

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(G) FACS analysis of cell cycle using DAPI. Numbers indicate the percentage of cells in G1, S and G2/M respectively.


( H ) Alkaline phosphatase for ESCs , XGFP + PGCLCs XGFP - PGCLCs grown for 7 days in 2i / LIF medium on immortalised mouse embryonic fibroblasts . ( I ) Barplot indicating absolute numbers of Alkaline Phosphatase ( AP ) positive colonies in each type after 7 days of culture in 2i / LIF medium on immortalised mouse embryonic fibroblasts . Y - axis is in square root scale ( sqrt ) for better plot visualisation . Each dot represents one technical replicate .

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(H) Alkaline phosphatase staining for ESCs, XGFP+ PGCLCs and XGFP- PGCLCs grown for 7 days in 2i/LIF medium on immortalised mouse embryonic fibroblasts. (I) Barplot indicating the absolute numbers of Alkaline Phosphatase (AP) positive colonies in each cell type after 7 days of culture in 2i/LIF medium on immortalised mouse embryonic fibroblasts. Y-axis is in square root scale (sqrt) for better plot visualisation. Each white dot represents one technical replicate.


( H ) Immunofluorescence of SYCP3 ( red ) , DAZL ( yellow ) DAPI at agg11 of IVDi tissue maturation from XGFP - PGCLCs . IVDi = in vitro differentiation . squares indicate positions of magnified section shown below . Top panel scale bar = 100 μm . Middle panel scale bar = 10 μm . Bottom panel scale bar = 50 μm . ( I ) of SYCP3 + ( oocytes in cyst follicles ) in IVDi tissues at agg11 . Each dot represents one IVDi tissue performed in 3 biological replicates .

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(H) Immunofluorescence images of SYCP3 (red), DAZL (yellow) and DAPI at agg11 of IVDi tissue maturation from XGFP- PGCLCs. IVDi = in vitro differentiation. White squares indicate the positions of the magnified section shown below. Top panel scale bar = 100 μm. Middle panel scale bar = 10 μm. Bottom panel scale bar = 50 μm. (I) Quantification of SYCP3+ cells (oocytes in cyst and primary follicles) in IVDi tissues at agg11. Each dot represents one IVDi tissue performed in 3 biological replicates.


( A ) Schematic representation of m220 stromal feeders expansion culture to compare meiotic capacity of XGFP - XGFP + PGCLCs . Meiosis is induced via addition of Retinoic Acid ( RA ) Bone Morphogenetic 2 ( BMP2 ) . c0 = starting day of culture , c9 = culture day 9 last day of culture . ( B ) Representative for expression of XGFP ( green ) SYCP3 ( red ) in germ at c5 , c7 c9 from XGFP + XGFP - PGCLCs . with SYCP3 signal irrespective of localization pattern or were scored as SYCP3 + . were counterstained with DAPI ( grey ) . Scale bars = 10 μm . ( C ) Number of SYCP3 + per m220 culture day originating from XGFP + XGFP - PGCLCs . Each dot represents a biological replicate ( n = 3 ) . P - values shown are from a two - sided Mann - Whitney U . n . s . = not significant . ( D ) of XGFP + among SYCP3 + at indicated m220 culture day , originating from XGFP + or XGFP - PGCLCs . Green bars show constitutive GFP expression in XGFP + PGCLC - derived , while striped bars signify XGFP reactivation in XGFP - . Each dot represents a biological replicate ( n = 3 ) .

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(A) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic capacity of XGFP- and XGFP+ PGCLCs. Meiosis is induced via addition of Retinoic Acid (RA) and Bone Morphogenetic Protein 2 (BMP2). c0 = starting day of culture, c9 = culture day 9 and last day of culture. (B) Representative images for the expression of XGFP (green) and SYCP3 (red) in germ cells at c5, c7 and c9 from XGFP+ and XGFP- PGCLCs. All cells with SYCP3 signal irrespective of localization pattern or intensity were scored as SYCP3+ cells. Cells were counterstained with DAPI (grey). Scale bars = 10 μm. (C) Number of SYCP3+ cells per m220 culture day originating from XGFP+ and XGFP- PGCLCs. Each white dot represents a biological replicate (n = 3). P-values shown are from a two-sided Mann-Whitney U test. n.s. = not significant. (D) Percentage of XGFP+ cells among SYCP3+ cells at the indicated m220 culture day, originating from XGFP+ or XGFP- PGCLCs. Green bars show constitutive GFP expression in XGFP+ PGCLC-derived cells, while striped bars signify XGFP reactivation in XGFP- cells. Each white dot represents a biological replicate (n = 3).


( E ) Representative showing stages of meiotic prophase I from culture day 9 ( c9 ) germ from XGFP + XGFP - PGCLCs . c9 germ were spread immunostained for SYCP3 ( red ) , γH2AX ( grey ) . Scale bars = 10 µm . ( F ) of meiotic progression in culture day ( c9 ) expanded germ derived from XGFP + XGFP - PGCLCs . graphs show percentages of meiotic stage for SYCP3 + . Lepto , leptotene ; Zygo , zygotene ; Pachy , pachytene ; Diplo , diplotene . Numbers indicate detected per meiotic stage original XGFP status .

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(E) Representative images showing stages of meiotic prophase I from culture day 9 (c9) germ cells from XGFP+ and XGFP- PGCLCs. c9 germ cells were spread and immunostained for SYCP3 (red), and γH2AX (grey). Scale bars = 10 µm. (F) Quantification of meiotic progression in culture day (c9) expanded germ cells derived from XGFP+ and XGFP- PGCLCs. The graphs show the percentages of the meiotic stage for SYCP3+ cells. Lepto, leptotene; Zygo, zygotene; Pachy, pachytene; Diplo, diplotene. Numbers indicate detected cells per meiotic stage and original XGFP status.


( G ) Representative tile scan merged of XGFP ( green ) Xtomato ( red ) in germ at c7 from XGFP + XGFP - PGCLCs . were counterstained with DAPI ( grey ) . Scale Bar = 1 mm . squares contain a magnified image of region depicted by dotted squares ( Scale Bar = 1 µm ) . ( H ) Density ridge plot showing of in ( G ) measured using XTomato signal . X - Axis shows in µm2 scaled as log2 . Dashed represents mean . XFGP - mean = 1320 µm2 , XGFP + mean = 5514 µm2 . XGFP - n = 297 , XGFP + n = 373 . P - value from two - sample unpaired Wilcoxon - Mann - Whitney with R defaults .

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(G) Representative tile scan merged images of XGFP (green) and Xtomato (red) in germ cells at c7 from XGFP+ and XGFP- PGCLCs. Cells were counterstained with DAPI (grey). Scale Bar = 1 mm. The white squares contain a magnified image of the region depicted by the white dotted squares (Scale Bar = 1 µm). (H) Density ridge plot showing size of cellular aggregates in (G) measured using the XTomato signal. X-Axis shows area in µm2 scaled as log2. Dashed line represents the mean. XFGP- mean = 1320 µm2, XGFP+ mean = 5514 µm2. XGFP- n = 297 aggregates, XGFP+ n = 373 aggregates. P-value from two-sample unpaired Wilcoxon-Mann-Whitney test with R defaults.


( B ) EC50 values ( 95 % confidence interval ) for VHH - B9 , VHH - 10C4 VHH - 4A2 are 99 . 2 nM ( 83 . 8 - 117 . 5 nM ) , 112 . 5 nM ( 101 . 1 - 125 . 2 nM ) 788 . 7 nM ( 682 . 7 - 911 . 2 nM ) , respectively . Values are mean ± S . D . , n = 3 , technical replicates .

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(B) The EC50 values (95% confidence interval) for VHH-B9, VHH-10C4 and VHH-4A2 are 99.2 nM (83.8-117.5 nM), 112.5 nM (101.1-125.2 nM) and 788.7 nM (682.7-911.2 nM), respectively. Values are mean ± S.D., n=3, technical replicates.


( C , D ) cultured neurons were transduced by Semliki Forest ( SFV ) expressing wild type human APP695 treated with 3 µM of indicated VHHs . PBS anti - GFP VHH were used as controls . ( C ) Western blot of conditioned media for sAPP ⍺ sAPPβ , as well as extracts for full APP , CTF ⍺ CTFβ . ( D ) ELISA measurements of Aβ1 - 40 Aβ1 - 42 in conditioned media . Values are mean ± S . E . M . , n = 3 cultures for each . One - way ANOVA , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 0001 .

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(C, D) Primary cultured neurons were transduced by Semliki Forest virus (SFV) expressing wild type human APP695 and treated with 3 µM of the indicated VHHs. PBS and anti-GFP VHH were used as controls. (C) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (D) ELISA measurements of Aβ1-40 and Aβ1-42 in conditioned media. Values are mean ± S.E.M., n=3 cultures for each analysis. One-way ANOVA, *p<0.5, **p<0.01, *** p<0.0001.


( E , F ) Dose - dependent inhibition of BACE1 in cultured neurons by VHH - B9 . Cultured neurons were transduced by SFV expressing wild type human APP695 treated with PBS ( control : CT ) or decreasing concentrations of VHH - B9 , ranging from 10 µM to 0 . 7 nM . ( E ) Western blot of conditioned media for sAPP ⍺ sAPPβ , as well as extracts for full APP , CTF ⍺ CTFβ . ( F ) Conditioned media was analyzed by ELISA to assess of Aβ1 - 40 . Values are mean ± S . D . , n = 3 cultures for each . EC50 value ( 95 % confidence interval ) was estimated as 85 . 4 nM ( 52 . 5 - 125 . 6 nM ) .

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(E, F) Dose-dependent inhibition of BACE1 in primary cultured neurons by VHH-B9. Cultured neurons were transduced by SFV expressing wild type human APP695 and treated with PBS (control: CT) or decreasing concentrations of VHH-B9, ranging from 10 µM to 0.7 nM. (E) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (F) Conditioned media was analyzed by ELISA to assess levels of Aβ1-40. Values are mean ± S.D., n=3 cultures for each analysis. The EC50 value (95 % confidence interval) was estimated as 85.4 nM (52.5-125.6 nM).


( A ) Co - with anti - N - Terminal Aβ 82E1 ( green ) Thioflavin - S ( magenta ) reveals Aβ plaques ( arrow heads ) in regions of interest . Representative are shown . Scale bar : 50 μm . ( B , C ) number of Aβ plaques in regions of interest was significantly reduced by treatment AAV - VHH - B9 ( B ) , as was total plaque ( C ) . (

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(A) Co-staining with anti-N-Terminal Aβ 82E1 (green) and Thioflavin-S (magenta) reveals Aβ plaques (arrow heads) in regions of interest. Representative images are shown. Scale bar: 50 μm. (B, C) The number of Aβ plaques in regions of interest was significantly reduced by treatment AAV-VHH-B9 (B), as was total plaque area (C). (


( A ) ( left ) Representative of anti - GFAP immunostaining in Prefrontal Cortex Amygdala from AppNL - G - F mice receiving either AAV - VHH - B9 or AAV - GFP ; ( right ) of GFAP + number in indicated brain regions across experimental cohorts .

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(A) (left) Representative images of anti-GFAP immunostaining in Prefrontal Cortex and Amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the GFAP+ cell number in the indicated brain regions across experimental cohorts.


( B ) ( left ) Representative of anti - IBA1 immunostaining in dentate gyrus amygdala from AppNL - G - F mice receiving either AAV - VHH - B9 or AAV - GFP ; ( right ) of IBA1 + number in indicated brain regions across experimental cohorts .

(B) (left) Representative images of anti-IBA1 immunostaining in dentate gyrus and amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the IBA1+ cell number in the indicated brain regions across experimental cohorts.


( a ) Δ133p53α expression is induced by cycloheximide ( CHX ) . MRC - 5 fibroblasts ( at population doubling ( PDLs ) 51 ) were treated with CHX ( 25 μg ml − 1 ) for indicated time periods examined in immunoblot for Δ133p53α full - p53 . β - actin was a loading control for normalization . relative expression of Δ133p53α full - p53 are shown below .

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(a) Δ133p53α expression is induced by cycloheximide (CHX). MRC-5 fibroblasts (at population doubling levels (PDLs) 51) were treated with CHX (25 μg ml−1) for indicated time periods and examined in immunoblot for Δ133p53α and full-length p53. β-actin was a loading control for normalization. The relative expression levels of Δ133p53α and full-length p53 are shown below the images.


( b ) Δ133p53α expression is induced by phosphoinositide 3 - kinase inhibitors . MRC - 5 fibroblasts ( PDLs 51 ) were treated for 4 h with LY303511 ( 10 μM ) , LY294002 ( 10 μM ) , Wortmannin ( 50 nM ) or dimethylsulphoxide alone ( as control ) examined in immunoblot for Δ133p53α β - actin expression .

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(b) Δ133p53α expression is induced by phosphoinositide 3-kinase inhibitors. MRC-5 fibroblasts (PDLs 51) were treated for 4 h with LY303511 (10 μM), LY294002 (10 μM), Wortmannin (50 nM) or dimethylsulphoxide alone (as control) and examined in immunoblot for Δ133p53α and β-actin expression.


( c ) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1 . immunoblot analyses were performed in early - passage ( Y ) replicatively senescent ( S ) human fibroblast strains MRC - 5 WI - 38 . examined PDLs were 30 ( Y ) 60 ( S ) for MRC - 5 ; 30 ( Y ) 54 ( S ) for WI - 38 . Bafilomycin A 1 treatment was at 100 nM for 4 h . relative expression of Δ133p53α , full - p53 p62 / SQSTM1 ( normalized with β - actin ) are shown . Anti - LC3B antibody detected LC3 - I LC3 - II , which was used in quantitative shown in d . ( d ) Replicative senescence is not associated with enhanced autophagy in general . LC3 - II expression were normalized with β - actin ( data in c ) are shown in bar graphs as relative values to replicatively senescent without bafilomycin A1 ( S / − ; defined as 1 ) . LC3 - II in replicatively senescent were higher than those in early - passage whether in presence or absence of bafilomycin A1 , which may suggest increased synthesis of autophagosomes at replicative senescence . However , differences in LC3 - II between presence absence of bafilomycin A1 were similar in early - passage replicatively senescent : in MRC - 5 , 1 . 9 for Y ( 2 . 1 − 0 . 2 ) 2 . 0 for S ( 3 . 0 − 1 ) ; in WI - 38 , 1 . 6 for Y ( 2 . 2 − 0 . 6 ) 1 . 6 for S ( 2 . 6 − 1 ) . These data indicate that early - passage replicatively senescent have similar rates of autophagic flux , that is , number of autophagosomes that are delivered to lysosomes for degradation .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1. The immunoblot analyses were performed in early-passage (Y) and replicatively senescent (S) human fibroblast strains MRC-5 and WI-38. The examined PDLs were 30 (Y) and 60 (S) for MRC-5; and 30 (Y) and 54 (S) for WI-38. Bafilomycin A 1 treatment was at 100 nM for 4 h. The relative expression levels of Δ133p53α, full-length p53 and p62/SQSTM1 (normalized with β-actin) are shown. Anti-LC3B antibody detected LC3-I and LC3-II, which was used in quantitative analysis shown in d. (d) Replicative senescence is not associated with enhanced autophagy in general. LC3-II expression levels were normalized with β-actin (data in c) and are shown in the bar graphs as relative values to replicatively senescent cells without bafilomycin A1 (S/−; defined as 1). The LC3-II levels in replicatively senescent cells were higher than those in early-passage cells whether in the presence or absence of bafilomycin A1, which may suggest increased synthesis of autophagosomes at replicative senescence. However, the differences in LC3-II levels between the presence and absence of bafilomycin A1 were similar in early-passage and replicatively senescent cells: in MRC-5, 1.9 for Y (2.1−0.2) and 2.0 for S (3.0−1); in WI-38, 1.6 for Y (2.2−0.6) and 1.6 for S (2.6−1). These data indicate that early-passage and replicatively senescent cells have similar rates of autophagic flux, that is, the number of autophagosomes that are delivered to lysosomes for degradation.


( a - c ) Knockdown of pro - autophagic restores Δ133p53α expression . MRC - 5 fibroblasts at late - passage ( PDLs 51 ) were transfected with three independent siRNA oligonucleotides ( lanes 1 - 3 ) against ATG5 ( a ) , ATG7 ( b ) Beclin - 1 ( c ) , along with their control siRNA ( lane C ) , for 4 days . Knockdown of ATG5 was confirmed by loss of ATG5 - ATG12 conjugate ( detected by anti - ATG5 antibody ) . Knockdown of other two was confirmed using corresponding antibodies . Δ133p53α , full - p53 LC3B ( LC3 - I LC3 - II ) were examined in samples . relative expression of Δ133p53α full - p53 ( normalized with β - actin ) are shown below .

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

(a-c) Knockdown of pro-autophagic proteins restores Δ133p53α expression. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with three independent siRNA oligonucleotides (lanes 1-3) against ATG5 (a), ATG7 (b) and Beclin-1 (c), along with their control siRNA (lane C), for 4 days. Knockdown of ATG5 was confirmed by the loss of ATG5-ATG12 conjugate (detected by anti-ATG5 antibody). Knockdown of the other two proteins was confirmed using the corresponding antibodies. Δ133p53α, full-length p53 and LC3B (LC3-I and LC3-II) were examined in all samples. The relative expression levels of Δ133p53α and full-length p53 (normalized with β-actin) are shown below the images.


( d ) Knockdown of pro - autophagic treatment with bafilomycin A1 show similar effects on Δ133p53α p62 / SQSTM1 . MRC - 5 fibroblasts at late - passage ( PDLs 51 ) were transfected with control siRNA ( c ) , ATG5 siRNA ( A5 , no . 1 in a ) , ATG7 siRNA ( A7 , no . 1 in b ) Beclin - 1 siRNA ( B1 , no . 2 in c ) as in a - c , untreated ( − ) or treated with bafilomycin A1 ( + , 100 nM for 4 h ) , examined in immunoblots for indicated . relative expression of Δ133p53α p62 / SQSTM1 ( normalized with β - actin ) are shown below .

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(d) Knockdown of pro-autophagic proteins and treatment with bafilomycin A1 show similar effects on Δ133p53α and p62/SQSTM1. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with control siRNA (c), ATG5 siRNA (A5, no. 1 in a), ATG7 siRNA (A7, no. 1 in b) and Beclin-1 siRNA (B1, no. 2 in c) as in a-c, untreated (−) or treated with bafilomycin A1 (+, 100 nM for 4 h), and examined in immunoblots for indicated proteins. The relative expression levels of Δ133p53α and p62/SQSTM1 (normalized with β-actin) are shown below the images.


( a ) Endogenous STUB1 is downregulated at replicative senescence . Immunoblot was performed using early - passage ( Y ) replicatively senescent ( S ) fibroblasts ( same set of without treatment as in Fig . 1c ) .

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(a) Endogenous STUB1 protein is downregulated at replicative senescence. Immunoblot was performed using early-passage (Y) and replicatively senescent (S) fibroblasts (the same set of cells without treatment as in Fig. 1c).


( b ) STUB1 mRNA is downregulated at replicative senescence . same set of as in a were examined by qRT - PCR . Data were from triplicate experiments ( mean ± s . d . ) . * P0 . 01 ; * * P0 . 001 ( Student ' s t - ) .

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(b) STUB1 mRNA is downregulated at replicative senescence. The same set of cells as in a were examined by qRT-PCR. Data were from triplicate experiments (mean±s.d.). *P0.01; **P0.001 (Student's t-test).


( c ) Knockdown of STUB1 represses Δ133p53α . MRC - 5 fibroblasts ( 30 PDLs ) were transfected with two siRNAs against STUB1 ( no . 1 no . 2 ) control siRNA . At 4 days after transfection , immunoblots were performed using anti - STUB1 , anti - Δ133p53α , DO - 1 ( detecting full - p53 ) , CM1 ( detecting both Δ133p53α full - p53 ) , anti - p62 / SQSTM1 anti - β - actin antibodies .

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(c) Knockdown of STUB1 represses Δ133p53α. MRC-5 fibroblasts (30 PDLs) were transfected with two siRNAs against STUB1 (no. 1 and no. 2) and control siRNA. At 4 days after transfection, immunoblots were performed using anti-STUB1, anti-Δ133p53α, DO-1 (detecting full-length p53), CM1 (detecting both Δ133p53α and full-length p53), anti-p62/SQSTM1 and anti-β-actin antibodies.


( d ) STUB1 knockdown inhibits proliferation . MRC - 5 fibroblasts ( 30 PDLs ) were seeded on six - well plates on day 0 ( 4 × 104 per well in 6 - well plate ; nine wells for each siRNA ) . were counted on days 1 , 4 7 ( three wells each ; mean ± s . d . ) , while siRNA transfection was performed on days 1 4 . * P0 . 01 ( Student ' s t - ) .

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(d) STUB1 knockdown inhibits cell proliferation. MRC-5 fibroblasts (30 PDLs) were seeded on six-well plates on day 0 (4 × 104 cells per well in 6-well plate; nine wells for each siRNA). Cells were counted on days 1, 4 and 7 (three wells each; mean±s.d.), while siRNA transfection was performed on days 1 and 4. *P0.01 (Student's t-test).


( e ) STUB1 knockdown induces senescence . siRNA transfection was performed as in c repeated 4 days later , followed by incubation for 3 days SA - β - Gal . Percentages of SA - β - Gal - positive were from triplicate experiments ( mean ± s . d . ) . * * P0 . 001 ( Student ' s t - ) .

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(e) STUB1 knockdown induces cellular senescence. The siRNA transfection was performed as in c and repeated 4 days later, followed by incubation for 3 days and SA-β-Gal staining. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).


( f , g ) STUB1 knockdown upregulates p21WAF1 IL - 8 . same set of as in e were examined in qRT - PCR for p21WAF1 mRNA ( f ) IL - 8 mRNA ( g ) . Data were from triplicate experiments ( mean ± s . d . ) . * * P0 . 001 ( Student ' s t - ) .

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(f,g) STUB1 knockdown upregulates p21WAF1 and IL-8. The same set of cells as in e were examined in qRT-PCR for p21WAF1 mRNA (f) and IL-8 mRNA (g). Data were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).


( h ) Δ133p53α rescues STUB1 knockdown - induced senescence . MRC - 5 fibroblasts were transfected with STUB1 siRNA ( no . 1 ) as in e . At next day after second transfection ( on day 5 ) , were transduced with a lentiviral vector driving Δ133p53α or its vector control . At 5 days after lentiviral transduction , were stained for SA - β - Gal activity . Representative pictures ( left ) quantitative data ( right ) are shown . Percentages of SA - β - Gal - positive were from triplicate experiments ( mean ± s . d . ) . * * P0 . 001 ( Student ' s t - ) .

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(h) Δ133p53α rescues STUB1 knockdown-induced senescence. MRC-5 fibroblasts were transfected with STUB1 siRNA (no. 1) as in e. At the next day after the second transfection (on day 5), the cells were transduced with a lentiviral vector driving Δ133p53α or its vector control. At 5 days after the lentiviral transduction, the cells were stained for SA-β-Gal activity. Representative pictures (left) and quantitative data (right) are shown. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).


( a ) STUB1 knockdown - induced repression of Δ133p53α is abrogated by bafilomycin A1 treatment . siRNA transfection in MRC - 5 fibroblasts was performed as in Fig . 3e . At 7 days after initial transfection , were untreated , treated with bafilomycin A1 ( 100 nM for 4 h ) or treated with MG - 132 ( 15 μM for 4 h ) , examined in immunoblots as indicated . results of Δ133p53α , full - p53 p62 / SQSTM1 in untreated replicate those shown in Fig . 3c . inhibition of autophagy by bafilomycin A1 was confirmed by increased of p62 / SQSTM1 LC3B . inhibition of proteasomal degradation by MG - 132 was confirmed by increased full - p53 .

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(a) STUB1 knockdown-induced repression of Δ133p53α is abrogated by bafilomycin A1 treatment. The siRNA transfection in MRC-5 fibroblasts was performed as in Fig. 3e. At 7 days after the initial transfection, the cells were untreated, treated with bafilomycin A1 (100 nM for 4 h) or treated with MG-132 (15 μM for 4 h), and examined in immunoblots as indicated. The results of Δ133p53α, full-length p53 and p62/SQSTM1 in untreated cells replicate those shown in Fig. 3c. The inhibition of autophagy by bafilomycin A1 was confirmed by increased levels of p62/SQSTM1 and LC3B. The inhibition of proteasomal degradation by MG-132 was confirmed by increased full-length p53.


( b ) STUB1 knockdown - induced repression of Δ133p53α is abrogated by knockdown of pro - autophagic . MRC - 5 fibroblasts ( PDLs 30 ) were transfected for 4 days with control siRNA ( − ) or STUB1 siRNA ( no . 1 , + ) , in combination with control siRNA ( c ) , p62 / SQSTM1 siRNA ( 62 ) , ATG5 siRNA ( A5 , no . 1 in Fig . 2a ) , ATG7 siRNA ( A7 , no . 1 in Fig . 2b ) or Beclin - 1 siRNA ( B1 , no . 2 in Fig . 2c ) , examined in immunoblots for indicated .

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(b) STUB1 knockdown-induced repression of Δ133p53α is abrogated by knockdown of pro-autophagic proteins. MRC-5 fibroblasts (PDLs 30) were transfected for 4 days with control siRNA (−) or STUB1 siRNA (no. 1, +), in combination with control siRNA (c), p62/SQSTM1 siRNA (62), ATG5 siRNA (A5, no. 1 in Fig. 2a), ATG7 siRNA (A7, no. 1 in Fig. 2b) or Beclin-1 siRNA (B1, no. 2 in Fig. 2c), and examined in immunoblots for indicated proteins.


( c ) Δ133p53α is recruited to autophagosomes upon STUB1 downregulation . MRC - 5 fibroblasts at early passage ( PDLs 30 ; Y ) , those at or close to replicative senescence ( PDLs 58 ; S ( Rep ) ) those induced to senesce by STUB1 knockdown ( S ( KD ) ) were used in immunoprecipitation ( IP ) with anti - STUB1 , anti - Hsp70 anti - LC3B antibodies , followed by immunoblots ( IB ) as indicated . Whole - lysates without IP ( input ) were also examined for Δ133p53α β - actin .

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(c) Δ133p53α is recruited to autophagosomes upon STUB1 downregulation. MRC-5 fibroblasts at early passage (PDLs 30; Y), those at or close to replicative senescence (PDLs 58; S (Rep)) and those induced to senesce by STUB1 knockdown (S (KD)) were used in immunoprecipitation (IP) with anti-STUB1, anti-Hsp70 and anti-LC3B antibodies, followed by immunoblots (IB) as indicated. Whole-protein lysates without IP (input) were also examined for Δ133p53α and β-actin levels.


( d ) Bafilomycin A1 stabilizes LC3B - interacting Δ133p53α in senescent . same set of as in c were treated with bafilomycin A1 ( + , 100 nM for 4 h ) or untreated ( − ) . These were used in IP with anti - LC3B antibody , followed by IB with anti - Δ133p53 antibody . Whole - lysates without IP were also examined in IB with anti - LC3B antibody . While untreated samples replicate result shown in c , differences between early passage senescent become more evident with bafilomycin A1 treatment .

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(d) Bafilomycin A1 stabilizes LC3B-interacting Δ133p53α in senescent cells. The same set of cells as in c were treated with bafilomycin A1 (+, 100 nM for 4 h) or untreated (−). These cells were used in IP with anti-LC3B antibody, followed by IB with anti-Δ133p53 antibody. Whole-protein lysates without IP were also examined in IB with anti-LC3B antibody. While untreated samples replicate the result shown in c, the differences between early passage and senescent cells become more evident with bafilomycin A1 treatment.


( a ) Δ133p53α is polyubiquitinated . FLAG - tagged versions of wild - type Δ133p53α ( wt ) mutant Δ133p53α ( mut ; from c below ) , along with vector control ( − ) , were retrovirally transduced into MRC - 5 fibroblasts . wild - type Δ133p53α - expressing had a pair of STUB1 siRNA - transfected ( + , siRNA no . 1 for 4 days ) untransfected ( − ) counterparts . These were maintained for 8 h under acid - serum - starved conditions with bafilomycin A1 ( 100 nM ) . lysates were used either in IP with anti - ubiquitin ( Ub ) antibody , followed by IB with anti - FLAG antibody ( top ) , or directly in IB for FLAG - Δ133p53α , STUB1 β - actin ( lower three panels ) . Smear signals indicate polyubiquitinated FLAG - Δ133p53α ( Poly - Ub ) . Asterisk indicates a nonspecific band in negative control . These experimental conditions resulted in similar amounts of FLAG - Δ133p53α in presence absence of STUB1 knockdown , allowing quantitative of ubiquitination . relative densitometric values of wild - type FLAG - Δ133p53α polyubiquitination in presence absence of STUB1 knockdown ( normalized with total FLAG - Δ133p53α ) are shown . (

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(a) Δ133p53α protein is polyubiquitinated. FLAG-tagged versions of wild-type Δ133p53α (wt) and mutant Δ133p53α (mut; from c below), along with vector control (−), were retrovirally transduced into MRC-5 fibroblasts. The wild-type Δ133p53α-expressing cells had a pair of STUB1 siRNA-transfected (+, siRNA no. 1 for 4 days) and untransfected (−) counterparts. These cells were maintained for 8 h under amino acid- and serum-starved conditions with bafilomycin A1 (100 nM). Protein lysates were used either in IP with anti-ubiquitin (Ub) antibody, followed by IB with anti-FLAG antibody (top), or directly in IB for FLAG-Δ133p53α, STUB1 and β-actin (lower three panels). Smear signals indicate polyubiquitinated FLAG-Δ133p53α protein (Poly-Ub). Asterisk indicates a nonspecific band in negative control. These experimental conditions resulted in similar amounts of FLAG-Δ133p53α in the presence and absence of STUB1 knockdown, allowing quantitative analysis of ubiquitination. The relative densitometric values of wild-type FLAG-Δ133p53α polyubiquitination in the presence and absence of STUB1 knockdown (normalized with total FLAG-Δ133p53α) are shown. (


( b ) Δ133p53α is ubiquitinated at C - terminal lysine residues . MRC - 5 fibroblasts expressing wild - type FLAG - Δ133p53α in absence of STUB1 knockdown were used in IP with anti - FLAG antibody , resulting immunoprecipitates were analysed using mass spectrometry . Representative MS / MS spectrum of an [ M + 3H ] 3 + peptide from FLAG - Δ133p53α shows sites of ubiquitin modification . peptide for spectrum is shown , with sites of modification marked as asterisks : - acid positions 248 249 , which correspond to positions 381 382 in full - p53 . same peptide was also observed in unmodified state .

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(b) Δ133p53α is ubiquitinated at the C-terminal lysine residues. MRC-5 fibroblasts expressing wild-type FLAG-Δ133p53α in the absence of STUB1 knockdown were used in IP with anti-FLAG antibody, and the resulting immunoprecipitates were analysed using mass spectrometry. Representative MS/MS spectrum of an [M+3H]3+ peptide from FLAG-Δ133p53α shows sites of ubiquitin modification. The peptide sequence for the spectrum is shown, with the sites of modification marked as asterisks: amino-acid positions 248 and 249, which correspond to positions 381 and 382 in full-length p53. The same peptide was also observed in the unmodified state.


( c ) Lysine ( K ) - to - arginine ( R ) substitutions at - acid residues 248 249 .

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(c) Lysine (K)-to-arginine (R) substitutions at amino-acid residues 248 and 249.


( d ) Mutation of ubiquitination sites renders Δ133p53α resistant to degradation . same three as in a were cultured under - acid - serum - starved conditions for 8 h ( without bafilomycin A1 ) , transfected with STUB1 siRNA ( no . 1 for 4 days ) or untreated , examined in IBs for FLAG - Δ133p53α , STUB1 , p62 / SQSTM1 β - actin . effect of starvation was confirmed by decreased p62 / SQSTM1 in each . upregulation of p62 / SQSTM1 by wild - type mutant FLAG - Δ133p53α is likely associated with enhanced proliferation by Δ133p53α overexpression .

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(d) Mutation of the ubiquitination sites renders Δ133p53α resistant to degradation. The same three cells as in a were cultured under amino-acid- and serum-starved conditions for 8 h (without bafilomycin A1), transfected with STUB1 siRNA (no. 1 for 4 days) or untreated, and examined in IBs for FLAG-Δ133p53α, STUB1, p62/SQSTM1 and β-actin. The effect of starvation was confirmed by decreased p62/SQSTM1 in each cell. The upregulation of p62/SQSTM1 by wild-type and mutant FLAG-Δ133p53α is likely associated with enhanced cell proliferation by Δ133p53α overexpression.


( a ) Δ133p53α is colocalized with p62 / SQSTM1 upon autophagy induction . MRC - 5 fibroblasts expressing FLAG - Δ133p53α ( wild - type ) were cultured under - acid - serum - starved conditions for 4 h , co - immunostained with anti - FLAG antibody ( Δ133p53α , green ) anti - p62 / SQSTM1 antibody ( red ) . Two representative sets of , including with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) merged , are shown . Scale bars , 10 μm .

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(a) Δ133p53α is colocalized with p62/SQSTM1 upon autophagy induction. MRC-5 fibroblasts expressing FLAG-Δ133p53α (wild-type) were cultured under amino-acid- and serum-starved conditions for 4 h, and co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-p62/SQSTM1 antibody (red). Two representative sets of images, including nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and merged images, are shown. Scale bars, 10 μm.


( b ) Knockdown of p62 / SQSTM1 stabilizes endogenous Δ133p53α . MRC - 5 fibroblasts at late passage ( PDLs 51 ) were transfected with p62 / SQSTM1 siRNA ( p62 ) or control siRNA ( C ) for 3 days , examined in immunoblots for Δ133p53α p62 / SQSTM1 expression . relative expression of Δ133p53α ( normalized with β - actin ) are shown .

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(b) Knockdown of p62/SQSTM1 stabilizes endogenous Δ133p53α. MRC-5 fibroblasts at late passage (PDLs 51) were transfected with p62/SQSTM1 siRNA (p62) or control siRNA (C) for 3 days, and examined in immunoblots for Δ133p53α and p62/SQSTM1 expression. The relative expression levels of Δ133p53α (normalized with β-actin) are shown.


( c ) Δ133p53α is colocalized with LC3B upon autophagy induction . MRC - 5 fibroblasts with FLAG - Δ133p53α under starved conditions ( as in a ) were co - immunostained with anti - FLAG antibody ( Δ133p53α , green ) anti - LC3B antibody ( red ) . Two representative sets of , including DAPI merged , are shown . Scale bars , 10 μm . ( d ) Quantitative of Δ133p53α - LC3B colocalization . number of colocalized signals out of total number of Δ133p53α signals or out of total number of LC3B signals was counted in 26 . Data ( colocalization ) are mean ± s . d . Almost Δ133p53α signals were colocalized with LC3B , while ~ 80 % of LC3B signals were colocalized with Δ133p53α .

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(c) Δ133p53α is colocalized with LC3B upon autophagy induction. MRC-5 fibroblasts with FLAG-Δ133p53α under starved conditions (as in a) were co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-LC3B antibody (red). Two representative sets of images, including DAPI and merged images, are shown. Scale bars, 10 μm. (d) Quantitative analysis of Δ133p53α-LC3B colocalization. The number of colocalized signals out of total number of Δ133p53α signals or out of total number of LC3B signals was counted in 26 cells. Data (percent colocalization) are mean±s.d. Almost all Δ133p53α signals were colocalized with LC3B, while ~80% of LC3B signals were colocalized with Δ133p53α.


( A ) β - Catenin is hyperphosphorylated during mitotic phase . HeLa CCL2 were synchronized at G1 / S phase using a double thymidine block released using medium containing 200 ng / mL nocodazole ( Noc . ) . were harvested at indicated time points treated with ( + ) or without ( - ) λ - phosphatase ( λ - PPase ) . resulting lysates were used for immunoblot . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE . Asterisk indicates phosphorylated form of Cdc25C .

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(A) β-Catenin is hyperphosphorylated during the mitotic phase. HeLa CCL2 cells were synchronized at the G1/S phase using a double thymidine block and released using medium containing 200 ng/mL nocodazole (Noc.). Cells were harvested at the indicated time points and treated with (+) or without (-) λ-phosphatase (λ-PPase). The resulting cell lysates were used for immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. Asterisk indicates the phosphorylated form of Cdc25C.


( F ) Total lysates from nocodazole ( Noc . ) - treated HeLa CCL2 were immunoprecipitated with two different Plk1 antibodies . Each antibody ( gifted from Kyung S . Lee , NIH / NCI ) recognized either Plk1 N - terminus ( N - term . Ab . ) or C - terminus ( C - term . Ab . ) . resulting precipitates were subjected to immunoblot . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE .

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(F) Total cell lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were immunoprecipitated with two different Plk1 antibodies. Each antibody (gifted from Kyung S. Lee, NIH/NCI) recognized either Plk1 N-terminus (N-term. Ab.) or C-terminus (C-term. Ab.). The resulting precipitates were subjected to immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE.


( G ) Lysates from nocodazole ( Noc . ) - treated HeLa CCL2 were mixed with either control ( phosphate - buffered saline only , + PBS ) , 10 μg / ml of high - affinity Plk1 PBD binding phospho - peptide ( PLHSpT , + P ) , or 10 μg / ml of non - phospho - peptide ( PLHST , + NP ) . resulting lysates were immunoprecipitated with anti - Plk1 antibody . Precipitates were subjected to immunoblot . Immunoblotting with anti - Dvl2 antibody represents a positive control of phospho - peptide ( PLHSpT , + P ) competition for Plk1 binding . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE .

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(G) Lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were mixed with either control (phosphate-buffered saline only, +PBS), 10 μg/ml of high-affinity Plk1 PBD binding phospho-peptide (PLHSpT, +P), or 10 μg/ml of non-phospho-peptide (PLHST, +NP). The resulting cell lysates were immunoprecipitated with anti-Plk1 antibody. Precipitates were subjected to immunoblot analysis. Immunoblotting with anti-Dvl2 antibody represents a positive control of phospho-peptide (PLHSpT, +P) competition for Plk1 binding. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE.


( C ) Bacterially produced β - catenin T1 WT , T3 WT , corresponding alanine ( " A " ) mutants were purified reacted with Flag - Plk1 WT in presence of [ γ - 32P ] ATP . Incorporation of 32P onto each " A " mutant of β - catenin was confirmed by SDS - PAGE followed by autoradiography .

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(C) Bacterially produced β-catenin T1 WT, T3 WT, and corresponding alanine ("A") mutants were purified and reacted with Flag-Plk1 WT in the presence of [γ-32P]ATP. Incorporation of 32P onto each "A" mutant of β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis.


( H ) HeLa CCL2 were treated for 18 h with 200 ng / mL nocodazole followed by treatment with either DMSO ( control ) or BI 2536 for an additional 1 h . resulting were lysed with 2 × Laemmli SDS sample buffer used for immunoblot with indicated antibodies .

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(H) HeLa CCL2 cells were treated for 18 h with 200 ng/mL nocodazole followed by treatment with either DMSO (control) or BI 2536 for an additional 1 h. The resulting cells were lysed with 2× Laemmli SDS sample buffer and used for immunoblot analysis with the indicated antibodies.


( C ) Asynchronously growing HeLa CCL2 were immunostained with indicated antibodies as described in ( A ) , midbody localization of β - catenin / β - catenin p - S60 in late telophase was observed using a confocal microscope ( Zeiss LSM 700 ) . Scale bars , 10 μm .

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(C) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies as described in (A), and the midbody localization of β-catenin/β-catenin p-S60 in late telophase cells was observed using a confocal laser microscope (Zeiss LSM 700). Scale bars, 10 μm.


( A ) Asynchronously growing HeLa CCL2 were immunostained with indicated antibodies , early telophase were observed using a super - resolution confocal scanning microscope ( LSM - 880 with Airyscan ) ( left panel ) . Scale bars , 10 μm . Linear signal profiles of β - catenin / Plk1 / β - catenin p - S60 / F - actin in contractile ring region from XY plane of immunostained early telophase ( right panel ) . immunofluorescence linear signal profiles show representative data of more than 50 telophase obtained from three independent experiments . value of relative neck thickness is shown in lower left corner of merged ( A ) .

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(A) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies, and early telophase cells were observed using a super-resolution confocal laser scanning microscope (LSM-880 with Airyscan) (left panel). Scale bars, 10 μm. Linear signal profiles of β-catenin/Plk1/β-catenin p-S60/F-actin in the contractile ring region from the XY plane of immunostained early telophase cells (right panel). All immunofluorescence images and linear signal profiles show representative data of more than 50 telophase cells obtained from three independent experiments. The value of the relative neck thickness is shown in the lower left corner of the merged images (A).


HeLa CCL2 stably expressing control vector only ( + vector ) , β - catenin WT ( + WTR ) , S60A mutant ( + S60AR ) , or S60D mutant ( + S60DR ) were generated using lentiviral infection then infected with β - catenin knockdown lentivirus ( shβ - catenin ) . A dot density graph with a box plot shows ( % ) of with cytokinesis - defective ( C ) . box plot represents : central blue band , Median ; box , Interquartile range ; upper whisker , Maximum observation below upper fence ; lower whisker , Minimum observation above lower fence ; dot , Outlier . Each symbol in graph represents a of more than 30 from three independent experiments . * * * , p < 0 . 001 ( one - way ANOVA ) ; Scale bars , 10 μm .

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HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). A dot density graph with a box plot shows the percentage (%) of cells with cytokinesis-defective phenotypes (C). The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Each symbol in the graph represents a percentage of more than 30 cells from three independent experiments. ***, p<0.001 (one-way ANOVA); Scale bars, 10 μm.


HeLa CCL2 transiently transfected with either GFP - β - catenin WT or GFP - β - catenin S60A were monitored during cycle progression by live imaging ( E ) . subcellular localizations of GFP - tagged β - catenin WT or S60A mutant construct were confirmed by immunofluorescence with anti - GFP antibody . were additionally stained for Plk1 ( see Appendix Figure S5A ) . specific localization of GFP - β - catenin WT at midbody was observed under a confocal microscope with high magnification ( E , left panel ) . Cytokinesis - defective in each transfected HeLa were monitored ( E , right panel )

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HeLa CCL2 cells transiently transfected with either GFP-β-catenin WT or GFP-β-catenin S60A were monitored during cell cycle progression by live cell imaging (E). The subcellular localizations of GFP-tagged β-catenin WT or S60A mutant construct were confirmed by immunofluorescence with anti-GFP antibody. The cells were additionally stained for Plk1 (see Appendix Figure S5A). The specific localization of GFP-β-catenin WT at the midbody was observed under a confocal laser microscope with high magnification (E, left panel). Cytokinesis-defective cellular phenotypes in each transfected HeLa cell were monitored (E, right panel)


( E , F ) HeLa CCL2 infected with indicated lentiviruses were co - transfected with HA - Ect2 Flag - Plk1 . were then treated with nocodazole ( 200 ng / mL ) for 18 h released from cycle arrest by incubation in fresh medium for 6 h . resulting were used for immunoprecipitation using anti - Flag ( E ) or anti - HA ( F ) antibodies . Precipitated beads were used for immunoblot using indicated antibodies .

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(E, F) HeLa CCL2 cells infected with the indicated lentiviruses were co-transfected with HA-Ect2 and Flag-Plk1. Cells were then treated with nocodazole (200 ng/mL) for 18 h and released from cell cycle arrest by incubation in fresh medium for 6 h. The resulting cells were used for immunoprecipitation analysis using anti-Flag (E) or anti-HA (F) antibodies. Precipitated beads were used for immunoblot analysis using the indicated antibodies.


( G , H ) HeLa CCl2 infected with either sh - control luciferase ( shGL ) or shβ - catenin lentiviruses were co - immunostained with anti - β - catenin anti - Ect2 antibodies . Localization of β - catenin Ect2 in midbody of telophase was observed using a confocal microscope ( G ) signal of Ect2 was monitored in midbody of each knockdown telophase ( H ) . Arrows indicate total β - catenin Ect2 located at midbody in telophase HeLa CCL2 ( G ) . A dot density graph with a box plot shows signal of midbody - localized Ect2 in telophase . box plot represents : central blue band , Median ; box , Interquartile range ; upper whisker , Maximum observation below upper fence ; lower whisker , Minimum observation above lower fence ; dot , Outlier . Approximately 70 from three independent experiments were measured . * * * , p < 0 . 001 ( unpaired two - t ) ; Scale bars , 10 μm . value of relative neck thickness is shown in lower left corner of merged ( G ) .

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(G, H) HeLa CCl2 cells infected with either sh-control luciferase (shGL) or shβ-catenin lentiviruses were co-immunostained with anti-β-catenin and anti-Ect2 antibodies. Localization of β-catenin and Ect2 in the midbody of telophase cells was observed using a confocal microscope (G) and the signal intensity of Ect2 was monitored in the midbody of each knockdown telophase cell (H). Arrows indicate total β-catenin and Ect2 located at the midbody in telophase HeLa CCL2 cells (G). A dot density graph with a box plot shows the signal intensity of midbody-localized Ect2 in telophase cells. The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Approximately 70 cells from three independent experiments were measured. ***, p<0.001 (unpaired two-tailed t test); Scale bars, 10 μm. The value of the relative neck thickness is shown in the lower left corner of the merged images (G).


HeLa CCL2 treated with DMSO ( control ) or Plk1 inhibitor , BI 2536 ( 1 μM ) for 20 min were immunostained with a combination of either anti - total β - catenin anti - Ect2 antibodies or anti - β - catenin p - S60 anti - Ect2 antibodies ( A ) . Scale bars , 10 μm .

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HeLa CCL2 cells treated with DMSO (control) or the Plk1 inhibitor, BI 2536 (1 μM) for 20 min were immunostained with a combination of either anti-total β-catenin and anti-Ect2 antibodies or anti-β-catenin p-S60 and anti-Ect2 antibodies (A). Scale bars, 10 μm.


( C ) After 18 h of nocodazole ( 200 ng / mL ) - induced cycle arrest , HeLa CCL2 transfected with HA - Ect2 were released by incubation in fresh medium for 2 h treated with either DMSO ( control ) or Plk1 inhibitor ( BI 2536 ) for indicated times . resulting were used for immunoprecipitation using anti - HA antibody , immunoprecipitates were separated by SDS - PAGE followed by immunoblot with indicated antibodies . band of β - catenin bound to HA - Ect2 was quantified with ImageJ normalized to signal of immunoprecipitated HA - Ect2 , relative values are shown below bands .

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(C) After 18 h of nocodazole (200 ng/mL)-induced cell cycle arrest, HeLa CCL2 cells transfected with HA-Ect2 were released by incubation in fresh medium for 2 h and treated with either DMSO (control) or Plk1 inhibitor (BI 2536) for the indicated times. The resulting cells were used for immunoprecipitation analysis using anti-HA antibody, and immunoprecipitates were separated by SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The band intensity of β-catenin bound to HA-Ect2 was quantified with ImageJ and normalized to the signal intensity of immunoprecipitated HA-Ect2, and the relative values are shown below the bands.


( G ) HeLa CCL2 were transfected with either wild type Flag − β - catenin ( + WT ) , Flag - β - catenin S60A mutant ( + S60A ) , or Flag - β - catenin S60D mutant ( + S60D ) , then treated with 200 ng / mL nocodazole for 18 h ( metaphase ) released by incubation in fresh medium for 2 h ( telophase ) . lysates were used for pull - down with rhotekin - RBD beads , pulled down beads were separated by 12 % SDS - PAGE followed by immunoblot with indicated antibodies . was then stained with Coomassie ( CBB ) to determine amount of RBD precipitated .

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

(G) HeLa CCL2 cells were transfected with either wild type Flag−β-catenin (+WT), Flag-β-catenin S60A mutant (+S60A), or Flag-β-catenin S60D mutant (+S60D), then treated with 200 ng/mL nocodazole for 18 h (metaphase) and released by incubation in fresh medium for 2 h (telophase). Cell lysates were used for pull-down analysis with rhotekin-RBD beads, and the pulled down beads were separated by 12 % SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The membrane was then stained with Coomassie (CBB) to determine the amount of RBD precipitated.