( N ) Representative confocal of Irgm1 - / - mouse BMDMs processed for IF with RIG - I ( red ) dsRNA ( green ) antibodies . profile : Co - localization using profiles .

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(N) Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with RIG-I (red) and dsRNA (green) antibodies. Line profile: Co-localization analysis using line intensity profiles.


Representative confocal of Irgm1 - / - mouse BMDMs processed for IF with ( O ) Mda5 ( red ) dsRNA ( green ) antibodies Co - localization using profiles .

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Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (O) Mda5 (red) and dsRNA (green) antibodies Co-localization analysis using line intensity profiles.


Representative confocal of Irgm1 - / - mouse BMDMs processed for IF with ( P ) Mda5 ( red ) , dsRNA ( green ) Tia1 ( blue ) antibodies ( Scale bar , 3 µm ) . profile : Co - localization using profiles .

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Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (P) Mda5 (red), dsRNA (green) and Tia1 (blue) antibodies (Scale bar, 3 µm). Line profile: Co-localization analysis using line intensity profiles.


( A ) qRT - PCR with total isolated from Irgm1 + / + Irgm1 - / - mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG - I siRNA or cGAS siRNA RIG - I siRNA as indicated .

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(A) qRT-PCR analysis with total RNA isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG-I siRNA or cGAS siRNA and RIG-I siRNA as indicated.


( B ) qRT - PCR with total isolated from HT - 29 transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA cGAS siRNA or IRGM siRNA RIG - I siRNA or transfected with three siRNAs as indicated .

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(B) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG-I siRNA or transfected with three siRNAs as indicated.


( C ) qRT - PCR with total isolated from HT - 29 transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA STING siRNA or IRGM siRNA MAVS siRNA or transfected with three siRNAs as indicated .

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(C) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated.


( D ) qRT - PCR with total isolated from HT - 29 transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA MDA5 siRNA as indicated .

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(D) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated.


( E ) qRT - PCR with total isolated from HT - 29 transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA IRF3 siRNA as indicated .

(E) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated.


( F ) qRT - PCR with total isolated from HT - 29 transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA STAT1 siRNA or IRGM siRNA STAT2 siRNA as indicated .

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(F) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated.


RAW264 . 7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h , Ca WT or Ca YL , Lm WT or Lm Δhly for 60 min . ( A ) of Rab8A Rab8A pT72 phosphorylation were analysed by Western blot quantified by densitometry . Data represent mean ± of three independent biological experiments . One - way ANOVA followed by Dunnett ' s compared to WT strains . ns = non - significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h, Ca WT or Ca YL, and Lm WT or Lm Δhly for 60 min. (A) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry. Data represent the mean ± SEM of three independent biological experiments. One-way ANOVA followed by Dunnett's test compared to WT strains. ns= non-significant; *p ≤0.05; **p ≤0.01.


( B ) Recruitment of endogenous LRRK2 EGFP - Rab8A to M . tuberculosis ( Mtb ) was visualised by confocal microscopy . Scale bar = 5 μm .

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(B) Recruitment of endogenous LRRK2 and EGFP-Rab8A to M. tuberculosis (Mtb) was visualised by confocal microscopy. Scale bar = 5 μm.


( C ) C . albicans ( Ca ) was analysed by confocal microscopy . Scale bar = 5 μm . Bottom panels show quantitative of marker association to respective pathogen . Data represents mean ± of two to four independent biological replicates .

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(C) C. albicans (Ca) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.


( D ) L . monocytogenes ( Lm ) was analysed by confocal microscopy . Scale bar = 5 μm . Bottom panels show quantitative of marker association to respective pathogen . Data represents mean ± of two to four independent biological replicates .

(D) L. monocytogenes (Lm) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.


( A ) RAW264 . 7 macrophages were treated with 1 mM LLOMe for indicated time Rab8A Rab8A pT72 were analysed by Western blot .

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(A) RAW264.7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot.


( B ) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min Rab8A Rab8A pT72 were analysed by Western blot .

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(B) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot.


( C ) Macrophages were pre - treated with either 1 μM GSK inh or 0 . 1 μM MLi - 2 then treated with 1 mM LLOMe for 30 min . Rab8A Rab8A pT72 were analysed by Western blot .

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(C) Macrophages were pre-treated with either 1 μM GSK inh or 0.1 μM MLi-2 and then treated with 1 mM LLOMe for 30 min. Rab8A and Rab8A pT72 levels were analysed by Western blot.


( D - F ) RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . ( D ) Endogenous LRRK2 ( E ) Rab8A recruitment to LAMP - 1 positive compartments was visualised by immunofluorescence . Scale bar = 10 μm . ( F ) of D - E . Data represents mean ± of three independent biological experiments .

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(D-F) RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (D) Endogenous LRRK2 and (E) Rab8A recruitment to LAMP-1 positive compartments was visualised by immunofluorescence. Scale bar = 10 μm. (F) Quantification of D-E. Data represents the mean ± SEM of three independent biological experiments.


LRRK2 WT or LRRK2 KO macrophages were pre - treated or not with 1 μM GSK2578215A treated with 1 mM LLOMe for 30 min . number of LRRK2 per were monitored by immunofluorescence high - content imaging . Scale bar = 10 μm . Data represent mean ± of three to four independent biological experiments . One - way ANOVA followed by Dunnett ' s against untreated WT control . ns = non - significant ; * * p ≤ 0 . 01 .

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LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of LRRK2 per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.


LRRK2 WT or LRRK2 KO macrophages were pre - treated or not with 1 μM GSK2578215A treated with 1 mM LLOMe for 30 min . number of Rab8A positive vesicles per were monitored by immunofluorescence high - content imaging . Scale bar = 10 μm . Data represent mean ± of three to four independent biological experiments . One - way ANOVA followed by Dunnett ' s against untreated WT control . ns = non - significant ; * * p ≤ 0 . 01 .

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LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of Rab8A positive vesicles per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.


( A ) RAW264 . 7 macrophages were electroporated with EGFP - Rab8A treated with 1 mM LLOMe for 30 min . Intracellular distribution of Galectin - 3 , CHMP4B or LC3B was visualised by immunofluorescence . Scale bar = 5 μm .

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(A) RAW264.7 macrophages were electroporated with EGFP-Rab8A and treated with 1 mM LLOMe for 30 min. Intracellular distribution of Galectin-3, CHMP4B or LC3B was visualised by immunofluorescence. Scale bar = 5 μm.


( B ) Macrophages were electroporated with EGFP - Rab8A RFP - Galectin - 3 . were then treated with 1 mM of LLOMe monitored by live imaging . Snapshots at indicated time points after LLOMe addition are shown . Scale bar = 5 μm .

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(B) Macrophages were electroporated with EGFP-Rab8A and RFP-Galectin-3. Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging. Snapshots at the indicated time points after LLOMe addition are shown. Scale bar = 5 μm.


( C ) RAW264 . 7 macrophages were treated with 1 mM of LLOMe LRRK2 , Rab8A , CHMP4B , Galectin - 3 LC3B foci over time was analysed by high - content imaging . Data shows mean ± of five biological replicates .

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(C) RAW264.7 macrophages were treated with 1 mM of LLOMe and LRRK2, Rab8A, CHMP4B, Galectin-3 and LC3B foci over time was analysed by high-content imaging. Data shows the mean ± SEM of five biological replicates.


RAW264 . 7 macrophages were pre - treated with 10 μM BAPTA - AM for 1 hr , treated with 1 mM LLOMe for 30 min . ( D ) CHMP4B recruitment was monitored by immunofluorescence high - content imaging . Scale bar = 10 μm .

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RAW264.7 macrophages were pre-treated with 10 μM BAPTA-AM for 1 hr, and treated with 1 mM LLOMe for 30 min. (D) CHMP4B recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm.


( E ) Rab8A pT72 phosphorylation was analysed by Western blot .

(E) Rab8A pT72 phosphorylation was analysed by Western blot.


( F ) LRRK2 ( G ) Rab8A recruitment was monitored by immunofluorescence high - content imaging . Scale bar = 10 μm . ( H ) of E F .

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(F) LRRK2 and (G) Rab8A recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. (H) Quantification of E and F.


RAW264 . 7 WT , LRRK2 KO or Rab8A macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) were treated with 1 mM LLOMe for 30 min . CHMP4B vesicle numbers were analysed by immunofluorescence high content imaging . Scale bar = 20 μm . Right panels show of number of CHMP4B positive vesicles per . Mean ± of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons .

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RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of CHMP4B positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


RAW264 . 7 WT , LRRK2 KO or Rab8A macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) were treated with 1 mM LLOMe for 30 min . Galectin - 3 vesicle numbers were analysed by immunofluorescence high content imaging . Scale bar = 20 μm . Right panels show of number of Galectin - 3 positive vesicles per . Mean ± of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons .

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RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. Galectin-3 vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of Galectin-3 positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


Live imaging of LysoTracker positive spots in WT , LRRK2 KO macrophages treated with 1 mM LLOMe , followed by lysosomal recovery after LLOMe wash - out . One representative experiment out of three shown . Differences between slopes in LLOMe treatment window were estimated using linear regression .

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Live cell imaging of LysoTracker positive spots in WT, LRRK2 KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.


Live imaging of LysoTracker positive spots in WT , Rab8A KO macrophages treated with 1 mM LLOMe , followed by lysosomal recovery after LLOMe wash - out . One representative experiment out of three shown . Differences between slopes in LLOMe treatment window were estimated using linear regression .

Live cell imaging of LysoTracker positive spots in WT, Rab8A KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.


WT or LRRK2 KO RAW264 . 7 macrophages , RAW264 . 7 macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) WT or Rab8A KO RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . LC3B positive vesicle numbers were analysed by immunofluorescence high content imaging . Scale bar = 20 μm . Right panels show of number of positive vesicles per . Data show mean ± of three to four biological replicates . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons .

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WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. LC3B positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT or LRRK2 KO RAW264 . 7 macrophages , RAW264 . 7 macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) WT or Rab8A KO RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence high content imaging . Scale bar = 20 μm . Right panels show of number of positive vesicles per . Data show mean ± of three to four biological replicates . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons .

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WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


( C ) Representative of LC3B / LAMP - 1 double positive endolysosomes in control LLOMe treated macrophages . Scale bar = 10 μm . ( D ) of % of LC3B / LAMP - 1 double positive endolysosomes in LLOMe treated macrophages . Data shows mean ± of three independent biological experiments . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 by Student ' s t - .

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(C) Representative images of LC3B/LAMP-1 double positive endolysosomes in control and LLOMe treated macrophages. Scale bar = 10 μm. (D) Analysis of the % of LC3B/LAMP-1 double positive endolysosomes in LLOMe treated macrophages. Data shows mean ± SEM of three independent biological experiments. *p≤0.05, ***p≤0.001 by Student's t-test.


WT LRRK2 KO RAW264 . 7 macrophages were infected with Mtb for 24 h , Galectin - 3 LC3B recruitment was analysed by immunofluorescence high content imaging . Scale bar = 10 μm . Data show mean ± of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - .

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WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb for 24 h, Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT LRRK2 KO RAW264 . 7 macrophages were infected with Ca for 60 min Galectin - 3 LC3B recruitment was analysed by immunofluorescence high content imaging . Scale bar = 10 μm . Data show mean ± of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - .

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WT and LRRK2 KO RAW264.7 macrophages were infected with Ca for 60 min Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT LRRK2 KO RAW264 . 7 macrophages were infected with Lm for 60 min . Galectin - 3 LC3B recruitment was analysed by immunofluorescence high content imaging . Scale bar = 10 μm . Data show mean ± of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - .

WT and LRRK2 KO RAW264.7 macrophages were infected with Lm for 60 min. Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT LRRK2 KO RAW264 . 7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show mean ± of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons .

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WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT LRRK2 KO RAW264 . 7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h . Data show mean ± of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons .

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WT and LRRK2 KO RAW264.7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT LRRK2 KO RAW264 . 7 macrophages were infected with Lm WT or Lm Δhly . Growth measured for 2 h . Data show mean ± of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons .

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WT and LRRK2 KO RAW264.7 macrophages were infected with Lm WT or Lm Δhly. Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


Monocyte - derived macrophages from healthy controls PD carrying G2019S ( Donor 2 - 3 ) or R1441C ( Donor 1 ) LRRK2 mutation were pre - treated with 0 . 1 μM MLi - 2 treated with 1 mM LLOMe for 30 min . ( A ) Rab8A Rab8A pT72 were analysed by Western blot .

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Monocyte-derived macrophages from healthy controls and PD patients carrying the G2019S (Donor 2-3) or R1441C (Donor 1) LRRK2 mutation were pre-treated with 0.1 μM MLi-2 and treated with 1 mM LLOMe for 30 min. (A) Rab8A and Rab8A pT72 levels were analysed by Western blot.


( B ) Rab8A Galectin - 3 positive vesicle numbers were analysed by immunofluorescence . Data show mean ± of biological replicates . Scale bar = 20 μm . ns = non - significant , * * p ≤ 0 . 01 , by One - way ANOVA followed by Sidak ' s multiple comparisons .

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(B) Rab8A and Galectin-3 positive vesicle numbers were analysed by immunofluorescence. Data show the mean ± SEM of biological replicates. Scale bar = 20 μm. ns=non-significant, **p≤0.01, by One-way ANOVA followed by Sidak's multiple comparisons test.


( A ) Diauxic growth of WT E . coli ( NCM3722 ) in minimal media containing glucose acetate , density measured as optical density ( OD600 ) . Growth on glucose is captured by exponential fit ( blue dashed ) , proceeds until glucose runs out ( black dashed ) at time = 0 h . This is followed by a period of growth - lag lasting ~ 3 . 5h before exponential growth resumes on acetate ( red dashed ) .

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(A) Diauxic growth of WT E. coli (NCM3722) in minimal media containing glucose and acetate, bacterial density measured as optical density (OD600). Growth on glucose is captured by the exponential fit (blue dashed line), and proceeds until glucose runs out (black dashed line) at time = 0 h. This is followed by a period of growth-lag lasting ~3.5h before exponential growth resumes on acetate (red dashed line).


( D ) Lag - times for controlled titration of aceBA expression using an inducer construct in NQ1350 ( inset ) . Addition of chlorotetracycline ( cTc ) removes tetR repression induces aceBA expression . As expression of aceB / aceA during response ( inducer added at time = 0h ) is increased , lag times decrease . Bar plot shows mean lag - times ( N = 3 biological repeats ) for different inducer with error bars denoting Standard Deviations ( SD ) .

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(D) Lag-times for controlled titration of aceBA expression using an inducer construct in strain NQ1350 (inset). Addition of chlorotetracycline (cTc) removes the tetR repression and induces aceBA expression. As the expression of aceB/aceA during the response (inducer added at time = 0h) is increased, lag times decrease. Bar plot shows mean lag-times (N=3 biological repeats) for different inducer concentration with error bars denoting the Standard Deviations (SD).


( A ) A plasmid system ( inset ) is used to control expression of non - required gene lacZ using NQ1389 . lacZ expression was induced using cTc to varying degrees at moment of glucose depletion ( time = 0 h ) using indicated range of cTc concentrations . Diauxic growth conditions with glucose acetate , same as in Fig . 1 .

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(A) A plasmid system (inset) is used to control the expression of the non-required gene lacZ using the strain NQ1389. lacZ expression was induced using cTc to varying degrees at the moment of glucose depletion (time = 0 h) using the indicated range of cTc concentrations. Diauxic growth conditions with glucose and acetate, same as in Fig. 1.


( B ) Lag - times fall reciprocally with allocation towards required ( fraction of mRNA encoding for required ) during shift . Allocation of translation activity towards different are represented by of red gray arrows ( top ) .

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(B) Lag-times fall reciprocally with the allocation towards required proteins (the fraction of mRNA encoding for required proteins) during the shift. Allocation of translation activity towards different proteins are represented by the weight of red and gray arrows (top).


( B ) For various gene categories in panel A , temporal kinetics of expression during diauxic shift is plotted . Time 0 indicates time when glucose runs out . series of - seq through growth transition was performed once .

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(B) For the various gene categories in panel A, the temporal kinetics of expression during the diauxic shift is plotted. Time 0 indicates time when glucose runs out. The series of RNA-seq through the growth transition was performed once.


( C ) Steady - state growth in glucose acetate for WT motility deletion strains ΔfliC Δflh . Growth rate differences between WT mutants are not significant in glucose ( p - value > 0 . 05 ) , but are significant for growth in acetate ( p - values < 0 . 02 ) . Additional growth conditions are shown in Fig . EV6 .

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(C) Steady-state growth in glucose and acetate for the WT and the motility deletion strains ΔfliC and Δflh. Growth rate differences between WT and the mutants are not significant in glucose (p-value >0.05), but are significant for growth in acetate (p-values<0.02). Additional growth conditions are shown in Fig. EV6.


( D , E ) Growth transitions for motility deletion strains ΔfliC ΔflhD are substantially faster than that for WT , with ( D ) showing representative growth kinetics of three strains ( E ) showing mean lag - times standard error of four independent biological replicates . Lag - times for WT are significantly different from that of ∆ fliC ( p - value 0 . 01 ) ∆ flhD ( p - value 0 . 004 ) .

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(D, E) Growth transitions for the motility deletion strains ΔfliC and ΔflhD are substantially faster than that for the WT strain, with (D) showing representative growth kinetics of the three strains and (E) showing mean lag-times and standard error of four independent biological replicates. Lag-times for WT are significantly different from that of ∆fliC (p-value 0.01) and ∆flhD (p-value 0.004).


( F ) ∆ fliC shows a 2 - fold increase ( p - value 0 . 17 ) ∆ flhD shows a 3 - fold increase ( p - value 0 . 02 ) in aceB expression compared to WT , as measured by qPCR .

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(F) ∆fliC shows a 2-fold increase (p-value 0.17) and ∆flhD shows a 3-fold increase (p-value 0.02) in aceB expression compared to WT, as measured by qPCR.


Diauxic growth kinetics for growth on glucose a different secondary carbon sources for WT ( black ) motility deletion strains ΔfliC ΔflhD ( magenta green ) .

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Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT (black) and the motility deletion strains ΔfliC and ΔflhD strain (magenta and green).


qRT - PCR of Foxp1 - 4 in murine ECs freshly isolated from P11 mesentery ( n = 6 mice , data points shown ) . Data are presented as mean relative expression ( normalized to Gapdh ) ± SD . Transcript for each transcript are presented relative to in LECs . P , Student ' s t - . nd , not detected .

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qRT-PCR analysis of Foxp1-4 in murine ECs freshly isolated from P11 mesentery (n = 6 mice, individual data points shown). Data are presented as mean relative expression (normalized to Gapdh) ± SD. Transcript levels for each transcript are presented relative to levels in LECs. P, Student's t-test. nd, not detected.


Immunofluorescence ( C ) qRT - PCR ( D ) of HDLECs grown under static conditions or exposed to OSS for 48 h ( n = 3 independent experiments ) . Data are presented as mean ± SD . P , Student ' s t - .

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Immunofluorescence (C) and qRT-PCR analysis (D) of HDLECs grown under static conditions or exposed to OSS for 48 h (n = 3 independent experiments). Data are presented as mean ± SD. P, Student's t-test.


Whole - mount immunofluorescence of P7 mesenteries , showing efficient FOXP2 depletion in Foxp2flox / - ; Tie2 - Cre vessels . Dotted indicates vessel width . of mesenteric collecting lymphatic vessel width in P7 Foxp2flox / - ; Tie2 - Cre littermate controls ( n = vessels [ mice ] , as indicated ) .

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Whole-mount immunofluorescence of P7 mesenteries, showing efficient FOXP2 depletion in Foxp2flox/-;Tie2-Cre vessels. Dotted line indicates vessel width. Quantification of mesenteric collecting lymphatic vessel width in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = vessels [mice], as indicated).


Lymphatic valves in P7 mesenteric vessels of control Foxp2flox / - ; Tie2 - Cre mice . A spectrum of defects in mutants ranging from shortened integrin - α9 + FN - EIIIA + valve leaflets ( middle panels ) to a complete loss of leaflets ( right panels ) are shown . of proportion of valves with integrin - α9 + leaflets ( G ) , leaflet ( H ) in P7 Foxp2flox / - ; Tie2 - Cre littermate controls ( n = valves [ mice ] , as indicated ) . Leaflet = 0 corresponds to valves with no leaflets .

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Lymphatic valves in P7 mesenteric vessels of control and Foxp2flox/-;Tie2-Cre mice. A spectrum of defects in the mutants ranging from shortened integrin-α9+FN-EIIIA+ valve leaflets (middle panels) to a complete loss of leaflets (right panels) are shown. Quantification of the proportion of valves with integrin-α9+ leaflets (G), and leaflet length (H) in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = valves [mice], as indicated). Leaflet length = 0 corresponds to valves with no leaflets.


Whole - mount immunofluorescence ( A ) of NRP2 ( B ) in P6 mesenteric lymphatic vessels of Foxp2flox / - ; Tie2 - Cre littermate control mice ( n = vessels [ mice ] , as indicated ) .

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Whole-mount immunofluorescence (A) and quantification of NRP2 staining intensity (B) in P6 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).


RNAscope - whole - mount in situ hybridization ( C ) of Efnb2 transcript ( D ) in P7 mesenteric lymphatic vessels of Foxp2flox / - ; Tie2 - Cre littermate control mice ( n = vessels [ mice ] , as indicated ) .

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RNAscope-based whole-mount in situ hybridization (C) and quantification of Efnb2 transcript levels (D) in P7 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).


Whole - mount immunofluorescence of P6 mesenteries , showing valve defects in mutant vessels . of lymphatic valve numbers ( C ) , valve leaflet ( D ) vessel width ( E ) in Foxp2flox / flox ; Prox1 - CreERT2 littermate control mice ( n = vessels ( C , E ) or valves ( D ) [ mice ] , as indicated ) .

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Whole-mount immunofluorescence of P6 mesenteries, showing valve defects in the mutant vessels. Quantification of lymphatic valve numbers (C), valve leaflet length (D) and vessel width (E) in Foxp2flox/flox;Prox1-CreERT2 and littermate control mice (n = vessels (C, E) or valves (D) [mice], as indicated).


qRT - PCR of FOXP2 , FOXC2 GJA4 expression in control ( siCTRL ) FOXP2 or FOXC2 siRNA - treated HDLECs ( n = 6 ( FOXP2 , FOXC2 ) or n = 3 ( GJA4 ) independent experiments ) .

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qRT-PCR analysis of FOXP2, FOXC2 and GJA4 expression in control (siCTRL) and FOXP2 or FOXC2 siRNA-treated HDLECs (n = 6 (FOXP2, FOXC2) or n = 3 (GJA4) independent experiments).


Top panels : whole - mount immunofluorescence of E18 Foxc2flox / flox ; Prox1 - CreERT2 littermate control mesenteries , showing downregulation of FOXP2 in Foxc2 deficient vessels . Bottom panels : extraction of FOXP2 ( grey ) using IMARIS surface mask generated on PROX1 ( green ) . Unmasked FOXP2 is shown in magenta . of FOXP2 in mesenteric lymphatic vessels of Foxc2flox / flox ; Prox1 - CreERT2 control on FOXP2 signals as in ( B ) ( n = vessels [ mice ] , as indicated ) .

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Top panels: whole-mount immunofluorescence of E18 Foxc2flox/flox;Prox1-CreERT2 and littermate control mesenteries, showing downregulation of FOXP2 in Foxc2 deficient vessels. Bottom panels: extraction of nuclear FOXP2 staining (grey) using IMARIS surface mask generated based on PROX1 staining (green). Unmasked FOXP2 staining is shown in magenta. Quantification of FOXP2 levels in mesenteric lymphatic vessels of Foxc2flox/flox;Prox1-CreERT2 and control littermates based on nuclear FOXP2 signals as in (B) (n = vessels [mice], as indicated).


Whole - mount immunofluorescence of P8 mesenteric lymphatic vessels of Foxp2flox / - ; Tie2 - Cre littermate control mice showing loss of organized FOXC2high valve regions but unaltered expression of FOXC2 in Foxp2 deficient vessels . Whole - mount immunofluorescence of lymphatic valves in P7 mesenteries of control Foxp2flox / - ; Tie2 - Cre mice showing NFATc1 subcellular localization . Note predominantly localization of NFATc1 in control valves ( left panels ) , but localization ( middle panels , 3 out of 5 mice ) or downregulation of NFATc1 ( right panels , 2 out of 5 mice ) in mutant valves . of localization in NFATc1 + lymphatic valves from ( D ) ( n = valves [ mice ] , as indicated ) .

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Whole-mount immunofluorescence of P8 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice showing loss of organized FOXC2high valve regions but unaltered expression of FOXC2 in Foxp2 deficient vessels. Whole-mount immunofluorescence of lymphatic valves in P7 mesenteries of control and Foxp2flox/-;Tie2-Cre mice showing NFATc1 subcellular localization. Note predominantly nuclear localization of NFATc1 in control valves (left panels), but cytoplasmic localization (middle panels, 3 out of 5 mice) or downregulation of NFATc1 (right panels, 2 out of 5 mice) in the mutant valves. Quantification of protein localization in NFATc1+ lymphatic valves from (D) (n = valves [mice], as indicated).


( A ) Western blot of Caspase 8 , FLIP FADD recruitment to TRAIL - R2 DISC in A549 , HCT116 DU145 lines following incubation with increasing concentrations ( 1x , 2x , or 4x ) of anti - TRAIL - R2 ( AMG655 ) - conjugated magnetic beads for 90 minutes .

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(A) Western blot analysis of Caspase 8, FLIP and FADD recruitment to the TRAIL-R2 DISC in A549, HCT116 and DU145 cell lines following incubation with increasing concentrations (1x, 2x, or 4x) of anti-TRAIL-R2 (AMG655)-conjugated magnetic beads for 90 minutes.


( B ) Western blot of unbound soluble fraction An untreated input of each is included for comparison .

(B) Western blot analysis of the unbound soluble fraction An untreated input of each cell line is included for comparison.


( C ) of Western blot for FLIP , FADD , Procaspase 8 their respective cleavage fragments at TRAIL - R2 DISC . were quantified by densitometry normalised to known

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(C) Quantification of Western blot analysis for FLIP, FADD, Procaspase 8 and their respective cleavage fragments at the TRAIL-R2 DISC. Proteins were quantified by densitometry and normalised to known protein standards


( D ) Caspase - 8 - like ( IETDase ) activity in unbound soluble fraction from DISC IP An untreated control ( 0 ) was used for each to show basal caspase - 8 activity .

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(D) Caspase-8-like (IETDase) activity assay in the unbound soluble fraction from the DISC IP An untreated control (0) was used for each cell line to show basal caspase-8 activity.


( E ) Ratios of DED quantified at TRAIL - R2 DISC in A549 , HCT116 DU145 ; increasing concentrations of AMG655 are indicated by colour .

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(E) Ratios of DED proteins quantified at the TRAIL-R2 DISC in A549, HCT116 and DU145 cells; increasing concentrations of AMG655 are indicated by colour intensity.


( F ) Correlation between caspase - 8 activity of FADD : FLIP ( L ) .

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(F) Correlation between caspase-8 activity and ratio of FADD:FLIP(L).


( A ) Western blot of FADD , FLIP Caspase 8 at TRAIL - R2 DISC in HCT116 treated with escalating doses ( 0 - 30nM ) of caspase - 8 siRNA for 48 hours . Samples were incubated with anti - TRAIL - R2 ( AMG655 ) beads ( 4x ) for 90 minutes prior to collection . A scrambled control siRNA ( SC ) was transfected ( 30nM ) for comparison . ( B ) of Caspase 8 ( p55 , p43 / 41 p26 / 24 ) FLIP ( FLIP ( L ) , FLIP ( S ) p43 - FLIP ) at TRAIL - R2 DISC by densitometry normalised to known standards ( C ) of Caspase - 8 : FLIP calculated from values presented in ( B ) .

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(A) Western blot analysis of FADD, FLIP and Caspase 8 at the TRAIL-R2 DISC in HCT116 cells treated with escalating doses (0-30nM) of caspase-8 siRNA for 48 hours. Samples were incubated with anti-TRAIL-R2 (AMG655) beads (4x) for 90 minutes prior to collection. A scrambled control siRNA (SC) was transfected (30nM) for comparison. (B) Quantification of Caspase 8 (p55, p43/41 and p26/24) and FLIP (FLIP(L), FLIP(S) and p43-FLIP) at the TRAIL-R2 DISC by densitometry and normalised to known protein standards (C) Ratio of Caspase-8:FLIP calculated from values presented in (B).


( D ) Caspase - 8 ( IETDase ) - 3 / 7 ( DEVDase ) activity in unbound soluble

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(D) Caspase-8 (IETDase) and -3/7 (DEVDase) activity assay in the unbound soluble fraction


( E ) Western blot of FLIP , Caspase 8 , FADD recruitment to TRAIL - R2 DISC in PC3 stably expressing an empty vector ( EV ) or flag tagged FLIP L ( FL ) treated with 2 . 5µM MS - 275 for 48 hours , followed by a 90 minute DISC IP . * modified form of FLIP , potentially mono - ubiquitinated p43 - FLIP ( L ) .

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(E) Western blot analysis of FLIP, Caspase 8, and FADD recruitment to the TRAIL-R2 DISC in PC3 cells stably expressing an empty vector (EV) or flag tagged FLIP L (FL) treated with 2.5µM MS-275 for 48 hours, followed by a 90 minute DISC IP. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L).


( G ) Caspase - 8 ( IETDase ) activity assays of unbound soluble

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(G) Caspase-8 (IETDase) activity assays of the unbound soluble fraction


( A ) Western Blot of Caspase 8 , Caspase 10 , FLIP FADD recruitment to TRAIL - R2 DISC in procaspase 8 ( WT ) or procaspase 8 deficient ( Null ) A549 incubated with AMG655 beads ( 4x ) for either 30 , 60 or 180 minutes .

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(A) Western Blot analysis of Caspase 8, Caspase 10, FLIP and FADD recruitment to the TRAIL-R2 DISC in procaspase 8 (WT) or procaspase 8 deficient (Null) A549 cells incubated with AMG655 beads (4x) for either 30, 60 or 180 minutes.


( B ) Western blot of recruitment of FLIP , Caspase 8 , Caspase 10 FADD to TRAIL - R2 DISC in HCT116 Parental , caspase 8 null ( CASP8 ) , caspase 10 null ( CASP10 ) or CASP 8 / 10 treated with 4x AMG655 conjugated beads for 90 minutes .

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(B) Western blot analysis of the recruitment of FLIP, Caspase 8, Caspase 10 and FADD to the TRAIL-R2 DISC in HCT116 Parental, caspase 8 null (CASP8), caspase 10 null (CASP10) or CASP 8/10 cells treated with 4x AMG655 conjugated beads for 90 minutes.


( C ) AlphaScreen ® assessment of interaction of recombinant FLIP DED1 / 2 recombinant FADD DED .

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(C) AlphaScreen® assessment of the interaction of recombinant FLIP DED1/2 and recombinant FADD DED.


( D ) AlphaScreen ® assessment of impact on FLIP - FADD interaction of unstapled stapled peptides corresponding to FADD α1 helix .

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(D) AlphaScreen® assessment of the impact on the FLIP-FADD interaction of unstapled and stapled peptides corresponding to the FADD α1 helix.


( E ) NanoBiT ® to quantify FLIP / FADD interactions in a U2OS stably expressing NanoBiT ® constructs , FLIP ( LgBiT ) FADD ( smBiT ) following 48 hour silencing of FLIP , FADD , or caspase 8 . Western blot was used to confirm successful knockdown of target by siRNA .

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(E) NanoBiT® assay to quantify FLIP/FADD interactions in a U2OS cell line stably expressing the NanoBiT® constructs, FLIP (LgBiT) and FADD (smBiT) following 48 hour silencing of FLIP, FADD, or caspase 8. Western blot analysis was used to confirm successful knockdown of target proteins by siRNA.


( F ) NanoBiT ® of HCT116 U20S wild type ( WT ) procaspase 8 null ( Null ) transiently co - transfected with FLIP ( LgBiT ) FADD ( SmBiT ) NanoBiT ® constructs for 24 hours . Construct expression was analysed by Western blotting .

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(F) NanoBiT® assay of HCT116 and U20S wild type (WT) and procaspase 8 null (Null) cells transiently co-transfected with FLIP (LgBiT) and FADD (SmBiT) NanoBiT® constructs for 24 hours. Construct expression was analysed by Western blotting.


( A ) Western blot of FLIP , Caspase 8 FADD recruitment to TRAIL - R2 DISC in U20S parental ( EV ) 3 independent caspase 8 null clones ( # 1 , # 2 , # 3 ) after incubation with AMG655 conjugated beads for 30 , 60 or 180 minutes .

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(A) Western blot analysis of FLIP, Caspase 8 and FADD recruitment to the TRAIL-R2 DISC in U20S parental cells (EV) and 3 independent caspase 8 null clones (#1, #2, #3) after incubation with AMG655 conjugated beads for 30, 60 or 180 minutes.


( B ) Western blot of FLIP , Caspase 8 FADD in soluble unbound

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(B) Western blot analysis of FLIP, Caspase 8 and FADD in the soluble unbound fraction


( C ) of FADD recruitment to TRAIL - R2 DISCs FADD were normalised to TRAIL - R2 in pull - downs . Densitometry was performed using ImageJ ® .

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(C) Quantification of FADD recruitment to the TRAIL-R2 DISCs FADD levels were normalised to TRAIL-R2 levels in the pull-downs. Densitometry was performed using ImageJ®.


( E ) Western blot of FADD recruitment to TRAIL - R2 DISC in HCT116 transiently transfected with an empty vector ( EV ) , Flag - tagged FADD ( WT ) , or Flag tagged FADD with point mutations at either Phe25 ( F25A ) or His9 ( H9G ) . Western blotting of soluble unbound fraction was used to monitor transfection efficiency .

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(E) Western blot analysis of FADD recruitment to the TRAIL-R2 DISC in HCT116 cells transiently transfected with an empty vector (EV), Flag-tagged FADD (WT), or Flag tagged FADD with point mutations at either Phe25 (F25A) or His9 (H9G). Western blotting analysis of the soluble unbound fraction was used to monitor transfection efficiency.


( F ) NanoBiT ® of U20S transiently co - transfected with caspase 8 ( LgBiT ) either WT FADD smBiT or FADD ( smBiT ) constructs with a point mutation at either phenylalanine 25 ( F25A ) or histidine 9 ( H9G ) for 48 hours . Western blot was used to assess expression level of each construct .

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(F) NanoBiT® assay of U20S cells transiently co-transfected with caspase 8 (LgBiT) and either WT FADD smBiT or FADD (smBiT) constructs with a point mutation at either phenylalanine 25 (F25A) or histidine 9 (H9G) for 48 hours. Western blot analysis was used to assess the expression level of each construct.


( A ) Western blot of FLIP , caspase 8 FADD recruitment to TRAIL - R2 DISC in parental ( WT ) CFLAR deficient ( KO ) HAP - 1 treated with AMG655 conjugated beads for either 45 minutes ( 45 ' ) , 90 minutes ( 90 ' ) , 180 minutes ( 180 ' ) , 3 hours ( 3h ) , 6 hours ( 6h ) or 9 hours ( 9h ) .

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(A) Western blot analysis of FLIP, caspase 8 and FADD recruitment to the TRAIL-R2 DISC in parental (WT) and CFLAR deficient (KO) HAP-1 cells treated with AMG655 conjugated beads for either 45 minutes (45'), 90 minutes (90'), 180 minutes (180'), 3 hours (3h), 6 hours (6h) or 9 hours (9h).


( B ) Caspase activity assays of unbound soluble

(B) Caspase activity assays of the unbound soluble fraction


( C ) Caspase - 8 ( i ) - 3 / 7 ( ii ) activity assays high content microscopy assessment of ( iii ) in Parental ( PAR ) FLIP knockout ( CFLAR CRISPR ) treated with escalating doses of isoleucine zipper TRAIL ( IZ - TRAIL ) for 6 hours .

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(C) Caspase-8 (i) and -3/7 (ii) activity assays and high content microscopy assessment of cell death (iii) in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 6 hours.


( D ) Caspase - 8 ( i ) - 3 / 7 ( ii ) activity assays in Parental ( PAR ) FLIP knockout ( CFLAR CRISPR ) treated with escalating doses of isoleucine zipper TRAIL ( IZ - TRAIL ) for 24 hours . ( iii ) Flow cytometry assessment of induction in Parental ( PAR ) FLIP knockout ( CFLAR CRISPR ) treated with IZ - TRAIL for 24h .

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(D) Caspase-8 (i) and -3/7 (ii) activity assays in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 24 hours. (iii) Flow cytometry assessment of cell death induction in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with IZ-TRAIL for 24h.


( E ) Western Blot of Parental ( WT ) FLIP knockout ( KO ) HAP - 1 treated with 10ng / mL IZ - TRAIL for 0 , 3 , 6 or 24 hours .

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(E) Western Blot analysis of Parental (WT) and FLIP knockout (KO) HAP-1 cells treated with 10ng/mL IZ-TRAIL for 0, 3, 6 or 24 hours.


( F ) viability of Parental ( PAR ) FLIP knockout ( CFLAR CRISPR ) treated with an escalating dose ( 0 - 1000ng / mL ) of IZ - TRAIL for 72 hours .

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(F) Cell viability assay of Parental (PAR) and FLIP knockout (CFLAR CRISPR cells) treated with an escalating dose (0-1000ng/mL) of IZ-TRAIL for 72 hours.


( B ) NanoBiT ® performed on TRAIL - R2 DISC IPs ( 90 ' ) carried out in U20S Caspase - 8 null co - transfected with wild - type or F114A FLIP - LgBiT fusion wild - type or F122A caspase - 8 - SmBiT fusion .

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(B) NanoBiT® assay performed on TRAIL-R2 DISC IPs (90') carried out in U20S Caspase-8 null cells co-transfected with wild-type or F114A FLIP-LgBiT fusion proteins and wild-type or F122A caspase-8-SmBiT fusion proteins.


( E ) Western blot of exclusion chromatography experiments in which HCT116 transfected with Flag - tagged wild - type ( WT ) FLIP ( S ) were treated with 100ng / mL IZ - TRAIL for 3h prior to lysis separation of low high MW complexes using a exclusion column . fractions eluted at 2MDa > 2MDa are presented .

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(E) Western blot analysis of size exclusion chromatography experiments in which HCT116 cells transfected with Flag-tagged wild-type (WT) FLIP(S) were treated with 100ng/mL IZ-TRAIL for 3h prior to cell lysis and separation of low and high MW complexes using a size exclusion column. The fractions eluted at 2MDa and >2MDa are presented.


MST of binding interactions between Sir4 H ‑ BRCT Cy5 - labeled phospho ( filled circles ) non - phospho peptides ( empty circles ) from Esc1 , Ty5 Ubp10 . KD = equilibrium dissociation constant . ΔFnorm values were divided by amplitude of saturation level , resulting in fraction bound ( from 0 to 1 ) for each data point . Data are represented as mean ± from ≥ 3 independent measurements .

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MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1, Ty5 and Ubp10. KD = equilibrium dissociation constant. ΔFnorm values were divided by the amplitude of the saturation level, resulting in the fraction bound (from 0 to 1) for each data point. Data are represented as the mean ± SEM from ≥3 independent measurements.


Example of Nup49 - RFP Sir4 - GFP in wild - type ( wt ) ( GA - 10372 ; row 1 ) ; sir4 RKR ( GA - 10373 ; row 2 ) ; wt transformed with either empty plasmid ( row 3 ) , plasmid overexpressing wt Sir4 H ‑ BRCT ( row 4 ) , or plasmid overexpressing RKR mutant Sir4 H ‑ BRCT ( row 5 ) . bar indicates 2 μm .

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Example images of Nup49-RFP Sir4-GFP in wild-type (wt) cells (GA-10372; row 1); sir4 RKR cells (GA-10373; row 2); wt cells transformed with either empty plasmid (row 3), plasmid overexpressing wt Sir4 H‑BRCT (row 4), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 5). The bar indicates 2 μm.


of mean of Sir4 - GFP using > 100 . Error bars show SD from 3 independent experiments . of containing Sir4 clusters at envelope ( NE ) . Data are represented as mean ± SD from 3 independent experiments where > 100 were counted .

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Quantification of mean fluorescence intensity of Sir4-GFP using >100 cells. Error bars show the SD from 3 independent experiments. Quantification of cells containing Sir4 clusters at the nuclear envelope (NE). Data are represented as mean ± SD from 3 independent experiments where >100 cells were counted.


Top : Schematic representation of TEL7L : : URA3 reporter used to silencing at telomeres . Bottom : wt ( GA - 503 ) transformed with either an empty plasmid ( row 1 ) , plasmid overexpressing wt Sir4 H ‑ BRCT ( row 2 ) , or plasmid overexpressing RKR mutant Sir4 H ‑ BRCT ( row 3 ) . lacking endogenous sir4 ( GA - 5822 ) ( row 4 ) . were grown in absence of uracil plated as 1 : 5 serial dilutions onto control YPAD or YPAD lacking uracil plates imaged after 2 days . Top : Schematic representation of HMR - EΔB : : TRP1 reporter gene locus used for assaying silencing at HMR locus . Bottom : wt ( GA - 485 ) transformed with plasmids as in panel D . lacking endogenous sir4 ( GA - 6888 ) ( row 4 ) . were grown in absence of tryptophan plated as 1 : 5 serial dilutions onto YPAD or YPAD lacking tryptophan plates imaged after 2 days .

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Top: Schematic representation of the TEL7L::URA3 reporter used to assay silencing at telomeres. Bottom: wt cells (GA-503) transformed with either an empty plasmid (row 1), plasmid overexpressing wt Sir4 H‑BRCT (row 2), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 3). Cells lacking endogenous sir4 (GA-5822) (row 4). Cells were grown in the absence of uracil and plated as 1:5 serial dilutions onto control YPAD or YPAD lacking uracil plates and imaged after 2 days. Top: Schematic representation of the HMR-EΔB::TRP1 reporter gene locus used for assaying silencing at the HMR locus. Bottom: wt cells (GA-485) transformed with plasmids as in panel D. Cells lacking endogenous sir4 (GA-6888) (row 4). Cells were grown in the absence of tryptophan and plated as 1:5 serial dilutions onto YPAD or YPAD lacking tryptophan plates and imaged after 2 days.


Volcano plot showing that interact with wt Sir4 H ‑ BRCT but not with RKR mutant ( upper right quadrant ) .

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Volcano plot showing proteins that interact with wt Sir4 H‑BRCT but not with the RKR mutant (upper right quadrant).


MST of binding interactions between Sir4 H ‑ BRCT Cy5 - labeled phospho ( filled circles ) non - phospho peptides ( empty circles ) from Tom1 Cbf1 . KD represents equilibrium dissociation constant . KD for interactions with non - phospho peptides could not be determined ( n . d . ) . ΔFnorm [ 0 / 00 ] represents change in during thermophoresis normalized to initial . Data are represented as mean ± from at least 3 independent measurements .

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MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Tom1 and Cbf1. KD represents the equilibrium dissociation constant. The KD for interactions with the non-phospho peptides could not be determined (n.d.). ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are represented as the mean ± SEM from at least 3 independent measurements.


Extracts from asynchronous cultures of strains Sir4 - Myc Tom1 - HA ( GA - 10645 ) , Sir4 - RKR - Myc Tom1 - HA ( GA - 10646 ) , Tom1 - HA ( GA - 10647 ) Sir4 - Myc ( GA - 10199 ) were subjected to anti - Myc IP western blotting with indicated antibodies in presence absence of lambda phosphatase .

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Extracts from asynchronous cultures of strains Sir4-Myc Tom1-HA (GA-10645), Sir4-RKR-Myc Tom1-HA (GA-10646), Tom1-HA (GA-10647) and Sir4-Myc (GA-10199) were subjected to anti-Myc IP and western blotting with indicated antibodies in the presence and absence of lambda phosphatase.


MST of binding interactions between Dbf4 H ‑ BRCT Cy5 - labeled phospho ( filled circles ) non - phospho peptides ( empty circles ) from Esc1 Ubp10 . KD = equilibrium dissociation constant , which could not be determined ( n . d . ) for non - phospho peptides . ΔFnorm [ 0 / 00 ] represents change in during thermophoresis normalized to initial . Data are mean ± from ≥ 3 independent measurements .

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MST analysis of the binding interactions between Dbf4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1 and Ubp10. KD = equilibrium dissociation constant, which could not be determined (n.d.) for non-phospho peptides. ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are the mean ± SEM from ≥3 independent measurements.


Trajectory plots of siRNA - transfected HUVECs under VEGF / or Sema3E gradients for 12 h . Red trajectories indicate with displacement to negative y - axis at end of analyses .

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Trajectory plots of siRNA-transfected HUVECs under VEGF and/or Sema3E gradients for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses.


Rose plots of trajectories in ( A ) . Leaflets are grouped into 10 ° segments . radius of each leaflet represents cumulative number of migrating in corresponding angular bin . P values for Rayleigh represent non - random distributions of endpoints .

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Rose plots of cell trajectories in (A). Leaflets are grouped into 10° segments. The radius of each leaflet represents the cumulative number of cells migrating in the corresponding angular bin. P values for the Rayleigh test represent non-random distributions of cell endpoints.


Forward migration index ( FMI ) along y - x - axes during migration . n = 90 per group . Data represent mean ± . * * p < 0 . 01 ; * * * p < 0 . 001 ; NS , not significant , by Tukey - Kramer .

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Forward migration index (FMI) along the y- and x-axes during cell migration. n = 90 cells per group. Data represent mean ± SEM. **p < 0.01; ***p < 0.001; NS, not significant, by Tukey-Kramer test.


Confocal ( left three columns ) super - resolution ( right - most columns ) of subcellular distributions of ectopic RhoJ ( red ) , endogenous VEGFR2 ( green ) PlexinD1 ( left , ; right , blue ) in cultured HUVECs 6 h after transfection with indicated RhoJ constructs . Note colocalization of WT - RhoJ CA - RhoJ ( Q79L ) with PlexinD1 in perinuclear endosomes DN - RhoJ ( T35N ) with VEGFR2 in . - blue asterisks in PlexinD1 panels indicate nuclei . See also Appendix Fig S2 for full of super - resolution microscopy . Scale bar , 5 µm ( left ) ; 1 µm ( right ) .

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Confocal (left three columns) and super-resolution (right-most columns) images of subcellular distributions of ectopic RhoJ (red), endogenous VEGFR2 (green) and PlexinD1 (left, white; right, blue) in cultured HUVECs 6 h after transfection with the indicated RhoJ constructs. Note the colocalization of WT-RhoJ and CA-RhoJ (Q79L) with PlexinD1 in perinuclear endosomes and DN-RhoJ (T35N) with VEGFR2 in cytoplasmic and nuclear puncta. Light-blue asterisks in the PlexinD1 panels indicate nuclei. See also Appendix Fig S2 for the full images of super-resolution microscopy. Scale bar, 5 µm (left); 1 µm (right).


Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 / or PlexinD1 . n = 10 per group . Data represent mean .

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Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 and/or PlexinD1. n = 10 cells per group. Data represent mean.


Co - immunoprecipitation ( co - IP ) from 293T 48 h after transfection . WT - CA - RhoJ associated with PlexinD1 , while DN - RhoJ associated with VEGFR2 .

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Co-immunoprecipitation (co-IP) from 293T cells 48 h after transfection. WT- and CA-RhoJ associated with PlexinD1, while DN-RhoJ associated with VEGFR2.


Co - IP from 293T 48 h after transfection . RhoJ - PlexinD1 binding was disrupted by deleting RBD or ECD of PlexinD1 by 30 - min stimulation with Sema3E . Arrows indicate PD1 Full ( upper , 250 kDa ) PD1 △ RBD ( lower , 1 . 7 kDa smaller than PD1 Full ) .

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Co-IP from 293T cells 48 h after transfection. RhoJ-PlexinD1 binding was disrupted by deleting the RBD or ECD of PlexinD1 and by 30-min stimulation with Sema3E. Arrows indicate PD1 Full (upper, 250 kDa) and PD1△RBD (lower, 1.7 kDa smaller than PD1 Full).


PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation . Scale bar , 10 µm .

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PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation. Scale bar, 10 µm.


Super - resolution of subcellular distributions of ectopic WT - RhoJ ( red ) endogenous PlexinD1 ( green ) in HUVECs 24 h after transfection . RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation . Scale bar , 1 µm .

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Super-resolution images of subcellular distributions of ectopic WT-RhoJ (red) and endogenous PlexinD1 (green) in HUVECs 24 h after transfection. RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation. Scale bar, 1 µm.


Labeling of siRNA - transfected HUVECs with phalloidin ( ) 30 min after Sema3E stimulation . Scale bar , 50 µm .

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Labeling of siRNA-transfected HUVECs with phalloidin (white) 30 min after Sema3E stimulation. Scale bar, 50 µm.


of areas of HUVECs . n > 100 per group . Data represent mean ± . * * * p < 0 . 001 ; NS , not significant , by Tukey - Kramer .

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Quantification of areas of individual HUVECs. n > 100 cells per group. Data represent mean ± SEM. ***p < 0.001; NS, not significant, by Tukey-Kramer test.


Co - IP from HUVECs . Blots for β - actin shown in ( A ) were used to confirm equal loading in ( B ) , as same lysates were analyzed in both experiments .

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Co-IP from HUVECs. Blots for β-actin shown in (A) were used to confirm equal loading in (B), as the same cell lysates were analyzed in both experiments.


Immunoblots in siRNA - transfected HUVECs .

Immunoblots in siRNA-transfected HUVECs.


Co - IP from siRNA - transfected HUVECs . graph shows relative of PlexinD1 - VEGFR2 association , in which values at 0 min are set to 1 . n = 3 per group .

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Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.


Super - resolution of receptor colocalization in siRNA - transfected HUVECs 30 min after Sema3E stimulation . Left panels show immunolabeling of VEGFR2 ( green ) , PlexinD1 ( red ) , Nrp1 ( ) , with - blue asterisks indicating nuclei . Middle panels show magnified views of boxes in left panels . Right panels represent profiles along 5 μm arbitrary lines in middle panels . Scale bar , 10 µm . graph shows proportion of receptor complexes in PlexinD1 - positive vesicles . n = 10 per group .

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Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after Sema3E stimulation. Left panels show immunolabeling of VEGFR2 (green), PlexinD1 (red), and Nrp1 (white), with light-blue asterisks indicating nuclei. Middle panels show magnified views of white boxes in the left panels. Right panels represent fluorescence intensity profiles along 5 μm arbitrary lines in the middle panels. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in PlexinD1-positive vesicles. n = 10 cells per group.


Trajectory plots ( left ) rose plots ( right ) of HUVECs pre - treated with p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h . Red trajectories indicate with displacement to negative y - axis at end of analyses . P values for Rayleigh represent non - random distributions of endpoints . graphs show FMI along y - x - axes during migration . n = 90 per group .

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Trajectory plots (left) and rose plots (right) of HUVECs pre-treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses. P values for the Rayleigh test represent non-random distributions of cell endpoints. The graphs show FMI along the y- and x-axes during cell migration. n = 90 cells per group.


Super - resolution of receptor colocalization in siRNA - transfected HUVECs 30 min after VEGF stimulation . VEGFR2 ( green ) , PlexinD1 ( red ) , Nrp1 ( ) are shown as in Fig 4E . Scale bar , 10 µm . graph shows proportion of receptor complexes in PlexinD1 - positive vesicles . Data of " no stimuli " are same from Fig 4E . n = 10 per group .

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Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after VEGF stimulation. VEGFR2 (green), PlexinD1 (red), and Nrp1 (white) are shown as in Fig 4E. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in the PlexinD1-positive vesicles. Data of "no stimuli" are the same from Fig 4E. n = 10 cells per group.


Immunoblots in siRNA - transfected HUVECs . Blots for β - actin shown in ( C ) were used to confirm equal loading in ( D ) , as same lysates were analyzed in these experiments . graph shows changes of total VEGFR2 as a of values at 0 min . n = 3 per group .

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Immunoblots in siRNA-transfected HUVECs. Blots for β-actin shown in (C) were used to confirm equal loading in (D), as the same cell lysates were analyzed in these experiments. The graph shows changes of total VEGFR2 protein levels as a percentage of the values at 0 min. n = 3 per group.


Immunoblots in siRNA - transfected HUVECs . Note early transient activation of PLCγ , Erk1 / 2 , Akt , but not p38 MAPK , in RhoJ - knockdown ECs .

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Immunoblots in siRNA-transfected HUVECs. Note the early and transient activation of PLCγ, Erk1/2, and Akt, but not p38 MAPK, in RhoJ-knockdown ECs.


Immunoblots for ectopic RhoJ endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ - expressing vectors . graph shows of VEGFR2 relative to those of control set as 100 % . n = 3 per group .

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Immunoblots for ectopic RhoJ and endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ-expressing vectors. The graph shows protein levels of VEGFR2 relative to those of the control set as 100%. n = 3 per group.


Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model . Upper panels are snapshots of time - lapse imaging in which EC nuclei are labeled by SYTO dye ( green ) . In analyzed regions framed by dotted lines , centroids of first five EC nuclei from tip at 0 min are pseudocolored in magenta those of remaining EC nuclei are shown in cyan . Initial discrete labeling becomes intermixed over time ( see also Movie EV1 ) . Lower panels show trajectory analyses representing normalized positional changes of ECs during vessel elongation . Scale bar , 100

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Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model. Upper panels are snapshots of time-lapse imaging in which EC nuclei are labeled by SYTO dye (green). In analyzed regions framed by dotted lines, the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan. Initial discrete labeling becomes intermixed over time (see also Movie EV1). Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation. Scale bar, 100 µ


of kinetics of EC migration . Values of forward reverse migration for RhojWT / WT are normalized to 1 . n = 33 vessel branches per group .

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Quantification of the kinetics of EC migration. Values of forward and reverse migration for RhojWT/WT are normalized to 1. n = 33 vessel branches per group.


of vessel elongation ( left graph ) . n = 33 vessel branches per group . Scatter plot shows relationship between " vessel elongation " " reverse migration " in RhojGFP / GFP aortic rings as evaluated by a Pearson correlation coefficient .

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Quantification of vessel elongation (left graph). n = 33 vessel branches per group. Scatter plot shows relationship between "vessel elongation" and "reverse migration" in RhojGFP/GFP aortic rings as evaluated by a Pearson correlation coefficient.


Labeling for YFP / GFP ( green ) , CD31 ( red ) , ETS transcription factor ERG ( blue ) in retinal vascular fronts of P5 CAG - MerCreMer : R26R - EYFPflox / WT CAG - MerCreMer : Rhojfloxflox mice after intraperitoneal ( i . p . ) injections of 10 µg of 4 - hydroxytamoxifen ( 4OHT ) at P1 . Scale bar , 50 µm . Relative contribution of YFP / GFP - positive ECs to tip positions . n = 16 per group . Values for CAG - MerCreMer : R26R - EYPflox / WT are normalized to 1 .

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Labeling for YFP/GFP (green), CD31 (red), and ETS transcription factor ERG (blue) in retinal vascular fronts of P5 CAG-MerCreMer:R26R-EYFPflox/WT and CAG-MerCreMer:Rhojfloxflox mice after intraperitoneal (i.p.) injections of 10 µg of 4-hydroxytamoxifen (4OHT) at P1. Scale bar, 50 µm.   Relative contribution of YFP/GFP-positive ECs to the tip positions. n = 16 per group. Values for CAG-MerCreMer:R26R-EYPflox/WT are normalized to 1.


Labeling for GFP ( green ) CD31 ( red ) in retina of P4 RhojGFP / GFP mouse . Scale bar , 200 µm ( left ) ; 20 µm ( right ) .

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Labeling for GFP (green) and CD31 (red) in retina of P4 RhojGFP/GFP mouse. Scale bar, 200 µm (left); 20 µm (right).


Labeling for EdU ( green ) , ERG ( red ) , CD31 ( gray ) in retinas of P4 RhojWT / WT RhojGFP / GFP mice 2 h after i . p . EdU injections . Scale bar , 20 µm . graph shows EC proliferation index in retinal capillaries behind angiogenic fronts . n = 12 per group .

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Labeling for EdU (green), ERG (red), and CD31 (gray) in retinas of P4 RhojWT/WT and RhojGFP/GFP mice 2 h after i.p. EdU injections. Scale bar, 20 µm. The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts. n = 12 per group.


Labeling for CD31 in retinas of P4 P7 RhojWT / WT RhojGFP / GFP mice . Scale bars , 500 µm .

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Labeling for CD31 in retinas of P4 and P7 RhojWT/WT and RhojGFP/GFP mice. Scale bars, 500 µm.


Morphometric analyses of retinal vessels in RhojWT / WT ( P4 , n = 18 ; P7 , n = 8 ) RhojGFP / GFP ( P4 , n = 16 ; P7 , n = 11 ) mice .

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Morphometric analyses of retinal vessels in RhojWT/WT (P4, n = 18; P7, n = 8) and RhojGFP/GFP (P4, n = 16; P7, n = 11) mice.


Labeling for CD31 in retinas of P4 Rhojflox / flox Pdgfb - iCreERT2 : Rhojflox / flox ( RhojiΔEC ) mice after single i . p . injection of 100 µg of 4OHT at P1 . Scale bar , 500 µm .

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Labeling for CD31 in retinas of P4 Rhojflox/flox and Pdgfb-iCreERT2:Rhojflox/flox (RhojiΔEC) mice after single i.p. injection of 100 µg of 4OHT at P1. Scale bar, 500 µm.


Morphometric analyses of retinal vessels in P4 Rhojflox / flox ( n = 7 ) Rhoji ∆ EC ( n = 6 ) mice .

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Morphometric analyses of retinal vessels in P4 Rhojflox/flox (n = 7) and Rhoji∆EC (n = 6) mice.


Relative expression of Vegfa Sema3e mRNA from WT mouse retinas . n = 3 per group .

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Relative expression levels of Vegfa and Sema3e mRNA from WT mouse retinas. n = 3 per group.


Labeling for GFP ( green ) CD31 ( red ) in retina of P18 OIR - RhojGFP / WT mouse . Scale bar , 200

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Labeling for GFP (green) and CD31 (red) in retina of P18 OIR-RhojGFP/WT mouse. Scale bar, 200 µ


Labeling for CD31 in retinas of P18 OIR - Rhojflox / flox OIR - Rhoji ∆ EC mice after daily i . p . injection of 200 µg of 4OHT from P12 . Scale bar , 500 µm . of areas of neovascular tufts in P18 OIR - Rhojflox / flox OIR - Rhoji ∆ EC mice . n = 6 per group .

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Labeling for CD31 in retinas of P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice after daily i.p. injection of 200 µg of 4OHT from P12. Scale bar, 500 µm. Quantification of areas of neovascular tufts in P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice. n = 6 per group.


( A ) Western blot of Beclin ‐ 1 FACS profiles following siRNA treatment in HeLa stably expressing a HA ‐ Beclin ‐ 1 construct resistant to siRNA targeting endogenous Beclin ‐ 1 .

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(A) Western blot of Beclin‐1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA‐Beclin‐1 construct resistant to the siRNA targeting endogenous Beclin‐1.


( B ) FACS profiles of treated with indicated siRNA following nocodazole blockage release .

(B) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release.


( C , D ) of in mitosis ( C ) of each step of mitosis ( D ) by H3Ser10P for Control Beclin ‐ 1 siRNA ‐ treated . Error bars represent standard deviations ( s . d . ) ( n = number of counted ) calculated on three independent experiments . Two three asterisks indicate significant results ( P = 0 . 0028 P = 0 . 0006 , respectively ) .

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(C,D) Quantification of cells in mitosis (C) and quantification of each step of mitosis (D) by H3Ser10P staining for Control and Beclin‐1 siRNA‐treated cells. Error bars represent standard deviations (s.d.) (n=number of counted cells) calculated on three independent experiments. Two and three asterisks indicate significant results (P=0.0028 and P=0.0006, respectively).


( E ) Knockdown of Beclin ‐ 1 increases number of mitotic with misaligned chromosomes . siRNA ‐ treated HeLa were fixed in PFA , immunolabelled for α ‐ tubulin ( green ) stained with DAPI ( blue ) . arrows indicate misaligned chromosomes . of with misaligned chromosomes is shown at right ( n = number of counted ) . Three asterisks indicate significant results ( P = 0 . 0001 ) . Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; HA , haemagglutinin ; PFA , paraformaldehyde ; siRNA , small interfering .

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(E) Knockdown of Beclin‐1 increases the number of mitotic cells with misaligned chromosomes. siRNA‐treated HeLa cells were fixed in PFA, immunolabelled for α‐tubulin (green) and stained with DAPI (blue). White arrows indicate misaligned chromosomes. Quantification of cells with misaligned chromosomes is shown at the right (n=number of counted cells). Three asterisks indicate significant results (P=0.0001). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; HA, haemagglutinin; PFA, paraformaldehyde; siRNA, small interfering RNA.


Beclin ‐ 1 depletion prolongs prometaphase by delaying chromosome congression . ( A - H ) Time ‐ lapse films of HeLa stably expressing YFP ‐ α ‐ tubulin H2B ‐ Cherry treated with indicated siRNA . pictures are acquired every 10 min . Time lapse starts at envelope breakdown . ( A ) Mitosis in control . ( B ) Western blot of whole ‐ lysates from Control , Beclin ‐ 1 Beclin ‐ 1 + Mad2 siRNA ‐ treated . ( C - E ) Three different classes of mitotic progression were observed upon Beclin ‐ 1 depletion . Higher magnification of lagging chromatids in anaphase of Beclin ‐ 1 ‐ depleted is shown on right of ( D ) .

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Beclin‐1 depletion prolongs prometaphase by delaying chromosome congression. (A-H) Time‐lapse films of HeLa cells stably expressing YFP‐α‐tubulin and H2B‐Cherry and treated with the indicated siRNA. The pictures are acquired every 10 min. Time lapse starts at nuclear envelope breakdown. (A) Mitosis in control cells. (B) Western blot of whole‐cell lysates from Control, Beclin‐1 and Beclin‐1+Mad2 siRNA‐treated cells. (C-E) Three different classes of mitotic progression were observed upon Beclin‐1 depletion. Higher magnification of lagging chromatids in the anaphase of Beclin‐1‐depleted cells is shown on the right of (D).


( F ) prometaphase delay is dependent on MAD2 spindle checkpoint . treated simultaneously with siRNA targeting MAD2 Beclin ‐ 1 rapidly exit mitosis . ( G ) Average duration of mitosis in different classes of Beclin ‐ 1 ‐ depleted , compared to control MAD2 depletions . n = number of counted in three independent experiments . Error bars on graphs represent s . d .

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(F) The prometaphase delay is dependent on MAD2 and the spindle checkpoint. Cells treated simultaneously with siRNA targeting MAD2 and Beclin‐1 rapidly exit mitosis. (G) Average duration of mitosis in different classes of Beclin‐1‐depleted cells, compared to control and MAD2 depletions. n=number of counted cells in three independent experiments. Error bars on graphs represent s.d.


( H ) frequency of different Beclin ‐ 1 depended on efficiency of Beclin ‐ 1 knockdown . Mitotic progression in treated with increasing concentrations of Beclin ‐ 1 siRNA was followed by video ‐ microscopy . Left panel : western blot of Beclin ‐ 1 upon RNAi treatment ; right panel : of appearance of three different mitotic progression patterns , that is , prolonged mitosis with normal anaphase ( as C ) , with lagging chromatids ( as D ) mitotic exit without clear anaphase ( as E ) upon increased Beclin ‐ 1 knockdown experiments . NEB , envelop breakdown ; RNAi , interference ; siRNA , small interfering ; YFP , yellow .

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(H) The frequency of the different Beclin‐1 phenotypes depended on the efficiency of Beclin‐1 knockdown. Mitotic progression in cells treated with increasing concentrations of Beclin‐1 siRNA was followed by video‐microscopy. Left panel: western blot of Beclin‐1 upon RNAi treatment; right panel: quantification of the appearance of the three different mitotic progression patterns, that is, prolonged mitosis with normal anaphase (as C), with lagging chromatids (as D) and mitotic exit without clear anaphase (as E) upon increased Beclin‐1 knockdown experiments. NEB, nuclear envelop breakdown; RNAi, RNA interference; siRNA, small interfering RNA; YFP, yellow fluorescent protein.


( A ) Inter ‐ kinetochore is reduced in unaligned chromosomes of Beclin ‐ 1 ‐ depleted . between each kinetochore pair was quantified in siRNA ‐ treated control treated with nocodazole for 3 h before fixation . were stained with anti ‐ CREST antibody DAPI . of between kinetochore pairs ( indicated by coloured bars ) is reported at bottom done on sister kinetochores present in same focal plane . One asterisk indicates significant result ( P = 0 . 0365 ) . Graphs represent three independent experiments ( error bars = s . d . ) .

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(A) Inter‐kinetochore distance is reduced in unaligned chromosomes of Beclin‐1‐depleted cells. The distance between each kinetochore pair was quantified in siRNA‐treated and control cells treated with nocodazole for 3 h before fixation. Cells were stained with anti‐CREST antibody and DAPI. Quantification of the distance between kinetochore pairs (indicated by coloured bars) is reported at the bottom and done on sister kinetochores present in the same focal plane. One asterisk indicates significant result (P=0.0365). Graphs represent three independent experiments (error bars=s.d.).


( B ) Depletion of Beclin ‐ 1 leads to defects in K ‐ MT attachment . siRNA ‐ treated were cooled on ice before fixation to reveal stable k ‐ fibres then stained with anti ‐ α ‐ tubulin ( red ) anti ‐ CREST ( green ) antibodies . Numbers point to magnified areas indicate mode of attachment of k ‐ fibres to kinetochores ( 1 , bi ‐ oriented ; 2 , unattached ; 3 , mono ‐ oriented kinetochores ) . Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; NS , not significant ; siRNA , small interfering .

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(B) Depletion of Beclin‐1 leads to defects in K‐MT attachment. siRNA‐treated cells were cooled on ice before fixation to reveal stable k‐fibres and then stained with anti‐α‐tubulin (red) and anti‐CREST (green) antibodies. Numbers point to magnified areas and indicate the mode of attachment of k‐fibres to kinetochores (1, bi‐oriented; 2, unattached; 3, mono‐oriented kinetochores). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; NS, not significant; siRNA, small interfering RNA.


( A ) CENP ‐ E CREST . Two asterisks indicate significant results , P = 0 . 0053 .

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(A) CENP‐E and CREST. Two asterisks indicate significant results, P=0.0053.


( B ) CENP ‐ F CREST . Three asterisks indicate significant results , P = 0 . 0006 .

(B) CENP‐F and CREST. Three asterisks indicate significant results, P=0.0006.


( C ) ZW10 CREST . Three asterisks indicate significant results , P = 0 . 0007 .

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(C) ZW10 and CREST. Three asterisks indicate significant results, P=0.0007.


( D ) MAD2 CREST . Two asterisks indicate significant results , P = 0 . 0032 .

(D) MAD2 and CREST. Two asterisks indicate significant results, P=0.0032.


( F ) KNL ‐ 1 CREST . On right , magnified areas of of of interest normalized to of CREST are shown . Graphs represent three independent experiments ( error bars = s . d . ) . Scale bar , 10 μm .

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(F) KNL‐1 and CREST. On the right, magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown. Graphs represent three independent experiments (error bars=s.d.). Scale bar, 10 μm.


( G ) Western blot of whole ‐ lysates from control Beclin ‐ 1 siRNA ‐ treated . siRNA , small interfering .

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(G) Western blot of whole‐cell lysates from control and Beclin‐1 siRNA‐treated cells. siRNA, small interfering RNA.