( E F ) RAW264 . 7 were transiently transfected with control or miR - 155 mimic for 24 h , then incubated with a specific dye MDC ( 50 µM ) . MDC - labeled autophagic vacuoles were detected by confocal microscopy ( E ) with MDC - positive autophagic vacuoles were quantified ( F ) . treated with rapamycin were used as a positive control . Arrows indicate GFP - LC3 or autophagic vacuoles labeled with MDC ; scale bar = 5 µm . Data are shown as mean ± of three independent experiments ( n = 300 ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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 O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O

(E and F) RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with a specific fluorescent dye MDC (50 µM). The MDC-labeled autophagic vacuoles were detected by confocal microscopy (E) and cells with MDC-positive autophagic vacuoles were quantified (F). Cells treated with rapamycin were used as a positive control. Arrows indicate the GFP-LC3 puncta or autophagic vacuoles labeled with MDC; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 300 cells). **, p<0.01; ***, p<0.001.


( A ) RAW264 . 7 stably expressing GFP - LC3 were transiently transfected with control or miR - 155 mimic then infected with Texas Red - labeled BCG for 1 h . co - localization of BCG with LC3 was detected by confocal microscopy . ( B ) of co - localization of BCG with LC3 - positive autophagosomes is shown .

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

(A) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG with LC3 was detected by confocal microscopy. (B) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown.


( C ) RAW264 . 7 were transiently transfected with control or miR - 155 mimic , then infected with Texas Red - labeled BCG for 1 h . Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( Green ) . co - localization of BCG with endogenous LC3 was detected by confocal microscopy . ( D ) of co - localization of BCG with LC3 - positive autophagosomes is shown .

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 B-MEASURED_VAR 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 B-MEASURED_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

(C) RAW264.7 cells were transiently transfected with control or miR-155 mimic, and then infected with Texas Red-labeled BCG for 1 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green). The co-localization of BCG with endogenous LC3 was detected by confocal microscopy. (D) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown.


( E ) After transient transfection with control or miR - 155 mimic , RAW264 . 7 were infected with Texas Red - labeled BCG for 1 h , then were labeled with a specific dye MDC ( 50 µM ) for autophagic vacuoles . co - localization of BCG with MDC - positive autophagic vacuoles was detected by confocal microscopy . ( F ) of co - localization of BCG with MDC - positive autophagosomes is shown . treated with rapamycin were used as a positive control . Arrows indicate co - localization of BCG with autophagosomes ; scale bar = 5 µm . Data are shown as mean ± of three independent experiments ( n = 100 phagosomes ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) After transient transfection with control or miR-155 mimic, RAW264.7 cells were infected with Texas Red-labeled BCG for 1 h, and then were labeled with a specific fluorescent dye MDC (50 µM) for autophagic vacuoles. The co-localization of BCG with MDC-positive autophagic vacuoles was detected by confocal microscopy. (F) Quantification of the co-localization of BCG with MDC-positive autophagosomes is shown. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with autophagosomes; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01; ***, p<0.001.


( A B ) RAW 264 . 7 were transiently transfected with control or miR - 155 mimic for 24 h , then incubated with DMSO or 3 - MA for 2 h . of LC3 were detected by Western - blot in uninfected ( A )

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 O O O O O O O O O O O

(A and B) RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with DMSO or 3-MA for 2 h. Protein levels of LC3 were detected by Western-blot in uninfected cells (A)


Intracellular mycobacterial viability was determined at indicated timepoints by CFU after BCG challenge for 1 h ( B )

Intracellular mycobacterial viability was determined at the indicated timepoints by CFU assay after BCG challenge for 1 h (B)


( C D ) RAW 264 . 7 were transiently co - transfected with control or miR - 155 mimic together with a control siRNA or Atg7 siRNA . expression of Atg7 LC3 were detected by Western - blot ( C ) .

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

(C and D) RAW 264.7 cells were transiently co-transfected with control or miR-155 mimic together with a control siRNA or Atg7 siRNA. The expression levels of Atg7 and LC3 were detected by Western-blot (C).


Intracellular mycobacterial viability was determined by CFU at indicated time after challenging with BCG for 1 h ( D ) . Values of LC3 - II / β - actin ratios are indicated below representative blot . Data are shown as mean ± of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Intracellular mycobacterial viability was determined by CFU assay at the indicated time after challenging with BCG for 1 h (D). Values of LC3-II/β-actin ratios are indicated below the representative blot. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.


( A ) Sequences of mouse human miR - 155 their predicted interactions with conserved 7 - mer 1A miR - 155 seeds found within Rheb 3 ′ UTRs of different species are shown . of Rheb 3 ′ UTR seed mutant used for reporter assays predicted disruption of miR - 155 interaction are also shown .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O

(A) Sequences of mouse and human miR-155 and their predicted interactions with conserved 7-mer 1A miR-155 seeds found within the Rheb 3′UTRs of different species are shown. The sequence of the Rheb 3′UTR seed mutant used for the reporter assays and the predicted disruption of the miR-155 interaction are also shown.


B ) RAW264 . 7 were co - transfected with control or miR - 155 mimic a wild - type ( WT - Rheb ) or mutated Rheb 3 ′ UTR ( mut - Rheb ) luciferase reporter plasmid assessed for luciferase activity at 24 h after transfection . Data are shown as mean ± of three independent experiments . * * * , p < 0 . 001 ; NS , not significant .

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 B-MEASURED_VAR O O O B-MEASURED_VAR 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 O O O

B) RAW264.7 cells were co-transfected with control or miR-155 mimic and a wild-type (WT-Rheb) or mutated Rheb 3′UTR (mut-Rheb) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection. Data are shown as the mean ± SEM of three independent experiments. ***, p<0.001; NS, not significant.


( C - F ) RAW264 . 7 were transfected with miR - 155 mimic ( C E ) or inhibitor ( D F ) for 24 h either left uninfected or infected with BCG . expression of Rheb were detected by Western - blot . Values of Rheb / β - actin ratios are indicated below representative blot ( C D )

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 O O O O O O B-MEASURED_VAR 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

(C-F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D)


( C - F ) RAW264 . 7 were transfected with miR - 155 mimic ( C E ) or inhibitor ( D F ) for 24 h either left uninfected or infected with BCG . expression of Rheb were detected by Western - blot . Values of Rheb / β - actin ratios are indicated below representative blot ( C D ) , expression of Rheb mRNA were detected by RT - PCR ( E F ) .

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 O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O

(C-F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D), and expression levels of Rheb mRNA were detected by RT-PCR (E and F).


( A ) RAW264 . 7 were transfected with a plasmid expressing Rheb for 24 h , then incubated with DMSO or BafA . for 2 h . expression of Rheb LC3 were detected by Western - blot . Values of LC3 - II / β - actin ratios are indicated below representative blot .

O O O O O O 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 O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

(A) RAW264.7 cells were transfected with a plasmid expressing Rheb for 24 h, and then incubated with DMSO or BafA. for 2 h. The expression levels of Rheb and LC3 were detected by Western-blot. Values of LC3-II/β-actin ratios are indicated below the representative blot.


( B C ) RAW264 . 7 were co - transfected with control or miR - 155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h , then infected with Texas Red - labeled BCG for 1 h . co - localization of BCG MDC - labeled autophagosome was detected by confocal microscopy . Arrows indicate of BCG co - localization with autophagosomes described in B is shown ( C ) .

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 B-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

(B and C) RAW264.7 cells were co-transfected with control or miR-155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h, and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG and MDC-labeled autophagosome was detected by confocal microscopy. Arrows indicate Quantification of BCG co-localization with autophagosomes described in B is shown (C).


D ) RAW264 . 7 were co - transfected with control or miR - 155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h . were infected with BCG for 1 h , washed for three times to remove extracellular mycobacteria . Intracellular mycobacterial viability was determined by CFU at indicated timepoints . Data are shown as mean ± of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; NS , not significant .

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 B-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

D) RAW264.7 cells were co-transfected with control or miR-155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h. Cells were infected with BCG for 1 h, and washed for three times to remove extracellular mycobacteria. Intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.


( b , c ) Immunoblots of recruitment reactions performed as described in ( a ) , with indicated omitted . In ( c ) , a mid reaction wash in high salt ( 0 . 5 M NaCl ) buffer ( HSW ) was included following DDK phosphorylation as indicated .

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(b, c) Immunoblots of recruitment reactions performed as described in (a), with the indicated proteins omitted. In (c), a mid reaction wash in high salt (0.5 M NaCl) buffer (HSW) was included following DDK phosphorylation as indicated.


( d ) Recruitment reaction performed as in ( a ) , using wild type ( wt ) or a Cdc45 - binding mutant ( 3E1 ) of Sld3 .

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(d) Recruitment reaction performed as in (a), using wild type (wt) or a Cdc45-binding mutant (3E1) of Sld3.


( f ) Wild type or mutant FLAG - Sld3 was added to an S - phase extract tested for interaction with Cdc45 . Immunoprecipitated were analysed by immunoblot .

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(f) Wild type or mutant FLAG-Sld3 was added to an S-phase protein extract and tested for interaction with Cdc45. Immunoprecipitated proteins were analysed by immunoblot.


( h ) Reaction performed as in ( g ) . Nascent was separated in a 0 . 7 % alkaline agarose gel in this subsequent replication reactions .

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(h) Reaction performed as in (g). Nascent DNA was separated in a 0.7% alkaline agarose gel in this and all subsequent replication reactions.


( a ) Reaction performed as in Fig . 1a , except Cdc45 was omitted .

(a) Reaction performed as in Fig. 1a, except Cdc45 was omitted.


( b ) Reaction performed as in Fig . 1g , with indicated omitted .

(b) Reaction performed as in Fig. 1g, with the indicated proteins omitted.


( e ) Reaction performed as in ( a ) , except Sld3 / 7 was incubated with Rad53 before addition to reactions . ATP was omitted from this pre - incubation as indicated .

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

(e) Reaction performed as in (a), except Sld3/7 was incubated with Rad53 before addition to reactions. ATP was omitted from this pre-incubation as indicated.


( f ) Sld3 recruitment reaction performed as in ( a ) . Sld7 was omitted from this experiment .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(f) Sld3 recruitment reaction performed as in (a). Sld7 was omitted from this experiment.


( b ) Recruitment reaction performed as described in ( a ) .

(b) Recruitment reaction performed as described in (a).


( c ) Sld3 / 7 recruitment reaction conducted as in Fig . 2a .

O O O B-CONTROLLED_VAR O O O O O O O O O O O

(c) Sld3/7 recruitment reaction conducted as in Fig. 2a.


( d ) Replication reaction performed as in Fig . 1g .

(d) Replication reaction performed as in Fig. 1g.


( e ) Representative tetrads from dissection of SLD3 + / sld3 - 6E heterozygotes .

O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O

(e) Representative tetrads from dissection of SLD3+/sld3-6E heterozygotes.


( b , c , D ) Immunoblot of reactions performed as described in ( a ) . In ( B ) , either reconstituted Sld3 / 7 ( lanes 6 7 ) or Sld3 alone ( lanes 8 9 ) was coupled to beads . In ( C ) , MCM was treated with Lambda Phosphatase after dissociation from ( lanes 3 7 ) .

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

(b, c, D) Immunoblot of reactions performed as described in (a). In (B), either reconstituted Sld3/7 complex (lanes 6 and 7) or Sld3 alone (lanes 8 and 9) was coupled to beads. In (C), MCM was treated with Lambda Phosphatase after dissociation from DNA (lanes 3 and 7).


( b , c , D ) Immunoblot of reactions performed as described in ( a ) . In ( D ) , Sld7 was omitted .

O O O 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, c, D) Immunoblot of reactions performed as described in (a). In (D), Sld7 was omitted.


( E ) 30 - residue phosphorylated Mcm6 peptides were bound to streptavidin - coated beads used as bait to pulldown Sld3 . Peptide - coupled beads were dephosphorylated with Lambda Phosphatase as indicated .

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

(E) 30-residue phosphorylated Mcm6 peptides were bound to streptavidin-coated beads and used as bait to pulldown Sld3. Peptide-coupled beads were dephosphorylated with Lambda Phosphatase as indicated.


( F ) Reaction conducted as in ( a ) , using wild type ( wt ) or indicated S / T - A substitution mutant versions of Mcm4 Mcm6 . Positions of mutations in Mcm6 are summarised in Fig . EV5c .

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(F) Reaction conducted as in (a), using wild type (wt) or the indicated S/T-A substitution mutant versions of Mcm4 and Mcm6. Positions of mutations in Mcm6 are summarised in Fig. EV5c.


( a ) Sld3 / 7 recruitment reaction performed as in Fig . 2a .

O O O B-MEASURED_VAR O O O O O O O O O O O

(a) Sld3/7 recruitment reaction performed as in Fig. 2a.


( b ) Replication reaction performed as in Fig . 1g . Experiments in ( a ) ( b ) used MCM containing wild type ( wt ) or indicated S / T - A substitution mutant versions of Mcm4 Mcm6 .

O O O O O O O O O O O O O O O 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

(b) Replication reaction performed as in Fig. 1g. Experiments in (a) and (b) used MCM containing wild type (wt) or the indicated S/T-A substitution mutant versions of Mcm4 and Mcm6.


c ) of ( b ) , performed using ImageQuant software ( GE Healthcare ) .

c) Quantification of (b), performed using ImageQuant software (GE Healthcare).


( d ) Representative tetrads from diploids heterozygous for mcm4 - 25A mcm6ΔN + 11A .

O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O

(d) Representative tetrads from diploids heterozygous for mcm4-25A and mcm6ΔN+11A.


( b ) Immunoblots of reactions performed as in ( a ) part i ( lanes 1 - 3 ) or ii ( lanes 4 - 6 ) .

(b) Immunoblots of reactions performed as in (a) part i (lanes 1-3) or ii (lanes 4-6).


( c ) Reactions in i ii were performed as described in ( a ) parts i ii , respectively .

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(c) Reactions in i and ii were performed as described in (a) parts i and ii, respectively.


( a , b ) Recruitment reactions performed as in Fig . 2a . Sld7 was omitted in ( b ) .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

(a, b) Recruitment reactions performed as in Fig. 2a. Sld7 was omitted in (b).


( c ) Recruitment reaction conducted as in Fig . 3a , with washes performed at 0 . 3 M KCl .

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(c) Recruitment reaction conducted as in Fig. 3a, with washes performed at 0.3 M KCl.


( d ) Replication reaction performed as in Fig . 1g . Experiments in ( a ) - ( d ) used MCM containing wild type ( wt ) or S / T - D substitution mutants of Mcm4 Mcm6 as indicated . In ( d ) , MCM loading was equalised across samples by using 5 - fold more mutant than wild type MCM , as is shown in Fig . EV6c .

O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O

(d) Replication reaction performed as in Fig. 1g. Experiments in (a)-(d) used MCM containing wild type (wt) or S/T-D substitution mutants of Mcm4 and Mcm6 as indicated. In (d), MCM loading was equalised across samples by using 5-fold more mutant than wild type MCM protein, as is shown in Fig. EV6c.


B Representative TIRF - M of DCP1 - GFP ( DCP1pro : DCP1 - GFP transgene ) in lateral PM of epidermal showing transient attachment of DCP1 - positive to PM . Yellow arrowheads denote a PB that shows motility at PM focal plane ; blue arrowheads show a PB that transiently localizes to PM . Scale bars : 2 μm . corresponding kymographs are shown to right . Right : distribution of immobile mobile DCP1 molecules relative to motility log ( D ) value of - 0 . 75 ( threshold ; see Material methods for details ) , in NS or HS conditions ( D , diffusion coefficient ) . Inset : trajectories of mobile DCP1 - GFP in NS HS conditions ( 500 frames , n = 120 ) , showing a combination of directional Brownian motion for both NS / HS . green arrowheads denote beginning of NS HS tracks for DCP1 - GFP .

O O 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 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 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 O O O O

B Representative TIRF-M of DCP1-GFP (DCP1pro:DCP1-GFP transgene) in the lateral PM of epidermal cells showing the transient attachment of DCP1-positive puncta to the PM. Yellow arrowheads denote a PB that shows motility at the PM focal plane; blue arrowheads show a PB that transiently localizes to the PM. Scale bars: 2 μm. The corresponding kymographs are shown to the right. Right: distribution of immobile and mobile DCP1 molecules relative to the motility log(D) value of -0.75 (threshold; see Material and methods for details), in NS or HS conditions (D, diffusion coefficient). Inset: individual trajectories of mobile DCP1-GFP in NS and HS conditions (500 frames, n = 120), showing a combination of directional and Brownian motion for both NS/HS. The green arrowheads denote the beginning of the NS and HS tracks for DCP1-GFP.


C Representative confocal from lines co - expressing RPS5apro : HF - mScarlet - DCP1 PIN2pro : PIN2 - GFP ( epidermal , region 3 , Scale bars : 5 μm ) . Bottom : polarity index of DCP1 in root meristematic ( compared to propidium iodide ( PI ) tubulin of root ; N , biological replicates = 3 roots , n = 13 ) . Polarity index calculated as of average of apical basal VS lateral side of signal of root epidemies . arrowhead in PIN2 indicates plate or PM in DCP1 . Right : representative confocal showing that PM localization is independent of promoter used ( DCP1pro , 35Spro ; region 2 , epidermal , or RPS5apro on lower right ) . details from inset show increased localization at edge ( discussed later ) . mSc , mScarlet .

O O O O O O O O O O O O O O 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 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 B-MEASURED_VAR 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 O O O O O O O O O O O

C Representative confocal micrographs from lines co-expressing RPS5apro:HF-mScarlet-DCP1 and PIN2pro:PIN2-GFP (epidermal cells, region 3, Scale bars: 5 μm). Bottom: polarity index of DCP1 in root meristematic cells (compared to propidium iodide (PI) and tubulin staining of root cells; N, biological replicates = 3 roots, n = 13 cells). Polarity index calculated as the ratio of average of apical and basal VS lateral side of fluorescence signal intensity of the root epidemies cells. The arrowhead in PIN2 indicates the cell plate or PM in DCP1. Right: representative confocal micrographs showing that PM localization is independent of the promoter used (DCP1pro, 35Spro; region 2, epidermal cells, or RPS5apro on the lower right). The details from the inset show increased localization at the cell edge (discussed later). mSc, mScarlet.


B Representative confocal micrograph of DCP1 - GFP ( DCP1pro : DCP1 - GFP , transgene ) in root meristematic under NS / HS conditions ( region 2 ) . Scale bars , 5 μm . Note depletion of DCP1 from PM upon HS , but increased edge / vertex signal ( yellow arrowheads denote vertex signal ) . Right : DCP1 signal at PM or edge / vertex ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 18 - 23 PMs or edges / vertices ) .

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B Representative confocal micrograph of DCP1-GFP (DCP1pro:DCP1-GFP, transgene) in root meristematic cells under NS/HS conditions (region 2). Scale bars, 5 μm. Note the depletion of DCP1 from the PM upon HS, but the increased edge/vertex signal (yellow arrowheads denote the vertex signal). Right: DCP1 signal intensity at the PM or edge/vertex (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 18-23 PMs or edges/vertices).


C Representative confocal showing fusion ( coarsening ) , fission , growth of PBs ( DCP1 - positive ) at PM ( region 3 ) . Right : states of PBs ( dynamic : fusion fission non - dynamic : stable ; N , biological replicates = 2 , n ( pooled data of 3 biological replicates ) = 6 - 8 PBs ) . As a cautionary note , " stable " PBs may not show dynamicity in imaging time used ( ~ 3 - 5 min ) but later , may do .

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C Representative confocal images showing fusion (coarsening), fission, and growth of PBs (DCP1-positive) at the PM (region 3). Right: states of PBs (dynamic: fusion and fission and non-dynamic: stable; N, biological replicates = 2, n (pooled data of 3 biological replicates) = 6-8 PBs). As a cautionary note, the "stable" PBs may not show dynamicity in the imaging time used (~3-5 min) but later, may do.


Confocal showing single optical sections PLA - assays producing signal that resembles spots . antibodies used were anti - FLAG / anti - GFP detecting HF - mScarlet - DCP1 / DCP2 - YFP , respectively ( RPS5apro : HF - mScarlet - DCP1 35Spro : DCP2 - YFP ) . Inset 1 : magnification showing colocalization of PLA spots with DCP1 or DCP2 signals ( colocalization Pearson ' s correlation coefficient PCC value for spots shown ) . dotted in PLA channel corresponds to PM plane . Inset 2 : positive PLA signal for DCP1 DCP2 in nucleus . " n1 - n3 " correspond to nuclei regions ( green circles ) . On right , note PLA spot nearby nucleus ( " detail of PLA signal " ) . chart shows of PLA spots per at ( cytoplasm ) or on PM ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 16 - 33 ) . As a cautionary technical note , , or PM " spots " do not connote physiologically relevant , condensates or PM clusters . Scale bar : 20 μm / 10 μm for insets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal micrographs showing single optical sections PLA-assays producing signal that resembles spots. The antibodies used were anti-FLAG/anti-GFP detecting the HF-mScarlet-DCP1/ DCP2-YFP, respectively (RPS5apro:HF-mScarlet-DCP1 and 35Spro:DCP2-YFP). Inset 1: magnification showing the colocalization of PLA spots with DCP1 or DCP2 signals (colocalization and Pearson's correlation coefficient PCC value for the spots shown). The dotted white line in the PLA channel corresponds to the PM plane. Inset 2: positive PLA signal for DCP1 and DCP2 in the nucleus. "n1-n3" correspond to nuclei regions (green circles). On the right, note the PLA spot nearby the nucleus ("detail of PLA signal"). The chart shows the quantification of PLA spots per cell at puncta (cytoplasm) or on the PM (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 16-33 cells). As a cautionary technical note, the cytoplasmic, nuclear or PM "spots" do not connote physiologically relevant puncta, condensates or PM clusters. Scale bar: 20 μm / 10 μm for the insets.


B Representative confocal with acceptor photobleaching - FRET efficiency between SCAR2 - mCherry ( up ) or BRK1 - mRuby ( down ) DCP1 - GFP ( epidermal , regions 1 3 ) . Right : normalized FRET efficiency between SCAR2 or BRK1 DCP1 , respectively , among different developmental root regions ( N , biological replicates = 2 , n = 16 ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-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

B Representative confocal micrographs with acceptor photobleaching-FRET efficiency between SCAR2-mCherry (up) or BRK1-mRuby (down) and DCP1-GFP (epidermal cells, regions 1 and 3). Right: normalized FRET efficiency between SCAR2 or BRK1 and DCP1, respectively, among the different developmental root regions (N, biological replicates = 2, n = 16 cells).


D Representative confocal showing DCP1 localization detected by α - DCP1 in wild type ( WT ) or scar1 scar2 scar3 scar4 ( scar1234 ) quadruple mutant or in live - imaging of mScarlet - DCP1 ( RPS5apro : HF - mScarlet - DCP1 ) in WT or brk1 mutant ( bottom right ; epidermal , region 3 for α - DCP1 2 for live - imaging ) . arrowheads denote lack of robust DCP1 localization in scar1234 at edge / vertex . Small panels ( insets ) at right show details corresponding to regions delineated by dashed lines , where arrowhead denotes edge signal of DCP1 in WT ; scale bars , 1 μm . Bottom : of with proper edge / vertex localization of PB numbers ( DCP1 - foci ; N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 18 - 35 ) .

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

D Representative confocal micrographs showing DCP1 localization detected by α-DCP1 in the wild type (WT) or the scar1 scar2 scar3 scar4 (scar1234) quadruple mutant or in live-cell imaging of mScarlet-DCP1 (RPS5apro:HF-mScarlet-DCP1) in WT or brk1 mutant (bottom right; epidermal cells, region 3 for α-DCP1 and 2 for live-cell imaging). The arrowheads denote the lack of robust DCP1 localization in scar1234 at the edge/vertex. Small panels (insets) at right show details corresponding to the regions delineated by dashed lines, where arrowhead denotes the edge signal of DCP1 in WT; scale bars, 1 μm. Bottom: percentage of cells with proper edge/vertex localization and quantification of PB numbers (DCP1-foci; N, biological replicates = 3, n (pooled data of 3 biological replicates) = 18-35 cells).


A Representative confocal showing colocalization between DCP1 or two DCP1 phosphovariants with LifeAct - mCherry in lines co - expressing DCP1pro : DCP1 - GFP ( or variants ) UBQ10pro : LifeAct - mCherry ( edges are indicated by yellow arrowheads ) . Right : relative signal of actin at edges / vertices ( normalized to PM ) in epidermal ( N , biological replicates = 3 , n = 15 - 30 , regions 2 - 3 ) .

O O O O O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative confocal micrographs showing colocalization between DCP1 or two DCP1 phosphovariants with LifeAct-mCherry in lines co-expressing DCP1pro:DCP1-GFP (or variants) and UBQ10pro:LifeAct-mCherry (cell edges are indicated by yellow arrowheads). Right: relative signal intensity of actin at cell edges/vertices (normalized to the PM) in epidermal cells (N, biological replicates = 3, n = 15-30, regions 2-3).


B Representative confocal showing actin localization in WT , dcp1 - 1 , dcp1 - 3 , scar1234 , dcp2 - 1 , dcp5 pat1 upon phalloidin graph ( right ) indicating of in region 3 with an accumulation of actin at edges in various genotypes ( N , biological replicates = 3 , n = 7 - 9 roots , bars show means + s . d . ) .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

B Representative confocal micrographs showing actin localization in WT, dcp1-1, dcp1-3, scar1234, dcp2-1, dcp5 and pat1 upon phalloidin staining and graph (right) indicating the percentage of cells in region 3 with an accumulation of actin at edges in various genotypes (N, biological replicates = 3, n = 7-9 roots, bars show means + s.d.).


A SE - FRET efficiency between DCP1 - GFP or its phosphovariants with SCAR2 - mCherry ( among three different root regions ; mainly epidermal ) . Scale bar : 50 μm . arrowhead denotes high FRET efficiency at edges / vertices of region 3 . Right : signal of SE - FRET efficiency between indicated combinations at epidermis of 3 regions ( N , biological replicates = 6 , n ( pooled data of 3 biological replicates ) = 10 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A SE-FRET efficiency between DCP1-GFP or its phosphovariants with SCAR2-mCherry (among the three different root regions; mainly epidermal cells). Scale bar: 50 μm. The arrowhead denotes high FRET efficiency at edges/vertices of region 3. Right: signal quantification of SE-FRET efficiency between the indicated combinations at the epidermis of 3 regions (N, biological replicates = 6, n (pooled data of 3 biological replicates) = 10).


C Representative confocal showing SCAR2 - mCherry localization in WT dcp1 - 3 mutant , respectively ( root region 2 , epidermal ) of edge / vertex with SCAR2 a confined signal ( N , biological replicates = 3 , n = 5 - 8 ) .

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C Representative confocal micrographs showing SCAR2-mCherry localization in WT and dcp1-3 mutant, respectively (root region 2, epidermal cells) and quantification of edge/vertex with SCAR2 a confined signal (N, biological replicates = 3, n = 5-8 cells).


D Representative confocal showing colocalization between DCP1 - GFP or phosphovariants SCAR2 - mCherry in root meristematic ( root region 2 , epidermal ) . Scale bars : 10 μm . insets show details of colocalization ; arrowhead denotes expansion of SCAR2 / DCP1 domain , while yellow arrowheads restricted edge / vertex SCAR2 / DCP1 domains . Scale bars : 3 μm . graph indicates relative signal for indicated combinations ( as Pearson ' s correlation coefficient ; N , biological replicates = 3 , n = 5 at edges / vertices : spread edges were not considered in calculations ) .

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

D Representative confocal micrographs showing the colocalization between DCP1-GFP or phosphovariants and SCAR2-mCherry in root meristematic cells (root region 2, epidermal cells). Scale bars: 10 μm. The insets show details of colocalization; the white arrowhead denotes the expansion of the SCAR2/DCP1 domain, while the yellow arrowheads the restricted edge/vertex SCAR2/DCP1 domains. Scale bars: 3 μm. The graph indicates the relative signal intensity for the indicated combinations (as Pearson's correlation coefficient; N, biological replicates = 3, n = 5 at edges/vertices: spread edges were not considered in calculations).


A Representative showing of dcp1 mutants mutants in other PB components or SCAR - WAVE components ( 5 - day - old seedlings ) . arrowheads show growth defects of homozygous dcp1 - 1 or dcp2 - 1 mutants ( denoted - / - ; heterozygous denoted dcp1 - 1 / DCP1 ; details are also shown ) . Lower : graph showing relative root ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 3 - 4 roots , bars show means + s . d ) .

O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-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

A Representative images showing the phenotypes of dcp1 mutants and mutants in other PB core components or SCAR-WAVE components (5-day-old seedlings). The arrowheads show the growth defects of homozygous dcp1-1 or dcp2-1 mutants (denoted -/-; heterozygous denoted dcp1-1/DCP1; details are also shown). Lower: graph showing relative root length (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-4 roots, bars show means + s.d).


B DCP1 regulates expansion anisotropy . Representative confocal showing FM4 - 64 of WT , dcp1 - 1 dcp2 - 1 mutants ( 2 μM , 10 min ) . Scale bars , 20 μm . Right : of isotropic per root meristem ( % , epidermal ) in each genotype ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 3 - 5 roots , bars show means ± s . d ) . Examples of isotropic or anisotropic are shown , along with developmental axis offset at x - y - axes .

O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-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

B DCP1 regulates cell expansion anisotropy. Representative confocal micrographs showing FM4-64 staining of the WT, dcp1-1 and dcp2-1 mutants (2 μM, 10 min). Scale bars, 20 μm. Right: percentage of isotropic cells per root meristem (%, epidermal cells) in each genotype (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-5 roots, bars show means ± s.d). Examples of isotropic or anisotropic cells are shown, along with the developmental axis offset at the x- and y-axes.


( A , B C ) Polyphenols TA , PGG EGCG activate rhpR - lux in wild type but not rhpS deletion Psph strains . Data information : , * indicate significant differences between mock group strains under different conditions . ( Student ' s t . n . s . , not significant , * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) .

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(A, B and C) Polyphenols TA , PGG and EGCG activate rhpR-lux in wild type but not rhpS deletion Psph strains. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).


( D , E F ) Polyphenols repress T3SS induction in P . syringae in MM . Transcripts of indicated genes in presence of 100 - μM TA ( D ) , 100 - µM PGG ( E ) or 2 - mM EGCG ( F ) were determined by RT - qPCR 6 hours after culturing in MM . Data information : , * indicate significant differences between mock group strains under different conditions . ( Student ' s t . n . s . , not significant , * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E and F) Polyphenols repress T3SS induction in P. syringae in MM. Transcripts of the indicated genes in the presence of 100-μM TA (D), 100-µM PGG (E) or 2-mM EGCG (F) were determined by RT-qPCR 6 hours after culturing in MM. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).


( D , E F ) Polyphenols inhibit phosphatase activity of RhpS . GST - RhpR was phosphorylated by adding 5 - μM PSPPH _ 3550c as well as 10 - μCi [ γ - 32P ] - ATP for 10 minutes . RhpS - FL was treated in presence of different concentrations of TA ( D ) , PGG ( E ) , or EGCG ( F ) for 10 min . Then , full - RhpS was added into mixture phosphorylated GST - RhpR for 1 hour before stopping reaction . phosphorylated were separated by 12 % SDS - PAGE . After electrophoresis , SDS - PAGE gels were dried with gel - dryer then exposed to a medical X - ray film overnight . This experiment was repeated twice , yielding similar results . Data information : reaction was stopped by adding 5 × loading buffer before SDS - PAGE . Abbreviation is as follows : CBB , coomassie bright blue stain ; CFU , colony - forming unit .

O O O O O O O O O O O B-CONTROLLED_VAR 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 B-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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E and F) Polyphenols inhibit the phosphatase activity of RhpS. The GST-RhpR was phosphorylated by adding 5-μM PSPPH_3550c protein as well as 10-μCi [γ-32P]-ATP for 10 minutes. The RhpS-FL protein was treated in the presence of different concentrations of TA (D), PGG (E), or EGCG (F) for 10 min. Then, the full-length RhpS protein was added into the mixture phosphorylated GST-RhpR for 1 hour before stopping the reaction. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results. Data information: the reaction was stopped by adding 5 × loading buffer before SDS-PAGE. Abbreviation is as follows: CBB, coomassie bright blue stain; CFU, colony-forming unit.


( I , J K ) Polyphenols inhibits virulence in planta growth of Psph in bean . virulence were estimated by inoculating Psph strains with or without polyphenols onto host bean . Disease symptoms were recorded 5 days after inoculation . Leaf disks ( 1 cm2 ) were removed 5 days after inoculation diluted in sterile water to CFUs . experiment was repeated three times . Data information : , * indicate significant differences between mock group strains under different conditions as indicated . ( Student ' s t . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) .

(I, J and K) Polyphenols inhibits the virulence levels and in planta growth of Psph in bean plants. Bacterial virulence levels were estimated by inoculating Psph strains with or without polyphenols onto host bean plants. Disease symptoms were recorded 5 days after inoculation. Leaf disks (1 cm2) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).


( B , C D ) Polyphenols bind to RhpS - FL . of RhpS - FL was consistently measured as 0 . 6 - μM . binding curves of TA ( B ) , PGG ( C ) EGCG ( D ) are shown . data points were shown ( n = 2 biological replicates ) .

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(B, C and D) Polyphenols bind to the RhpS-FL protein. The concentration of RhpS-FL protein was consistently measured as 0.6-μM. The binding curves of TA (B), PGG (C) and EGCG (D) are shown. The individual data points were shown (n=2 biological replicates).


( E ) Alanine - scanning mutagenesis of RhpS sensor . RhpS containing a corresponding mutation was provided in rhpS deletion on a pHM1 vector . LUX activity was estimated in absence ( black frame ) presence TA ( 50 - μM ) , PGG ( 100 - μM ) or EGCG ( 2 - mM ) . Error bars represent standard deviations ( n = 3 biological replicates ) . Data information : , * indicate significant differences between mock group strains under different conditions as indicated . ( Student ' s t . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , * * * , P ≤ 0 . 001 ) .

O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Alanine-scanning mutagenesis of the RhpS sensor. RhpS containing a corresponding mutation was provided in the rhpS deletion strain on a pHM1 vector. LUX activity was estimated in the absence (black frame) and presence TA (50-μM), PGG (100-μM) or EGCG (2-mM). Error bars represent standard deviations (n=3 biological replicates). Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001).


( C D ) rhpS / PSPPH _ 3550 / PSPPH _ 3736 / PSPPH _ 5115 quadruple deletion restored T3SS induction of rhpS deletion . wild type , rhpS deletion quadruple deletion strains were cultured in MM ( C ) for 6 hours or KB ( D ) to OD600 = 0 . 6 . Transcripts of indicated genes were determined by RT - qPCR . Data information : , * indicate significant differences between rhpS mutant quadruple mutant . Error bars represent standard deviations ( n = 3 biological replicates ) . Student ' s t . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , * * * , P ≤ 0 . 001 .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR O O B-CONTROLLED_VAR 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 O O O O O 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 O O O O O O O O O O O O O O O O O O O O O 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 and D) The rhpS/PSPPH_3550/PSPPH_3736/PSPPH_5115 quadruple deletion strain restored the T3SS induction of rhpS deletion strain. The wild type, rhpS deletion and quadruple deletion strains were cultured in MM (C) for 6 hours or KB (D) to OD600=0.6. Transcripts of the indicated genes were determined by RT-qPCR. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001.


( F ) quadruple mutant restored pathogenicity of rhpS deletion . virulence were estimated by inoculating wild type , rhpS deletion quadruple deletion strains onto host bean . Disease symptoms were recorded 5 days after inoculation . experiment was repeated three times .

O O O O O O O O B-CONTROLLED_VAR O O O O 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 O O O O O O

(F) The quadruple mutant strain restored the pathogenicity of rhpS deletion strain. Bacterial virulence levels were estimated by inoculating wild type, rhpS deletion and quadruple deletion strains onto host bean plants. Disease symptoms were recorded 5 days after inoculation. The experiment was repeated three times.


( G ) Leaf disks ( 1 cm2 ) from ( G ) were removed 5 days after inoculation diluted in sterile water to CFUs . experiment was repeated three times . Data information : , * indicate significant differences between rhpS mutant quadruple mutant . Error bars represent standard deviations ( n = 3 biological replicates ) . Student ' s t . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , * * * , P ≤ 0 . 001 .

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(G) Leaf disks (1 cm2) from (G) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001.


A Cytokine release by PBMCs challenged with heat inactivated S . aureus ( hiSa ) , / or RNase A ( RA ) treated solution , or Sa19 ( TLR13 activating ORN ) upon TLR2 blockade ( T2 . 5 ; n . p . , not performed ; * * , p ≤ 0 . 01 ; n = 3 ) .

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A Cytokine release by PBMCs challenged with heat inactivated S. aureus (hiSa), and/or RNase A (RA) treated solution, or Sa19 (TLR13 activating ORN) upon TLR2 blockade (T2.5; n.p., not performed; **, p ≤ 0.01; n = 3).


C Cytokine release of PBMCs transfected with ORNs including TLR7 / 8 ligand RNA40 ( n = 3 ) .

C Cytokine release of PBMCs transfected with ORNs including TLR7/8 ligand RNA40 (n = 3).


D , E Activity of PBMCs upon challenge , pretreatment with chloroquine in ( D ) only ( S , Svedberg ; r , ribosomal ; tot , total ; n = 3 ) .

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D, E Activity of PBMCs upon bacterial RNA challenge, pretreatment with chloroquine in (D) only (S, Svedberg; r, ribosomal; tot, total; n = 3).


F Activity of PBMCs upon mitochondrial ( mt ) challenge ( Hs , human ; triangle , increasing doses ; LFA , Lipofectamine 2000 ; n = 3 ) .

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F Activity of PBMCs upon mitochondrial (mt) RNA challenge (Hs, human; triangle, increasing doses; LFA, Lipofectamine 2000; n = 3).


G Activity of PBMCs challenged with ORNs total S . aureus ( Sa ) ( Myd88d / d , mutant MyD88 expression not impairing LPS driven IL - 8 production ; n = 2 ) .

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G Activity of PBMCs challenged with ORNs and total S. aureus (Sa) RNA (Myd88d/d, mutant MyD88 expression not impairing LPS driven IL-8 production; n = 2).


H Responsiveness of undifferentiated ( undiff ) 3 - or 8 days PMA differentiated ( ddi ) THP1 to Sa19 challenge ( n = 3 ) .

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H Responsiveness of undifferentiated (undiff) and 3- or 8 days PMA differentiated (ddi) THP1 cells to Sa19 challenge (n = 3).


I Activity of parental Unc93b1 - / - - 3ddiTHP - 1 challenged with ORNs ( n = 3 ) .

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I Activity of parental and Unc93b1-/--3ddiTHP-1 cells challenged with ORNs (n = 3).


A Cytokine release of 3 days differentiated ( ddi ) THP1 toward stimulation with TLR ligands RNAs ( S , Svedberg ; r , ribosomal ; tot , total ; n = 3 ) .

A Cytokine release of 3 days differentiated (ddi) THP1 cells toward stimulation with TLR ligands and bacterial RNAs (S, Svedberg; r, ribosomal; tot, total; n = 3).


B , C Responsiveness of Unc93b1 - / - - 3ddiTHP1 treated with T2 . 5 challenged with heat inactivated ( hi ) ( triangle , decreasing doses ; n = 3 ) .

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B, C Responsiveness of Unc93b1-/--3ddiTHP1 cells treated with T2.5 and challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; n=3).


D Ratios of constitutive mRNA amounts in 3 - versus 8ddi ( grey bar ) of each of both versus undifferentiated THP1 ( black or bar , respectively ) according to a transcriptome profiling result ( n = 1 ) .

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D Ratios of constitutive mRNA amounts in 3- versus 8ddi (grey bar) and of each of both versus undifferentiated THP1 cells (black or white bar, respectively) according to a transcriptome profiling result (n = 1).


E , F NF - κB driven relative luciferase activity ( Rel . luc . act . ) of hTLR8 overexpressing HEK293 murine RAW264 . 7 , respectively , upon challenges ( vector , empty plasmid ; n = 3 ) .

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E, F NF-κB driven relative luciferase activity (Rel. luc. act.) of hTLR8 overexpressing HEK293 and murine RAW264.7 cells, respectively, upon challenges (vector, empty plasmid; n = 3).


G Transfection of TLR8 siRNA impairs 3ddiTHP1 responsiveness ( n = 3 ) .

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G Transfection of TLR8 siRNA impairs 3ddiTHP1 cell responsiveness (n = 3).


A , B Cytokine release of 3 days differentiated ( ddi ) THP1 challenged with ( A ) ORNs or ( B ) fractions ( tot , total ; S , Svedberg ; r , ribosomal ) .

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A, B Cytokine release of 3 days differentiated (ddi) THP1 cells challenged with (A) ORNs or (B) bacterial RNA fractions (tot, total; S, Svedberg; r, ribosomal).


C Responsiveness of Tlr8 - / - - 3ddiTHP1 challenged with heat inactivated ( hi ) ( triangle , decreasing doses ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ) upon TLR2 blockade ( T2 . 5 ) .

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C Responsiveness of Tlr8-/--3ddiTHP1 cells challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; *, p ≤ 0.05; **, p ≤ 0.01) upon TLR2 blockade (T2.5).


D Responsiveness of 3ddiTHP1 upon challenge with human ( Hs ) mitochondrial ( mt ) ( triangle , increasing doses ) .

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D Responsiveness of 3ddiTHP1 cells upon challenge with human (Hs) mitochondrial (mt) RNA (triangle, increasing doses).


E - H NF - B driven relative luciferase activity ( Rel . luc . act . ) of hTLR8 + HEK293 or cytokine release by PBMCs transfected with RNAs indicated additional uridine ( U ) if indicated ( n . p . , not performed ; vector , empty plasmid ) .

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E-H NF-B driven relative luciferase activity (Rel. luc. act.) of hTLR8+ HEK293 cells or cytokine release by PBMCs all transfected with RNAs indicated and additional uridine (U) if indicated (n.p., not performed; vector, empty plasmid).


A Cytokine release of 3 days differentiated THP - 1 of indicated genotypes upon TLR2 blockade ( T2 . 5 ) challenge with TLR ligands , heat inactivated ( hi ) or viable .

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A Cytokine release of 3 days differentiated THP-1 cells of indicated genotypes upon TLR2 blockade (T2.5) and challenge with TLR ligands, heat inactivated (hi) or viable bacteria.


B PBMC activity upon endosome function inhibition ( chloroquine ) TLR2 blockade ( T2 . 5 ) followed by TLR ligand challenge or infection .

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B PBMC activity upon endosome function inhibition (chloroquine) and TLR2 blockade (T2.5) followed by TLR ligand challenge or bacterial infection.


C Activity of whole blood culture upon endosome function inhibition ( chloroquine ) challenge with TLR ligands , heat inactivated ( hi ) , or viable .

C Activity of whole blood culture upon endosome function inhibition (chloroquine) and challenge with TLR ligands, heat inactivated (hi), or viable bacteria.


F , Representative immunoblot of pFAK in 5 % LPDS ( - LDL ) upon increasing LDL loading times . Mean ± SD , n = 3 , paired student ' s t - ( * p < 0 . 05 ) .

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F, Representative immunoblot and quantification of pFAK levels in 5% LPDS (-LDL) and upon increasing LDL loading times. Mean ± SD, n = 3, paired student's t-test (*p < 0.05).


G , Representative confocal of NPC1 , internalized integrin β1 pFAK localization . Stably NPC1 - GFP expressing were loaded with 2 μg / mL integrin β1 antibody for 30 min before fixation stained with pFAK antibody followed by secondary antibodies . Dashed indicates edge . H , of G using Mander ' s colocalization for indicated markers . Mean ± SD , n = 5 . were treated with 5 % LPDS for 1 day then without ( - ) or with ( + ) 50 μg / ml LDL for 1h .

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G, Representative confocal images of NPC1, internalized integrin β1 and pFAK localization. Stably NPC1-GFP expressing cells were loaded with 2 μg/mL integrin β1 antibody for 30 min before fixation and stained with pFAK antibody followed by secondary antibodies. Dashed line indicates the cell edge. H, Quantification of G using Mander's colocalization for the indicated markers. Mean ± SD, n = 5 cells. Cells were treated with 5 % LPDS for 1 day and then without (-) or with (+) 50 μg/ml LDL for 1h.


D , After 1 day LDL depletion in 5 % LPDS , were loaded with 50 μg / ml LDL for indicated times intracellular cholesterol was detected with D4H as in C by confocal microscopy . CM , complete medium . Dashed lines indicate outlines . E , of D . Mean ± SD , n = 25 - 26 pooled from 2 independent experiments .

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D, After 1 day LDL depletion in 5 % LPDS, cells were loaded with 50 μg/ml LDL for the indicated times and intracellular cholesterol was detected with D4H as in C by confocal microscopy. CM, complete medium. Dashed lines indicate cell outlines. E, Quantification of D. Mean ± SD, n = 25-26 cells pooled from 2 independent experiments.


F , were treated for 1 day in 5 % LPDS followed by 50 μg / ml LDL loading for indicated times . Representative confocal of triple labeled with intracellular accessible cholesterol ( D4H ) , NPC1 - GFP internalized integrin β1 . Dashed lines indicate outlines . G , of D4H positive for NPC1 or internalized integrin β1 as in F . H , Fraction of D4H overlapping with NPC1 or internalized integrin β1 in a peripheral region toward leading edge ( exemplary box shown as inset in F ) . Mean ± SD , n = 27 - 30 pooled from 2 independent experiments . Student ' s t - .

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F, Cells were treated for 1 day in 5 % LPDS followed by 50 μg/ml LDL loading for the indicated times. Representative confocal images of cells triple labeled with intracellular accessible cholesterol (D4H), NPC1-GFP and internalized integrin β1. Dashed lines indicate cell outlines. G, Quantification of D4H area positive for NPC1 or internalized integrin β1 as in F. H, Fraction of D4H area overlapping with NPC1 or internalized integrin β1 in a peripheral region toward the leading edge (exemplary box shown as inset in F). Mean ± SD, n = 27-30 cells pooled from 2 independent experiments. Student's t-test.


A , After 1 day in 5 % LPDS , NPC1 - GFP expressing were loaded with 50 μg / ml LDL with or without 10 μM PF228 for indicated times , stained for intracellular D4H . Representative confocal of intracellular D4H , NPC1 - GFP internalized integrin β1 labeling . Dashed lines indicate outlines . B , Fraction of D4H positive in as in A . C , Fraction of D4H overlapping with NPC1 in a peripheral region toward leading edge ( exemplary box shown as inset in A ) . D , of D4H positive for integrin β1 in a peripheral region toward leading edge . Mean ± SD , n = 26 - 30 pooled from 2 independent experiments . Student ' s t - .

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A, After 1 day in 5 % LPDS, NPC1-GFP expressing cells were loaded with 50 μg/ml LDL with or without 10 μM PF228 for the indicated times, and stained for intracellular D4H. Representative confocal images of intracellular D4H, NPC1-GFP and internalized integrin β1 labeling. Dashed lines indicate cell outlines. B, Fraction of D4H positive area in the cell as in A. C, Fraction of D4H area overlapping with NPC1 in a peripheral region toward the leading edge (exemplary box shown as inset in A). D, Quantification of D4H area positive for integrin β1 in a peripheral region toward the leading edge. Mean ± SD, n = 26-30 cells pooled from 2 independent experiments. Student's t-test.


I , After 1 day 5 % LPDS , were loaded with 50 μg / ml LDL with or without 10 μM PF228 for indicated times endomembrane PI ( 4 , 5 ) P2 was stained with antibody imaged by confocal microscopy .

I, After 1 day 5 % LPDS, cells were loaded with 50 μg/ml LDL with or without 10 μM PF228 for the indicated times and endomembrane PI(4,5)P2 was stained with antibody and imaged by confocal microscopy.


J , of mean PI ( 4 , 5 ) P2 immunoreactivity in . Mean ± SD , n = 16 - 26 pooled from 2 independent experiments . Student ' s t - .

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J, Quantification of mean PI(4,5)P2 immunoreactivity in cells. Mean ± SD, n = 16-26 cells pooled from 2 independent experiments. Student's t-test.


C , Confocal image of endo - GFP - ORP2 pFAK immunofluorescence in saponin pre - permeabilized loaded with LDL for 2 h before fixation . GFP antibody was used to enhance endo - GFP - ORP2 signal . Insets show examples of " no contact " , " contact " " overlap " of organelles . Dashed indicates edge . D , of pFAK , integrin β1 , NPC1 association with endo - GFP - ORP2 in C . n = 380 pFAK ; 353 integrin β1 ; 232 NPC1 organelles in 2 .

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C, Confocal image of endo-GFP-ORP2 and pFAK immunofluorescence staining in saponin pre-permeabilized cells loaded with LDL for 2 h before fixation. GFP antibody was used to enhance endo-GFP-ORP2 signal. Insets show examples of "no contact", "contact" and "overlap" of organelles. Dashed line indicates cell edge. D, Quantification of pFAK, integrin β1, and NPC1 association with endo-GFP-ORP2 in C. n = 380 pFAK; 353 integrin β1; 232 NPC1 organelles in 2 cells.


F , Schematic outline for plasma D4H labeling in combination with ORP2 degron system , representative widefield epifluorescent of co - plated after 4 h of LDL loading . Asterisk indicates ORP2 - depleted degron . Dashed lines separate ORP2 expressing depleted . G , of F . Mean ± SD , n = 20 fields of pooled from 2 independent experiments . Student ' s t - .

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F, Schematic outline for plasma membrane D4H labeling in combination with ORP2 degron system, and representative widefield epifluorescent images of co-plated cells after 4 h of LDL loading. Asterisk indicates ORP2-depleted degron cells. Dashed lines separate ORP2 expressing and depleted cells. G, Quantification of F. Mean ± SD, n = 20 fields of cells pooled from 2 independent experiments. Student's t-test.


A , Control degron ORP2 were co - plated , after 1 day incubation in 5 % LPDS , were loaded with LDL ( + LDL ) IAA ( + IAA ; ORP2 depletion ) for indicated times . Intracellular cholesterol was labeled with D4H imaged by widefield epifluorescence microscopy . Asterisk indicates region of ORP2 - depleted . CM , complete medium . Dashed lines separate ORP2 expressing depleted . B , of A . Mean ± SD , n = 15 - 27 pooled from 2 independent experiments . Student ' s t - .

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A, Control and degron ORP2 cells were co-plated, and after 1 day incubation in 5 % LPDS, cells were loaded with LDL (+LDL) and IAA (+IAA; ORP2 depletion) for the indicated times. Intracellular cholesterol was labeled with D4H and imaged by widefield epifluorescence microscopy. Asterisk indicates region of ORP2-depleted cells. CM, complete medium. Dashed lines separate ORP2 expressing and depleted cells. B, Quantification of A. Mean ± SD, n = 15-27 cells pooled from 2 independent experiments. Student's t-test.


C , E , Control degron ORP2 were co - plated , after 1 day in 5 % LPDS , treated with 50 μg / ml LDL IAA for indicated times . Asterisk indicates an ORP2 - depleted . Double labeling of intracellular D4H Lamp1 ( C ) or intracellular D4H internalized integrin β1 ( E ) . were acquired by confocal microscopy . Red box indicates a peripheral region toward leading edge in control , blue box a corresponding region in ORP2 - depleted .

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C, E, Control and degron ORP2 cells were co-plated, and after 1 day in 5 % LPDS, treated with 50 μg/ml LDL and IAA for the indicated times. Asterisk indicates an ORP2-depleted cell. Double labeling of intracellular D4H and Lamp1 (C) or intracellular D4H and internalized integrin β1 (E). Images were acquired by confocal microscopy. Red box indicates a peripheral region toward the leading edge in the control cell, and blue box a corresponding region in the ORP2-depleted cell.


B , stably expressing NPC1 - mCherry were treated with control or OCRL siRNAs for 2 days , or transfected with GFP - ORP2 or - ORP2 - mHHK ( PI ( 4 , 5 ) P2 binding - deficient mutant ) for 1 day , stained with PI ( 4 , 5 ) P2 antibody imaged by confocal microscopy . For FAK inhibition , GFP - ORP2 transfected were treated with 10 μM PF228 for 4 h . Dashed lines indicate edges . Arrowheads indicate colocalization of PI ( 4 , 5 ) P2 NPC1 . C , of B . Values from Ctrl siRNA Vector control were pooled are shown as Ctrl siRNA / Vector ctrl . Mean ± SD , n = 20 - 43 pooled from 2 independent experiments . Student ' s t - .

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B, Cells stably expressing NPC1-mCherry were treated with control or OCRL siRNAs for 2 days, or transfected with GFP-ORP2 or -ORP2-mHHK (PI(4,5)P2 binding-deficient mutant) for 1 day, stained with PI(4,5)P2 antibody and imaged by confocal microscopy. For FAK inhibition, GFP-ORP2 transfected cells were treated with 10 μM PF228 for 4 h. Dashed lines indicate cell edges. Arrowheads indicate colocalization of PI(4,5)P2 and NPC1. C, Quantification of B. Values from Ctrl siRNA and Vector control were pooled and are shown as Ctrl siRNA/Vector ctrl. Mean ± SD, n = 20-43 cells pooled from 2 independent experiments. Student's t-test.


D , E , stably expressing NPC1 - mCherry were transfected with GFP - ORP2 or GFP - ORP2 - mHHK for 1 day were indicated , treated with 10 μM PF228 for 4 h . For degron ORP2 , NPC1 - mCherry was transiently transfected for 2 days incubated without ( - IAA ; ORP2 present ) or with IAA ( + IAA ; ORP2 depleted ) for 1 h . Arrowheads indicate tubular NPC1 organelles . Live were acquired by widefield epifluorescence miroscopy longest NPC1 tubule per was measured . Mean ± SD , n = 102 - 122 . Students ' s t - . Please see also ctrl ( MovieEV4 ) PF228 treated ( MovieEV5 ) , both videos 1 min recordings with 1 s frame rate .

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D, E, Cells stably expressing NPC1-mCherry were transfected with GFP-ORP2 or GFP-ORP2-mHHK for 1 day and were indicated, treated with 10 μM PF228 for 4 h. For degron ORP2 cells, NPC1-mCherry was transiently transfected for 2 days and cells incubated without (-IAA; ORP2 present) or with IAA (+IAA; ORP2 depleted) for 1 h. Arrowheads indicate tubular NPC1 organelles. Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured. Mean ± SD, n = 102-122 cells. Students's t-test. Please see also ctrl (MovieEV4) and PF228 treated cell (MovieEV5), both videos 1 min recordings with 1 s frame rate.


A , B , Control degron ORP2 were co - plated , after 1 day 5 % LPDS , treated with 50 μg / ml LDL IAA ( + IAA ; ORP2 depletion ) for indicated times immunostained for pFAK to quantify its . For rescue experiments , degron ORP2 were plated transfected with GFP - ORP2 or - ORP2 - mHHK or - ORP2 - ∆ ELSK for 6 h . expressing GFP - ORP2 constructs at similar to endo - GFP - ORP2 were used for quantifying pFAK . Dashed lines indicate outlines . See also Fig EV6C . Asterisk indicates depleted of endogenous ORP2 . were acquired by confocal microscopy . Mean ± SD , n = 20 - 23 pooled from 2 independent experiments . Student ' s t - .

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A, B, Control and degron ORP2 cells were co-plated, and after 1 day 5 % LPDS, treated with 50 μg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron ORP2 cells were plated and transfected with GFP-ORP2 or -ORP2-mHHK or -ORP2-∆ELSK for 6 h. Cells expressing GFP-ORP2 constructs at levels similar to endo-GFP-ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20-23 cells pooled from 2 independent experiments. Student's t-test.


C , of FAK in fractions in degron ORP2 with or without IAA . were starved overnight in LPDS loaded with 50 µg / mL LDL + / - IAA for 2 h . proportion of FAK signal in fraction ( of total FAK in cytosol + membranes ) is presented ± SD . For no IAA n = 13 for IAA n = 11 , 2 independent experiments . Student ' s t - .

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C, Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/- IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol+membranes) is presented ± SD. For no IAA n=13 and for IAA n= 11, 2 independent experiments. Student's t-test.


( A ) Immunohistochemistry of Bcl6 ( red ) , Cux1 ( green ) , Satb2 ( blue ) in P2 Bcl11aF / F ; Emx1IRESCre control neocortex . Nuclei are stained with Dapi ( ) . ( B ) Relative of Bcl6 + , Satb2 + , Cux1 + in Bcl11aF / F ; Emx1IRESCre control neocortex ( n = 4 ) . ( C ) Numbers of Cux1 + or Satb2 + that coexpress Bcl6 are reduced in Bcl11aF / F ; Emx1IRESCre compared to control neocortex ( n = 4 ) . Data information : graphs represent mean ± s . e . m . ; Student ' s t ; * * * p < 0 . 001 . Scale bar , 50 µm .

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(A) Immunohistochemistry of Bcl6 (red), Cux1 (green), and Satb2 (blue) in P2 Bcl11aF/F;Emx1IRESCre and control neocortex. Nuclei are stained with Dapi (white). (B) Relative quantification of Bcl6+, Satb2+, and Cux1+ cells in Bcl11aF/F;Emx1IRESCre and control neocortex (n = 4). (C) Numbers of Cux1+ or Satb2+ cells that coexpress Bcl6 are reduced in Bcl11aF/F;Emx1IRESCre compared to control neocortex (n = 4). Data information: All graphs represent the mean ± s.e.m.; Student's t test; *** p < 0.001. Scale bar, 50 µm.


( C - E ) Immunohistochemistry of electroporated P2 Bcl11aF / F neurons in superficial cortical layers with GFP ( green ) Bcl6 ( red , C ) , Cux1 ( red , D ) , or Satb2 ( red , E ) antibodies . Bcl6 expression is specifically downregulated Bcl11aF / F neocortex upon electroporation of CAG - CreGFP in comparison to CAG - CtlGFP control plasmid . Nuclei are stained with Dapi ( ) . Data information : arrowheads point at GFP + that also express Bcl6 , Cux1 , Satb2 indicate expressing only GFP + . Scale bars , 20 µm ( C - E ) ,

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(C-E) Immunohistochemistry of electroporated P2 Bcl11aF/F neurons in superficial cortical layers with GFP (green) and Bcl6 (red, C), Cux1 (red, D), or Satb2 (red, E) antibodies. Bcl6 expression is specifically downregulated Bcl11aF/F neocortex upon electroporation of CAG-CreGFP in comparison to CAG-CtlGFP control plasmid. Nuclei are stained with Dapi (white). Data information: White arrowheads point at GFP+ cells that also express Bcl6, Cux1, Satb2 indicate cells expressing only GFP+. Scale bars, 20 µm (C-E),


( F ) of of electroporated expressing Bcl6 ( n = 3 ) , Satb2 ( n = 3 ) , Cux1 ( n = 5 ) . Results are expressed as mean ± s . e . m . ; Student ' s t ; * * * p < 0 . 001 .

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(F) Quantification of the percentage of electroporated cells expressing Bcl6 (n = 3), Satb2 (n = 3), and Cux1 (n = 5). Results are expressed as mean ± s.e.m.; Student's t test; *** p < 0.001.


( H ) Immunohistochemistry of electroporated P5 Bcl11a ∆ / F Bcl11a + / F neurons in superficial cortical layers with GFP ( green ) caspase 3 ( CC3 , magenta ) antibodies . Electroporation of Neurod - CreGFP plasmid together with CAG - LSL - Bcl6GFP into Bcl11aF / F neocortex reduces number of CC3 + to control . ( I ) of experiment shown in ( H ) ( n = 4 ) . Results are expressed as mean ± s . e . m . ; one - way ANOVA followed by Tukey ' s post hoc ; * * * p < 0 . 001 . Data information : arrowheads point at GFP + that express CC3 . Black arrowheads indicate expressing only GFP + . Scale bars , , 50 µm ( H ) .

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(H) Immunohistochemistry of electroporated P5 Bcl11a∆/F and Bcl11a+/F neurons in superficial cortical layers with GFP (green) and cleaved caspase 3 (CC3, magenta) antibodies. Electroporation of Neurod-CreGFP plasmid together with CAG-LSL-Bcl6GFP into Bcl11aF/F neocortex reduces the number of CC3+ cells to control levels. (I) Quantification of the experiment shown in (H) (n = 4). Results are expressed as mean ± s.e.m.; one-way ANOVA followed by Tukey's post hoc test; *** p < 0.001. Data information: White arrowheads point at GFP+ cells that express CC3. Black arrowheads indicate cells expressing only GFP+. Scale bars, , 50 µm (H).


( A ) Immunohistochemistry of caspase 3 ( CC3 ) shows that number of CC3 + ( marked by black arrowheads ) is increased in P5 Bcl6F / F ; NexCre compared to control neocortex . Insets are enlargements of boxed areas in corresponding panels . Data information : Scale bars , 500 µm ( A )

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(A) Immunohistochemistry of cleaved caspase 3 (CC3) shows that the number of CC3+ cells (marked by black arrowheads) is increased in P5 Bcl6F/F;NexCre compared to control neocortex. Insets are enlargements of the boxed areas in corresponding panels. Data information: Scale bars, 500 µm (A)


( C ) of experiment shown in ( A ) ( n = 3 ) . Results are expressed as mean ± s . e . m . ; Student ' s t ; * p < 0 . 05 .

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(C) Quantification of the experiment shown in (A) (n = 3). Results are expressed as mean ± s.e.m.; Student's t test; * p < 0.05.


( J ) Immunohistochemistry of electroporated P5 Bcl11aF / F neurons in superficial cortical layers with GFP ( green ) caspase 3 ( CC3 , magenta ) antibodies . Electroporation of CAG - CreGFP plasmid together with Foxo1 - shRNAGFP # 4 into Bcl11aF / F neocortex reduces number of CC3 + to control . black arrowheads indicate GFP + CC3 + GFP + , respectively . ( K ) of experiment shown in ( J ) ( n = 4 , CAG - CtlGFP / Ctl - shRNAGFP ; n = 3 , CAG - CreGFP / Ctl - shRNAGFP ; n = 5 , CAG - CreGFP / Foxo1 - shRNAGFP # 4 ) . Results are expressed as mean ± s . e . m . ; one - way ANOVA followed by Tukey ' s post hoc ; * p < 0 . 05 . Data information : Scale bars , , 50 µm J ) .

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(J) Immunohistochemistry of electroporated P5 Bcl11aF/F neurons in superficial cortical layers with GFP (green) and cleaved caspase 3 (CC3, magenta) antibodies. Electroporation of CAG-CreGFP plasmid together with Foxo1-shRNAGFP#4 into Bcl11aF/F neocortex reduces the number of CC3+ cells to control levels. White and black arrowheads indicate GFP+ CC3+ and GFP+ cells, respectively. (K) Quantification of the experiment shown in (J) (n = 4, CAG-CtlGFP/Ctl-shRNAGFP; n = 3, CAG-CreGFP/Ctl-shRNAGFP; n = 5, CAG-CreGFP/Foxo1-shRNAGFP#4). Results are expressed as mean ± s.e.m.; one-way ANOVA followed by Tukey's post hoc test; * p < 0.05. Data information: Scale bars, , 50 µm J).


locations of native virally mediated Kcnq1 expression in cochleaA Diagram showing major landmarks of cochlear section to facilitate comparison of data shown in ( B - F ) . B - F Immunolabeling results of Kcnq1 ( green ) in cochlear cryosections are shown for uninjected WT ( B ) , untreated Kcnq1 − / − ( C ) , injected WT ( D ) , Kcnq1 − / − mice given injections into ST ( E ) or SM ( F ) . nuclei were outlined by counterstaining with either DAPI ( B - D , F ) or Qnuclear deep red ( E ) . Scale bars represent approximately 100 μm . Meaning of arrows are given in text .

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Cellular locations of native and virally mediated Kcnq1 expression in the cochleaA Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in (B-F).B-F Immunolabeling results of Kcnq1 (green) in cochlear cryosections are shown for uninjected WT (B), untreated Kcnq1−/− (C), injected WT (D), and Kcnq1−/− mice given injections into the ST (E) or SM (F). Cell nuclei were outlined by counterstaining with either DAPI (B-D, F) or Qnuclear deep red (E). Scale bars represent approximately 100 μm. Meaning of white arrows are given in the text.


Polarized intracellular trafficking of native virally expressed Kcnq1 was found specifically in marginal cellsKcnq1 cellnuclei were , respectively , labeled green purple . SV consists of three layers of ; marginal are first layer of on side of endolymphatic space . Cryosection through SV of WT mice . large arrow points to native Kcnq1 ( labeled green ) in WT mice . Smaller arrows show nuclei of marginal . Cryosection through SV of a Kcnq1 − / − mouse injected with AAV expressing Kcnq1 . Labeled in green ( bigger arrow ) is AAV1 - expressed Kcnq1 , found only in apical of marginal . Smaller arrows show nuclei of marginal . Arrowheads show Kcnq1immunolabeling in fibrocytes outside SV . Cryosection through lateral wall of Kcnq1 − / − mice showing intracellular distribution of Kcnq1immunolabeling ( green ) in fibrocytes . Cryosection through spiral ganglia of cochlea of treated Kcnq1 − / − mice showing immunolabeling ( green ) in of spiral ganglia ( arrows ) . Data information : Purple in panels is counterstaining with DAPI showing locations of cellnuclei . Scale bars represent appropriately 50 μm .

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Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were, respectively, labeled green and purple. The SV consists of three layers of cells; marginal cells are the first layer of cells on the side of the endolymphatic space.Cryosection through the SV of WT mice. The large arrow points to the native Kcnq1 (labeled green) in WT mice. Smaller arrows show nuclei of the marginal cells.Cryosection through the SV of a Kcnq1−/−mouse injected with AAV expressing Kcnq1. Labeled in green (bigger arrow) is the AAV1-expressed Kcnq1, found only in the apical membrane of the marginal cells. Smaller arrows show nuclei of the marginal cells. Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV.Cryosection through the lateral wall of Kcnq1−/−mice showing intracellular distribution of Kcnq1immunolabeling (green) in fibrocytes.Cryosection through the spiral ganglia of cochlea of treated Kcnq1−/−mice showing immunolabeling (green) in the cells of spiral ganglia (arrows).Data information: Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei. Scale bars represent appropriately 50 μm.


of Kcnq1 - positive marginal effect of treatment on organization of marginal in SVThe membranes of marginal are labeled ( red ) by phalloidin conjugated with rhodamine . A Immunolabeling results ( Kcnq1 labeled in green ) in WT mice . B , C Immunolabeling results ( Kcnq1 labeled in green ) of treated Kcnq1 − / − mice , middle ( B ) apical ( C ) turns , respectively . D of marginal having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT ( gray bars , left ) , untreated Kcnq1 − / − ( middle ) , treated Kcnq1 − / − mice ( black bars , right ) . Data are given as mean ± SD ( n = 6 ) . E , F organization of marginal in SV is outlined by labeling with phalloidin conjugated with rhodamine . Results from WT ( E ) untreated Kcnq1 − / − mice ( F ) are compared . Data information : Scale bars represent approximately 50 μm .

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Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled (red) by phalloidin conjugated with rhodamine.A Immunolabeling results (Kcnq1 labeled in green) in WT mice.B, C Immunolabeling results (Kcnq1 labeled in green) of treated Kcnq1−/−mice, middle (B) and apical (C) turns, respectively.D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT (gray bars, left), untreated Kcnq1−/− (middle), and treated Kcnq1−/−mice (black bars, right). Data are given as mean ± SD (n = 6).E, F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine. Results from WT (E) and untreated Kcnq1−/−mice (F) are compared.Data information: Scale bars represent approximately 50 μm.


Comparison of cochlear morphologyA - C Cochlear sections obtained from WT ( A ) , nontreated Kcnq1 − / − ( B ) , treated Kcnq1 − / − mice ( C ) were compared . Major landmarks of cochlear sections are labeled pointed by arrows . Scale bars represent approximately 50 μm .

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Comparison of cochlear morphologyA-C Cochlear sections obtained from WT (A), nontreated Kcnq1−/− (B), and treated Kcnq1−/− mice (C) were compared. Major landmarks of the cochlear sections are labeled and pointed by arrows. Scale bars represent approximately 50 μm.


Waveforms of ABRs are compared in WT , untreated Kcnq1 − / − , treated Kcnq1 − / − mice , as labeled above data traces . Series of averaged ABR data traces were evoked from tone - burst sounds with intensities ranging from 20 to 90 dB SPL .

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Waveforms of ABRs are compared in WT, untreated Kcnq1−/−, and treated Kcnq1−/− mice, as labeled above data traces. Series of averaged ABR data traces were evoked from tone-burst sounds with intensities ranging from 20 to 90 dB SPL.


Summary of averaged ABR thresholds at various frequencies for different groups of mice ; untreated Kcnq1 − / − ( open triangles ) , treated Kcnq1 − / − ( filled triangles ) , injected WT ( filled circles ) , uninjected WT ( filled squares ) . Plot legends are given in figure . plot with open circles connected with dashed lines represents average data from five best cases of treated Kcnq1 − / − mice . Error bars represent standard error of mean .

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Summary of averaged ABR thresholds at various frequencies for different groups of mice; untreated Kcnq1−/− (open triangles), treated Kcnq1−/− (filled triangles), injected WT (filled circles), and uninjected WT (filled squares). Plot legends are given in the figure. The plot with open circles connected with dashed lines represents average data from the five best cases of treated Kcnq1−/− mice. Error bars represent standard error of the mean.


Click - evoked ABR thresholds of WT ( filled squares ) , treated Kcnq1 − / − mice ( filled triangles ) , untreated Kcnq1 − / − mice ( open squares ) measured 4 - 30 after mice were born . Error bars represent standard error of mean . Upward arrows indicate that click - ABR thresholds were at maximal sound level that could be reliably measured by system .

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Click-evoked ABR thresholds of WT (filled squares), treated Kcnq1−/− mice (filled triangles), untreated Kcnq1−/− mice (open squares) measured 4-30 weeks after mice were born. Error bars represent standard error of the mean. Upward arrows indicate that click-ABR thresholds were at the maximal sound level that could be reliably measured by the system.


Western blot verification of knockout of FASN , ACC1 , / or ACC2 in MDA - MB - 231 .

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Western blot verification of knockout of FASN, ACC1, and/or ACC2 in MDA-MB-231 cells.


proliferation rate of MDA - MB - 231 HeLa with knockout of FASN , ACC1 , / or ACC2 .

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Cell proliferation rate of MDA-MB-231 and HeLa cells with knockout of FASN, ACC1, and/or ACC2.


Colony formation ability of MDA - MB - 231 with knockout of FASN , ACC1 , / or ACC2 .

Colony formation ability of MDA-MB-231 cells with knockout of FASN, ACC1, and/or ACC2.


Invasion ability of MDA - MB - 231 with knockout of FASN , ACC1 , / or ACC2 . right panel shows pictures of invasive , numbers are quantified in left panel .

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Invasion ability of MDA-MB-231 cells with knockout of FASN, ACC1, and/or ACC2. The right panel shows the pictures of invasive cells, and the cell numbers are quantified in the left panel.


formation ability in nude mice of MDA - MB - 231 HeLa with gene knockout as indicated .

Tumor formation ability in nude mice of MDA-MB-231 and HeLa cells with gene knockout as indicated.


Western blot verification of knockout of KAR or TECR in MDA - MB - 231 .

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Western blot verification of knockout of KAR or TECR in MDA-MB-231 cells.


B , C . proliferation rate colony formation ability of MDA - MB - 231 with knockout of KAR or TECR . Error bars represent mean ± SD . Data are from triplicate experiments , experimental data were verified in at least two independent experiments . * * P < 0 . 01 ( t - ) ; asterisk shows comparison with wild type group .

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B,C. Cell proliferation rate and colony formation ability of MDA-MB-231 cells with knockout of KAR or TECR. Error bars represent mean ± SD. Data are from triplicate experiments, and all experimental data were verified in at least two independent experiments. **P < 0.01 (t-test); asterisk shows comparison with wild type group.


formation ability in nude mice of MDA - MB - 231 with gene knockout , as indicated .

Tumor formation ability in nude mice of MDA-MB-231 cells with gene knockout, as indicated.


Relative of fatty in MDA - MB - 231 with knockout of KAR or TECR . Each colored datum shows mean from three independent cultures .

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Relative cellular levels of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. Each colored datum shows the mean from three independent cultures.


G . Relative of lipid - containing fatty in MDA - MB - 231 with knockout of KAR or TECR . top curve plot indicates fraction of corresponding fatty acid . Each colored datum shows mean from three independent cultures .

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G. Relative cellular levels of lipid-containing fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. The top curve plot indicates the fraction of the corresponding fatty acid. Each colored datum shows the mean from three independent cultures.


H . Mass isotopomer of fatty in MDA - MB - 231 with knockout of KAR or TECR cultured with 10 mM of 13C6 - glucose for 48 h . Error bars represent mean ± SD . Data are from three independent cultures .

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H. Mass isotopomer analysis of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR cultured with 10 mM of 13C6-glucose for 48 h. Error bars represent mean ± SD. Data are from three independent cultures.


MDA - MB - 231 Wt or ACC - DKO were pretreated for 24 h then cultured in FBS medium or DFBS medium with indicated treatment for 5 days . Treatments were as follows : z - VAD - FMK ( z - VAD , 10 μM ) , necrostain - 1 ( Nec - 1 , 2 μM ) , Disulfiram ( 0 . 4 μM ) , ferrostain - 1 ( Ferr - 1 , 10 μM ) , Trolox ( 100 μM ) , N - Acetyl - L - cysteine ( NAC , 3 mM ) , deferoxamine mesylate ( DFOM , 10 μM ) . Data are presented relative to values obtained for control cultured in FBS medium . Dotted shows survival level of in DFBS medium without treatment ( control ) .

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MDA-MB-231 Wt cells or ACC-DKO cells were pretreated for 24 h and then cultured in FBS medium or DFBS medium with the indicated treatment for 5 days. Treatments were as follows: z-VAD-FMK (z-VAD, 10 μM), necrostain-1 (Nec-1, 2 μM), Disulfiram (0.4 μM), ferrostain-1 (Ferr-1, 10 μM), Trolox (100 μM), N-Acetyl-L-cysteine (NAC, 3 mM), and deferoxamine mesylate (DFOM, 10 μM). Data are presented relative to the values obtained for the control cells cultured in FBS medium. Dotted line shows the survival level of cells in DFBS medium without treatment (control).


Relative of ROS in MDA - MB - 231 / ACC - DKO Wt cultured in FBS medium or DFBS medium for 5 days with or without exogenous VLCFA supplement .

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Relative cellular levels of ROS in MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium for 5 days with or without exogenous VLCFA supplement.


inhibitory effects of H2O2 PMS on MDA - MB - 231 ACC - DKO or Wt cultured in FBS medium for 24 h with indicated concentrations .

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The inhibitory effects of H2O2 and PMS on MDA-MB-231 ACC-DKO or Wt cells cultured in FBS medium for 24 h with indicated concentrations.


of PARP1 caspase - 3 in MDA - MB - 231 / ACC - DKO Wt cultured in FBS or DFBS medium .

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The protein levels of PARP1 and caspase-3 in MDA-MB-231/ACC-DKO and Wt cultured in FBS or DFBS medium.