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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 ) .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 ) .

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

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

(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 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 B-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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 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

(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 ) .

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 B-MEASURED_VAR 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

(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 O O O O O 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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(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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

O O O O O O O O O O O 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 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 ) .

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 .

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 .

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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 .

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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 .

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 .

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 .

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.


Annexin - V & Dead ( 7 - AAD ) flow cytometry of MDA - MB - 231 / ACC - DKO Wt cultured in DFBS medium . right panel shows of annexin V - positive in left panels .

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Annexin-V & Dead cell (7-AAD) flow cytometry analysis of MDA-MB-231/ACC-DKO and Wt cultured in DFBS medium. The right panel shows the quantification of annexin V-positive cells in the left panels.


relative survival of MDA - MB - 231 / ACC - DKO or Wt with control vector or Bcl - 2 or Bcl - xL overexpression cultured in FBS medium or DFBS medium for 5 days . left panels show survival , right panels verify overexpression of Bcl - 2 Bcl - xL .

The relative cell survival of MDA-MB-231/ACC-DKO or Wt with control vector or Bcl-2 or Bcl-xL overexpression cultured in FBS medium or DFBS medium for 5 days. The left panels show the cell survival, and the right panels verify the overexpression of Bcl-2 and Bcl-xL.


transmembrane potential in mitochondria of MDA - MB - 231 / ACC - DKO Wt cultured in FBS medium or DFBS medium was detected by FACS . bar graphs show of JC - 1 monomer .

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The transmembrane potential in mitochondria of MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium was detected by FACS analysis. The bar graphs show the percentage of JC-1 monomer.


MDA - MB - 231 / ACC - DKO Wt were stained for Mito Tracker to visualize mitochondria . Scale bar , 10 μm . Mitochondrial was scored in types indicated . Data represent three independent experiments , at least 100 were counted per type .

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MDA-MB-231/ACC-DKO and Wt cells were stained for Mito Tracker to visualize mitochondria. Scale bar, 10 μm. Mitochondrial morphology was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type.


MDA - MB - 231 / ACC - DKO were in FBS medium with or without exogenous LCFA or VLCFA supplementation , were stained for MitoTracker to visualize mitochondria . Scale bar , 10 μm . Mitochondrial in ( D ) was scored in types indicated . Data represent three independent experiments , at least 100 were counted per type . Error bars represent mean ± SD , data are from triplicate experiments .

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MDA-MB-231/ACC-DKO cells were in FBS medium with or without exogenous LCFA or VLCFA supplementation, and were stained for MitoTracker to visualize mitochondria. Scale bar, 10 μm.   Mitochondrial morphology in (D) was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type. Error bars represent mean ± SD, and data are from triplicate experiments.


A . Equal aliquots ( 2 mg ) of a Triton X - 100 solubilized lysate from rat brain were incubated with GST or GST - ROC . Specifically bound were detected by Western blot with antibodies recognizing indicated .

A. Equal aliquots (2 mg) of a Triton X-100 solubilized lysate from rat brain were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.


B . Brain lysate as in ( A ) was incubated with GST or GST - nCLCb . Specifically bound were detected by Western blot with antibodies recognizing indicated .

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B. Brain lysate as in (A) was incubated with GST or GST-nCLCb. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.


C . Full - GST - nCLCb ( 1 - 228 ) various truncation mutants were purified from . Equal aliquots of purified were incubated with GST or GST - ROC . Specifically bound were detected by Western blot with antibody against CLCs .

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C. Full-length GST-nCLCb (1-228) and various truncation mutants were purified from bacteria. Equal aliquots of purified protein were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibody against CLCs.


D . Brain lysate as in ( A ) was incubated with GST or GST - nCLCb with boundaries as indicated . Specifically bound were detected by Western blot with antibody against CLCs .

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D. Brain lysate as in (A) was incubated with GST or GST-nCLCb proteins with boundaries as indicated. Specifically bound proteins were detected by Western blot with antibody against CLCs.


F . Highly enriched triskelia were stripped from purified CCVs . triskelia were incubated with GST or GST - ROC . Specifically bound were detected by Western blot with antibodies recognizing indicated . For A - D / F , aliquots of starting material ( SM ) equal to 10 % of that added to beads , was analyzed in parallel .

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F. Highly enriched triskelia were stripped from purified CCVs. The triskelia were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins. For A-D/F, aliquots of the starting material (SM) equal to 10% of that added to the beads, was analyzed in parallel.


PCR results of LRRK2 - WT from clone E1 ( 1 ) using primers that detect endogenous LRRK2 . Clone E1 is positive for 3xHA - LRRK2 ( 2 ) using a primer pair with antisense in 3xHA insert sense primer in endogenous LRRK2 . ( 3 ) Control unedited COS - 7 using same primer combination as in ( 2 ) . ( 4 ) 1Kb marker .

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PCR results of LRRK2-WT from clone E1 (1) using primers that detect endogenous LRRK2. Clone E1 is positive for 3xHA-LRRK2 (2) using a primer pair with the antisense in the 3xHA insert and the sense primer in endogenous LRRK2. (3) Control unedited COS-7 cells using the same primer combination as in (2). (4) 1Kb marker.


COS - 7 clone E1 were serum starved followed by 20 min incubation with Alexa488 - EGF , after which were fixed processed for immunofluorescence using HA antibody . Scale bar = 10 m for bottom 6 panels 25 m for top 3 panels .

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COS-7 clone E1 cells were serum starved followed by 20 min incubation with Alexa488-EGF, after which the cells were fixed and processed for immunofluorescence using HA antibody. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.


COS - 7 clone E1 were fixed processed for immunofluorescence using HA CLC antibodies . Scale bar = 10 m .

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COS-7 clone E1 cells were fixed and processed for immunofluorescence using HA and CLC antibodies. Scale bar = 10 m.


COS - 7 clone E1 were fixed an processed for immunofluorescence using HA EEA1 antibodies . Scale bar = 10 m for bottom 6 panels 25 m for top 3 panels .

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COS-7 clone E1 cells were fixed an processed for immunofluorescence using HA and EEA1 antibodies. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.


Lysates from COS - 7 transfected with siRNA as indicated were processed for Western blot with antibodies recognizing indicated . Band intensities of blots as in A are presented as % control siRNA . Two - Student t - , Mann - Whiteney post hoc , * * * p < 0 . 001 , N = 3 .

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Lysates from COS-7 cells transfected with siRNA as indicated were processed for Western blot with antibodies recognizing the indicated proteins.Band intensities of blots as in A are presented as % control siRNA. Two-tailed Student t-test, Mann-Whiteney post hoc test, ***p < 0.001, N = 3.


NP - 40 soluble lysates were prepared from COS - 7 transfected with siRNA as indicated equal aliquots ( 1 . 5 mg ) were incubated with GST - PAK - CRIB domain . Specifically bound were detected by Western blot with antibody recognizing Rac1 . An aliquot of starting material ( SM ) equal to 5 % of that added to GST - PAK - CRIB was analyzed in parallel .

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NP-40 soluble lysates were prepared from COS-7 cells transfected with siRNA as indicated and equal protein aliquots (1.5 mg) were incubated with GST-PAK-CRIB domain. Specifically bound proteins were detected by Western blot with antibody recognizing Rac1. An aliquot of the starting material (SM) equal to 5% of that added to GST-PAK-CRIB was analyzed in parallel.


Band intensities of blots as in A were presented as % control siRNA . Bars represent mean . One - way ANOVA , Bonferroni ' s post hoc , * * * p < 0 . 001 , N = 3 .

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Band intensities of blots as in A were presented as % control siRNA. Bars represent mean sem. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3.


COS - 7 transfected with siRNA as indicated were fixed processed for immunofluorescence using Phalloidin - 647 antibody recognizing CLCs . Scale bar = 10 m .

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COS-7 cells transfected with siRNA as indicated were fixed and processed for immunofluorescence using Phalloidin-647 and antibody recognizing CLCs. Scale bar = 10 m.


Perimeter of as in A were measured a perimeter : was plotted as a % of control siRNA treated . One - way ANOVA , Bonferroni ' s post hoc , * * * p < 0 . 001 , N = 3 .

Perimeter and area of cells as in A were measured and a perimeter:area ratio was plotted as a % of the control siRNA treated cells. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3.


Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus ( at 7 days in vitro ) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa , CLCb , or both in combination . lysates were processed for Western blot with antibody that recognizes forms of CLCs .

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Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus (at 7 days in vitro) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa, CLCb, or both viruses in combination. The lysates were processed for Western blot with antibody that recognizes all forms of CLCs.


Hippocampal neurons transduced as in G were fixed at 21 days in vitro processed for immunofluorescence with polyclonal antibody specific for pre - synaptic synapsin monoclonal antibody specific for post - synaptic PSD95 . lentivirus drives expression of GFP . areas indicated by boxes in low power ( squares ) are shown enlarged immediately below . scale bars = 10 µm for low mag 2 . 5 µm for high mag .

Hippocampal neurons transduced as in G were fixed at 21 days in vitro and processed for immunofluorescence with polyclonal antibody specific for the pre-synaptic protein synapsin and monoclonal antibody specific for the post-synaptic protein PSD95. The lentivirus drives expression of GFP. The areas indicated by white boxes in the low power images (squares) are shown enlarged immediately below. The scale bars = 10 µm for the low mag and 2.5 µm for the high mag.


Ai - x . Lateral view of adult fly head . Scale bar = 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused ( arrows ) disorganized but still separated ommatidia ( arrowheads ) . ii / iii . Rac1 overexpression with KD of dlrrk ( UAS - dlrrk - RNAi ( v22139 or v22140 ) / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused . iv / v . Rac1 overexpression with KD of Clc ( UAS - Clc - RNAi ( v22318 or v106632 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , results in smaller eyes with some ommatidia fused . vi . Rac1 overexpression with KD of inwardly rectifying potassium channel 3 ( irk3 ) , a non - relevant RNAi control ( UAS - irk3 - RNAi ( v101174 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , has no effect . vii / viii . KD of dlrrk with GMR - GAL4 / + ; UAS - dlrrk - RNAi ( v22139 or 22140 ) / + , respectively as control have no effect on eye . ix / x . KD of Clc with GMR - GAL4 / UAS - Clc - RNAi ( v22318 or v106632 ) , respectively as control have no effect on eye .

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

Ai-x. Lateral view of the adult fly head. Scale bar = 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused (arrows) and disorganized but still separated ommatidia (arrowheads).ii/iii. Rac1 overexpression with KD of dlrrk (UAS-dlrrk-RNAi (v22139 or v22140)/longGMR-GAL4, UAS-Rac1W), results in smaller eyes where most ommatidia are fused.iv/v. Rac1 overexpression with KD of Clc (UAS-Clc-RNAi (v22318 or v106632)/+;longGMR-GAL4,UAS-Rac1W/+), results in smaller eyes with some ommatidia fused.vi. Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 (irk3), a non-relevant RNAi control (UAS-irk3-RNAi(v101174)/+; longGMR-GAL4,UAS-Rac1W/+), has no effect.vii/viii. KD of dlrrk with GMR-GAL4/+; UAS-dlrrk-RNAi(v22139 or 22140)/+, respectively as control have no effect on eye morphology.ix/x. KD of Clc with GMR-GAL4/UAS-Clc-RNAi (v22318 or v106632), respectively as control have no effect on eye morphology.


Bi - ii . Lateral view of adult fly head . Scale bar : 100 μm . i . Rac1 overexpression with removing one copy of dlrrk ( dlrrke03680 / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused together . ii . Removing one copy of dlrrke03680 in GMR - GAL4 background , as control has no effect on eye ( GMR - GAL4 / + ; dlrrke03680 / + ) .

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

Bi-ii. Lateral view of the adult fly head. Scale bar: 100 μm.i. Rac1 overexpression with removing one copy of dlrrk (dlrrke03680/longGMR-GAL4,UAS-Rac1W), results in smaller eyes where most ommatidia are fused together.ii. Removing one copy of dlrrke03680 in GMR-GAL4 background, as control has no effect on eye morphology (GMR-GAL4/+;dlrrke03680/+).


Ci - viii . Lateral view of adult fly head . Scale bar : 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused disorganized but still separated ommatidia ( as explained in Ai ) . ii . Rac1 co - expression with dlrrk - WT using UAS - dlrrk - WT / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye . iii . Rac1 co - expression with Clc using UAS - GFP - Clc / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye . iv . Rac1 co - expression with dlrrk - WT - 3KD using UAS - dlrrk - 3KD / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye . v . A wild - type eye of longGMR - GAL4 promoter as a control . vi - viii . Overexpression of dlrrk - WT , dlrrk - 3KD , or Clc alone with GMR - GAL4 as control , have no effect on eye .

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

Ci-viii. Lateral view of the adult fly head. Scale bar: 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia (as explained in Ai).ii. Rac1 co-expression with dlrrk-WT using UAS-dlrrk-WT/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iii. Rac1 co-expression with Clc using UAS-GFP-Clc/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iv. Rac1 co-expression with dlrrk-WT-3KD using UAS-dlrrk-3KD/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.v. A wild-type eye of the longGMR-GAL4 promoter as a control.vi-viii. Overexpression of dlrrk-WT, dlrrk-3KD, or Clc alone with GMR-GAL4 as control, have no effect on eye morphology.


( B ) flg22 - induced ROS production in Col - 0 fer - 4 mutant roots under Pi - starvation conditions . Seedlings grown in either HP or LP media were treated with 1 μM flg22 , RALF23 or both flg22 RALF23 for 15 mins . Roots were harvested stained with H2DCFDA . One representative root was shown . ( C ) Average H2DCFDA signal in roots shown in ( B ) . was quantified with ImageJ . data shown indicate means ± SDs ( n = 30 , n refers to number of roots per group ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

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 O O O O O O O O O O O O O O O O O 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) flg22-induced ROS production in Col-0 and fer-4 mutant roots under Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM flg22, RALF23 or both flg22 and RALF23 for 15 mins. Roots were harvested and stained with H2DCFDA. One representative root was shown. (C) Average H2DCFDA signal intensity in roots shown in (B). The fluorescence intensity was quantified with ImageJ. The data shown indicate the means ± SDs (n = 30, n refers to the number of roots per group); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( F ) RALF23 inhibits flg22 - induced MAMP - responsive FRK1 gene expression in a Pi content - dependent manner . Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins roots were harvested . relative expression of FRK1 was quantified via RT - qPCR . data shown indicate means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

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(F) RALF23 inhibits flg22-induced MAMP-responsive FRK1 gene expression in a Pi content-dependent manner. Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins and roots were harvested. The relative expression of FRK1 was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( A ) PSR induces cleavage of PRORALF23 - GFP into mature RALF23 - GFP . 35S promoter - driven RALF23 - GFP was grown in either HP or LP media for 5 days roots were harvested . Premature mature RALF23 were determined with anti - GFP . β - actin was used as a loading control . Data information : experiments were repeated three times with similar results .

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

(A) The PSR induces the cleavage of PRORALF23-GFP into mature RALF23-GFP. The 35S promoter-driven RALF23-GFP line was grown in either HP or LP media for 5 days and roots were harvested. Premature and mature RALF23 levels were determined with anti-GFP. β-actin was used as a loading control. Data information: All experiments were repeated three times with similar results.


( E ) PSR activates RALF expression in a PHR1 - dependent manner . Col - 0 phr1 seedlings were grown in LP media for 5 days roots were harvested . relative expression of RALFs was quantified via RT - qPCR . data shown indicate means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

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(E) The PSR activates RALF expression in a PHR1-dependent manner. Col-0 and phr1 seedlings were grown in LP media for 5 days and roots were harvested. The relative expression of RALFs was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( G ) PHR1 directly binds to RALF promoters , as shown via ChIP - PCR . Myc - labelled PHR1 overexpression was immunoprecipitated with anti - Myc . ChIP was performed on Col - 0 , CRY1 - Myc PHR1 - Myc transformed , with Col - 0 CRY1 - Myc transformed as negative controls . data shown indicate means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

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(G) PHR1 directly binds to RALF promoters, as shown via ChIP-PCR. The Myc-labelled PHR1 overexpression line was immunoprecipitated with anti-Myc. ChIP analysis was performed on Col-0, CRY1-Myc and PHR1-Myc transformed plants, with Col-0 and CRY1-Myc transformed plants as negative controls. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( B ) PHR1 negatively regulates FLS2 - BAK1 formation under Pi - starvation conditions . Col - 0 , phr1 mutant seedlings were grown in either HP or LP media for 5 days , then lysing roots into protoplasts , transformed with water or PHR1 - Myc plasmid treated with flg22 for 6 hours . amount of immunoprecipitated FLS2 coimmunoprecipitated BAK1 were determined using anti - FLS2 anti - BAK1 antibodies , respectively . Preimmune serum ( IgG ) was used as negative control . Data information : experiments were repeated three times with similar results .

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(B) PHR1 negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, phr1 mutant seedlings were grown in either HP or LP media for 5 days, then lysing roots into protoplasts, transformed with water or PHR1-Myc plasmid and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as negative control. Data information: All experiments were repeated three times with similar results.


( D ) Application of RALF23 / 34 disrupted FLS2 - BAK1 formation in phr1 mutants . phr1 mutants grown in LP media were treated with water , 1 μM flg22 , 1 μM RALF23 , 1 μM RALF34 or both flg22 RALF for 6 hours . amount of immunoprecipitated FLS2 coimmunoprecipitated BAK1 were determined using anti - FLS2 anti - BAK1 antibodies , respectively . IgG was used as a negative control . Data information : experiments were repeated three times with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Application of RALF23/34 disrupted FLS2-BAK1 complex formation in phr1 mutants. phr1 mutants grown in LP media were treated with water, 1 μM flg22, 1 μM RALF23, 1 μM RALF34 or both flg22 and RALF for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. IgG was used as a negative control. Data information: All experiments were repeated three times with similar results.


( E ) RALF23 / 34 promoted growth of Pto DC3000 in phr1 mutants . Col - 0 phr1 mutant seedlings grown in LP media were root inoculated ( by soaking ) with a suspension ( OD600 = 0 . 2 ) for 2 min , number of from root was quantified at 5 dpi . data shown indicate means ± SDs ( n = 18 , n refers to number of roots per group ) . different lowercase letters indicate statistical significance ( one - way ANOVA ) . Data information : experiments were repeated three times with similar results .

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(E) RALF23/34 promoted the growth of Pto DC3000 in phr1 mutants. Col-0 and phr1 mutant seedlings grown in LP media were root inoculated (by soaking) with a bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual root was quantified at 5 dpi. The data shown indicate the means ± SDs (n = 18, n refers to the number of roots per group). The different lowercase letters indicate statistical significance (one-way ANOVA). Data information: All experiments were repeated three times with similar results.


( F ) FER negatively regulates FLS2 - BAK1 formation under Pi - starvation conditions . Col - 0 , fer - 4 mutant FER / fer - 4 complementation lines seedlings were grown in either HP or LP media for 5 days treated with flg22 for 6 hours . amount of immunoprecipitated FLS2 coimmunoprecipitated BAK1 was determined using anti - FLS2 anti - BAK1 antibodies , respectively . Preimmune serum ( IgG ) was used as a negative control . Data information : experiments were repeated three times with similar results .

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

(F) FER negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, fer-4 mutant and FER/fer-4 complementation lines seedlings were grown in either HP or LP media for 5 days and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 was determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as a negative control. Data information: All experiments were repeated three times with similar results.