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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O B-TISSUE I-TISSUE O B-SUBCELLULAR O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O B-GENEPROD 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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR 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 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 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-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O 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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-TISSUE 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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

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 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 . ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-TISSUE 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE 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 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 ) .

O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 ) .

O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-ORGANISM O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

(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 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 ) .

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

O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM 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 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 .

O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) , 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 ) .

O O O O B-CELL_TYPE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 B-CELL_TYPE O O B-SMALL_MOLECULE O B-GENEPROD O O O B-SMALL_MOLECULE 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 bacterial RNA challenge , pretreatment with chloroquine in ( D ) only ( S , Svedberg ; r , ribosomal ; tot , total ; n = 3 ) .

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

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 ) RNA challenge ( Hs , human ; triangle , increasing doses ; LFA , Lipofectamine 2000 ; n = 3 ) .

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

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

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

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 ) and 3 - or 8 days PMA differentiated ( ddi ) THP1 cells to Sa19 challenge ( n = 3 ) .

O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O B-SMALL_MOLECULE 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 and Unc93b1 - / - - 3ddiTHP - 1 cells challenged with ORNs ( n = 3 ) .

O O O O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE 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 cells toward stimulation with TLR ligands and bacterial RNAs ( S , Svedberg ; r , ribosomal ; tot , total ; n = 3 ) .

O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O 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 cells treated with T2 . 5 and challenged with heat inactivated ( hi ) bacteria ( triangle , decreasing doses ; n = 3 ) .

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

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 ) and of each of both versus undifferentiated THP1 cells ( black or white bar , respectively ) according to a transcriptome profiling result ( n = 1 ) .

O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O

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 and murine RAW264 . 7 cells , respectively , upon challenges ( vector , empty plasmid ; n = 3 ) .

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

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 cell responsiveness ( n = 3 ) .

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


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 ) .

O O O O O O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

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 cells challenged with heat inactivated ( hi ) bacteria ( triangle , decreasing doses ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ) upon TLR2 blockade ( T2 . 5 ) .

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

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 cells upon challenge with human ( Hs ) mitochondrial ( mt ) RNA ( triangle , increasing doses ) .

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

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 cells or cytokine release by PBMCs all transfected with RNAs indicated and additional uridine ( U ) if indicated ( n . p . , not performed ; vector , empty plasmid ) .

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

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 cells of indicated genotypes upon TLR2 blockade ( T2 . 5 ) and challenge with TLR ligands , heat inactivated ( hi ) or viable bacteria .

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

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 ) and TLR2 blockade ( T2 . 5 ) followed by TLR ligand challenge or bacterial infection .

O B-TISSUE O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

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 ) and challenge with TLR ligands , heat inactivated ( hi ) , or viable bacteria .

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

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 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 ) .

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

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 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 .

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

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 , 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 .

O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O 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, 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 , 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 .

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

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 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 .

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

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 , 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 .

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

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 , 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 .

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

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 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 .

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

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 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 .

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

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 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 .

O O O O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

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

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 , 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 .

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

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 , 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 .

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

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 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 .

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

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 , 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 .

O O O O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

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 ) , 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 .

O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-TISSUE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) 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 ) ,

O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE O O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O

(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 ) 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 .

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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 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 ) .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O B-TISSUE O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O

(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 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 )

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

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

O O O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE O O B-TISSUE O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


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 .

O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-TISSUE O O O O B-TISSUE O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

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 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 .

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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.


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 .

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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 ) , 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 .

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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 − / − , 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 .

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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 ) , 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 .

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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 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 .

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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 , and / or ACC2 in MDA - MB - 231 cells .

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


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

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


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 .

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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.


Tumor formation ability in nude mice of MDA - MB - 231 and HeLa cells 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 cells .

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


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 .

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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.


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

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


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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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.


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 .

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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.


The protein levels of PARP1 and caspase - 3 in MDA - MB - 231 / ACC - DKO and 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 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 .

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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.


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 .

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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.


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 .

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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 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 .

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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 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 .

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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 proteins were detected by Western blot with antibodies recognizing the indicated proteins .

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

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 proteins were detected by Western blot with antibodies recognizing the indicated proteins .

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

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 - 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 .

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

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 proteins with boundaries as indicated . Specifically bound proteins were detected by Western blot with antibody against CLCs .

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

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 . 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 .

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

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 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 .

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

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 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 .

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

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 cells were fixed and processed for immunofluorescence using HA and CLC antibodies . Scale bar = 10 m .

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

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


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 .

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

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 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 .

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

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.