C . Overexpression of eIF2A induces the relative expression of PTENε . FLAG - tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN . GAPDH was used as a loading control .

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C. Overexpression of eIF2A induces the relative expression of PTENε. FLAG-tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN. GAPDH was used as a loading control.


C . The transfected Hela PTEN - / - cells were stained with an anti - β - catenin antibody and DAPI before being imaged with confocal microscopy .

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C. The transfected Hela PTEN-/- cells were stained with an anti-β-catenin antibody and DAPI before being imaged with confocal microscopy.


To determine the sequences that are critical for the membrane localization of PTENε . Hela were transfected with constructs Thirty - six hours after transfection , transfected Hela cells were stained with anti - β - catenin antibody and DAPI , followed by imaging with confocal microscopy . The scale bars represent 5 μm ( F ) .

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To determine the sequences that are critical for the membrane localization of PTENε. Hela were transfected with constructs Thirty-six hours after transfection, transfected Hela cells were stained with anti-β-catenin antibody and DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm (F).


The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP , E - cadherin , and β - tubulin antibodies ( G ) . C : cytoplasm . M : cell plasma membrane .

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The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP, E-cadherin, and β-tubulin antibodies (G). C: cytoplasm. M: cell plasma membrane.


D . Images of immunofluorescence staining for PTENε and its interacting proteins ( VASP and FSCN1 ) . Hela PTEN - / - cells were co - transfected by a plasmid expressing C - terminal GFP - tagged PTENε with FLAG - tagged targets ( FSCN1 , VASP ) separately , followed by staining with an anti - FLAG antibody and DAPI , and were imaged by confocal microscopy .

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D. Images of immunofluorescence staining for PTENε and its interacting proteins (VASP and FSCN1). Hela PTEN-/- cells were co-transfected by a plasmid expressing C-terminal GFP-tagged PTENε with FLAG-tagged targets (FSCN1, VASP) separately, followed by staining with an anti-FLAG antibody and DAPI, and were imaged by confocal microscopy.


E . The cell plasma membrane localization of PTENε is required for its interaction with downstream targets . FLAG - tagged CDC42 , VASP , ACTR2 , or FSCN1 plasmid was co - transfected with a plasmid expressing PTENε with or without 69 - 81aa deletion carrying C - terminal S - tag and HA tag in HEK293 cells . Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag .

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E. The cell plasma membrane localization of PTENε is required for its interaction with downstream targets. FLAG-tagged CDC42, VASP, ACTR2, or FSCN1 plasmid was co-transfected with a plasmid expressing PTENε with or without 69-81aa deletion carrying C-terminal S-tag and HA tag in HEK293 cells. Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag.


( H , I ) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His - PTENε or without purified PTENε protein as a control , followed by immunoblotting with antibodies against p - ACTR2 ( Thr237 and Thr238 ) and p - VASP ( Ser157 ) . Purified proteins ( PTENε , PKA , NIK , ACTR , and VASP ) were detected by anti - His antibody ( ZSGB - BIO , TA - 02 ) .

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(H, I) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His-PTENε or without purified PTENε protein as a control, followed by immunoblotting with antibodies against p-ACTR2 (Thr237 and Thr238) and p-VASP (Ser157). Purified proteins (PTENε, PKA, NIK, ACTR, and VASP) were detected by anti-His antibody (ZSGB-BIO, TA-02).


A . Immunofluorescence staining of F - actin in PTENε or PTENε Y210L overexpressed Hela PTEN - / - cells and control cells . PTENε - GFP , PTENε Y210L - GFP , or mock construct was introduced into HeLa PTEN - / - cells respectively . The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy . The scale bars represent 5 μm ( left panel ) . White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane . B . Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t - test . * * * * , p < 0 . 001 .

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A. Immunofluorescence staining of F-actin in PTENε or PTENε Y210L overexpressed Hela PTEN-/- cells and control cells. PTENε-GFP, PTENε Y210L-GFP, or mock construct was introduced into HeLa PTEN-/- cells respectively. The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy. The scale bars represent 5 μm (left panel). White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane. B. Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t-test. ****, p<0.001.


G . Left panel : immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells . Middle panel : representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model ( n = 4 ) . H & E , hematoxylin - eosin staining . The scale bars represent 600 mm ( upper panel ) and 500μm ( lower panel ) separately . Right panel : the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules ( data are presented as mean ± SD based on three independent replicates ) . The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group . * * * * , p < 0 . 0001 in the unpaired t - test .

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G. Left panel: immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells. Middle panel: representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model (n=4). H & E, hematoxylin-eosin staining. The scale bars represent 600 mm (upper panel) and 500μm (lower panel) separately. Right panel: the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules (data are presented as mean ± SD based on three independent replicates). The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group. ****, p<0.0001 in the unpaired t-test.


A Scheme of the lentiviral vector with inducible FlagOmomyc ( top ) and immunoblotting ( bottom ) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0 - 48 hours .

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A Scheme of the lentiviral vector with inducible FlagOmomyc (top) and immunoblotting (bottom) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0-48 hours.


B Proliferation curves of BT168FO and BT168GFPcells upon Dox treatment for 0 - 96 h ( n = 3 ; mean ± SD ) . Viable cells were counted using a haemocytometer .

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B Proliferation curves of BT168FO and BT168GFPcells upon Dox treatment for 0-96 h (n = 3; mean ± SD). Viable cells were counted using a haemocytometer.


C - D Self - renewal assay upon Dox treatment . C : histograms showing the percentage of cells capable of re - forming a neurosphere seven days after dissociation ( n = 3 ; mean ± SD ) . D : representative micrographs of BT168FO cell neurospheres .

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C-D Self-renewal assay upon Dox treatment. C: histograms showing the percentage of cells capable of re-forming a neurosphere seven days after dissociation (n = 3; mean ± SD). D: representative micrographs of BT168FO cell neurospheres.


E qRT - PCR of proliferation , stem cell and differentiation markers ( CCND1 , PTEN , SOX2 , NOTCH1 , NESTIN , MYC ) in BT168FO cells after 48 h Dox treatment , compared to uninduced cells ( n = 3 ; mean ± SD ) . Expression levels in non - induced cells were set as 1 .

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E qRT-PCR of proliferation, stem cell and differentiation markers (CCND1, PTEN, SOX2, NOTCH1, NESTIN, MYC) in BT168FO cells after 48 h Dox treatment, compared to uninduced cells (n = 3; mean ± SD). Expression levels in non-induced cells were set as 1.


F . Transwell migration assay of BT168FO cells after 3 days with or without Dox ( n = 3 ; mean ± SD ) . 10 fields were counted per assay .

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F. Transwell migration assay of BT168FO cells after 3 days with or without Dox (n = 3; mean ± SD). 10 fields were counted per assay.


G . Immunofluorescence analyses of GSC differentiation . To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days . The upper panel displays immunofluorescence images of NESTIN , GFAP , ß - tubulin III , SOX2 , and FlagOmomyc expression . FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels , while inducing the onset of ß - tubulin III . The lower panel shows the percentage of positive cells for each cell marker evaluated ( n = 4 ; mean ± SD ) . 16 fields for each assay were examined ; scale bar = 100 μm .

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G. Immunofluorescence analyses of GSC differentiation. To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days. The upper panel displays immunofluorescence images of NESTIN, GFAP, ß-tubulin III, SOX2, and FlagOmomyc expression. FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels, while inducing the onset of ß-tubulin III. The lower panel shows the percentage of positive cells for each cell marker evaluated (n = 4; mean ± SD). 16 fields for each assay were examined; scale bar = 100 μm.


A - H Immunostaining of representative xenografts derived from naïve ( control ) and Omomyc expressing BT168FO cells . A - D ) Flag , Ki67 , CD31 , and DCX ( doublecortin ) immunostaining of adjacent sections in control and Omomycxenografts . E - G ) Differentiation phenotype investigated by GFAP , ß - tubulin III , and OLIG2 staining . H ) Immunostaining for anti - human nuclei . Representative images are shown . Scale bar = 100μm .

A-H Immunostaining of representative xenografts derived from naïve (control) and Omomyc expressing BT168FO cells. A-D) Flag, Ki67, CD31, and DCX (doublecortin) immunostaining of adjacent sections in control and Omomycxenografts. E-G) Differentiation phenotype investigated by GFAP, ß-tubulin III, and OLIG2 staining. H) Immunostaining for anti-human nuclei. Representative images are shown. Scale bar = 100μm.


A - B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter - target genes in BT168FO ( A ) cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in units of tags per 50bp . The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows , in cells treated or not with Dox .

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A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in BT168FO (A) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.


A - B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter - target genes in U87FO ( B ) cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in units of tags per 50bp . The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows , in cells treated or not with Dox .

A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in U87FO (B) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.


A Heatmap of RNA Pol II levels around TSSs of MYC promoter - target genes in BT168FO cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in tags / 50bp .

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A Heatmap of RNA Pol II levels around TSSs of MYC promoter-target genes in BT168FO cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in tags/50bp.


B Tracks of Pol II binding signals ( ChIP - seq ) at NCL , miR - 17 - 92 , OLIG2 , HDAC1 , and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h . The y - axis shows Pol II binding signals as tags / 500bp / million reads . The x - axis displays genomic positions ; introns as lines , exons as boxes . Arrowheads denote direction of transcription . Blue and green bars denote the presence of MYC or Omomyc peaks . Grey boxes are TSS regions .

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B Tracks of Pol II binding signals (ChIP-seq) at NCL, miR-17-92, OLIG2, HDAC1, and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h. The y-axis shows Pol II binding signals as tags/500bp/million reads. The x-axis displays genomic positions; introns as lines, exons as boxes. Arrowheads denote direction of transcription. Blue and green bars denote the presence of MYC or Omomyc peaks. Grey boxes are TSS regions.


C Transcript levels ( FPKM from RNA - seq data ) in BT168FO cells of the genes shown in B . Data are means of 3 independent biological replicates .

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C Transcript levels (FPKM from RNA-seq data) in BT168FO cells of the genes shown in B. Data are means of 3 independent biological replicates.


D Ratio between fold enrichments ( specific antibody / IgG signal ) derived from qChIPs of Pol II - Ser2p and Pol II - Ser5p ( n = 3 ; mean ± SD ) .

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D Ratio between fold enrichments (specific antibody / IgG signal) derived from qChIPs of Pol II-Ser2p and Pol II-Ser5p (n = 3; mean ± SD).


E RNA Polymerase II traveling ratio distribution from the Pol II ChIP - seqs , in the presence or absence of Dox .

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E RNA Polymerase II traveling ratio distribution from the Pol II ChIP-seqs, in the presence or absence of Dox.


A Promoter occupancy by MYC correlates with transcript levels in cells untreated with doxycycline ( black line ) . In doxycycline treated cells promoter occupancy by MYC is lower , and no longer increases together with transcript levels . The scatter plot displays MYCChIP - seq reads at promoters ( - 1000 , + 100 regions with respect to TSS ) in BT168FO cells - untreated ( black ) or upon 48 h Dox treatment ( blue ) - versus transcript levels ( FPKM from RNA - seq data ) .

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A Promoter occupancy by MYC correlates with transcript levels in cells untreated with doxycycline (black line). In doxycycline treated cells promoter occupancy by MYC is lower, and no longer increases together with transcript levels. The scatter plot displays MYCChIP-seq reads at promoters (-1000, +100 regions with respect to TSS) in BT168FO cells - untreated (black) or upon 48 h Dox treatment (blue) - versus transcript levels (FPKM from RNA-seq data).


B Distribution of transcript level differences ( Δ FPKM ) of single genes classified as promoter - targets ( green ) , intragenic - targets ( blue ) , intergenic - targets ( red ) in Dox treated ( 24 h ) versus untreated cells , plotted against expression in untreated cells ( n = 3 ) . Δ FPKM = FPKM _ 24h Dox - FPKM _ no Dox .

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B Distribution of transcript level differences (Δ FPKM) of single genes classified as promoter-targets (green), intragenic-targets (blue), intergenic-targets (red) in Dox treated (24 h) versus untreated cells, plotted against expression in untreated cells (n=3). Δ FPKM = FPKM_24h Dox - FPKM_no Dox.


C The table first row shows the number of genes significantly up or down regulated at 24 h of Dox treatment , subdivided in promoter - , intragenic - , intergenic - targets ( ± 50 kbp from TSS ) , and the rest . The following rows depict - for each gene class - mean ( average ) values of FPKM in cells treated or not with Dox for 24 h and the corresponding Δ FPKM and Fold change ( Fc ) . Data are from 3 independent biological replicates .

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C The table first row shows the number of genes significantly up or down regulated at 24 h of Dox treatment, subdivided in promoter-, intragenic-, intergenic-targets (± 50 kbp from TSS), and the rest. The following rows depict - for each gene class - mean (average) values of FPKM in cells treated or not with Dox for 24 h and the corresponding Δ FPKM and Fold change (Fc). Data are from 3 independent biological replicates.


D Transcript level distribution of MYC promoter - targets significantly down ( red dots ) or up ( green dots ) modulated in cells treated with Dox for 24 h vs . untreated cells . Transcript levels , expressed as FPKM , represent averages of three independent experiments .

D Transcript level distribution of MYC promoter-targets significantly down (red dots) or up (green dots) modulated in cells treated with Dox for 24 h vs. untreated cells. Transcript levels, expressed as FPKM, represent averages of three independent experiments.


A RNA - seq expression values of select genes in BT168FO GSCs along a 48 h time course of Omomyc induction ( n = 3 independent biological replicates ) . The block on the left represents DUSP ( dual specificity phosphatase ) family genes . The middle block contains transcription factors ( TFs ) : the upper thirteen ( ASCL1 - SALL1 ) are GSC - specific and the remaining ones are oncogenic . The right block contains genes , including well - known MYC targets , with functions in proliferation , neurodifferentiation , and gliomagenesis . The first column of each block represents the average expression ( log2 FPKM ) in untreated cells ( 0 h ) in the colour scale illustrated by the lower bar : violet indicates low and blue high expression . The other columns depict relative expression versus untreated cells ( average log2 FC ) at different times ( 8 - 48h ) of Dox treatment , according to the scale shown by the upper bar : red indicating low and green high expression . The table in the upper left summarizes the GSEA score of the set 19 GSC - specific transcription factors [ 40 ] , showing a significant downregulation by Omomyc .

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A RNA-seq expression values of select genes in BT168FO GSCs along a 48 h time course of Omomyc induction (n = 3 independent biological replicates). The block on the left represents DUSP (dual specificity phosphatase) family genes. The middle block contains transcription factors (TFs): the upper thirteen (ASCL1-SALL1) are GSC-specific and the remaining ones are oncogenic. The right block contains genes, including well-known MYC targets, with functions in proliferation, neurodifferentiation, and gliomagenesis. The first column of each block represents the average expression (log2 FPKM) in untreated cells (0 h) in the colour scale illustrated by the lower bar: violet indicates low and blue high expression. The other columns depict relative expression versus untreated cells (average log2 FC) at different times (8-48h) of Dox treatment, according to the scale shown by the upper bar: red indicating low and green high expression. The table in the upper left summarizes the GSEA score of the set 19 GSC-specific transcription factors [40], showing a significant downregulation by Omomyc.


B Enrichment plots obtained by GSEA ( Gene Set Enrichment Analysis ) of RNA - seq data from BT168FO cells . Left : genes downregulated by Omomyc in BT168FO identify the set of genes targeted by the GSC core TFs [ 40 ] . Middle : Omomyc downregulated genes identify a gene set upregulated by MYC in cancer cells [ 66 ] . Right : genes upregulated by Omomyc identify genes downregulated in small cell lung cancers carrying MYC amplification [ 2 ] . .

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B Enrichment plots obtained by GSEA (Gene Set Enrichment Analysis) of RNA-seq data from BT168FO cells. Left: genes downregulated by Omomyc in BT168FO identify the set of genes targeted by the GSC core TFs [40]. Middle: Omomyc downregulated genes identify a gene set upregulated by MYC in cancer cells [66]. Right: genes upregulated by Omomyc identify genes downregulated in small cell lung cancers carrying MYC amplification [2]. .


B Immunoblots of EGFR , ZEB1 and GAPDH loading control expression in BT168FO and BT275FO after 2 days with ( + ) or without ( - ) Dox . Representative images are shown .

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B Immunoblots of EGFR, ZEB1 and GAPDH loading control expression in BT168FO and BT275FO after 2 days with (+) or without (-) Dox. Representative images are shown.


C RT - qPCR expression analysis of selected miRs in U87FO cells grown 48 h in the presence ( + ) or absence ( - ) of Dox ( n = 3 ; mean ± SD ) . Expression levels were normalized using U6RNA as control . Expression levels in non - induced cells were set as 1 .

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C RT-qPCR expression analysis of selected miRs in U87FO cells grown 48 h in the presence (+) or absence (-) of Dox (n = 3; mean ± SD). Expression levels were normalized using U6RNA as control. Expression levels in non-induced cells were set as 1.


D EGFR , ZEB1 , and GAPDHimmunoblots in the same cells as in panel C . Representative images are shown .

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D EGFR, ZEB1, and GAPDHimmunoblots in the same cells as in panel C. Representative images are shown.


E EGFR , ZEB1 , and GAPDHimmunoblots in uninduced U87FO cells ( - ) and in cells transfected with LNA oligos targeting miR - 200a - 200b - 429 ( LNA 200 ) and miR - 146b ( LNA 146b ) , or with a non - targeting LNA ( C ) , and induced with Dox for 48 h ( + Dox ) . Representative images are shown .

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E EGFR, ZEB1, and GAPDHimmunoblots in uninduced U87FO cells (-) and in cells transfected with LNA oligos targeting miR-200a-200b-429 (LNA 200) and miR-146b (LNA 146b), or with a non-targeting LNA (C), and induced with Dox for 48 h (+Dox). Representative images are shown.


F MTS cell proliferation assay of uninduced or Dox induced U87FO cells , transfected with either a non - targeting LNA ( LNA C ) , or LNA oligos targeting the miR - 200a - 200b - 429 cluster ( LNA 200 ) , miR - 146b ( LNA 146b ) , or both ( LNAmix ) and analysed at 24 , 48 and 72h ( n = 3 ; mean ± SD ) .

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F MTS cell proliferation assay of uninduced or Dox induced U87FO cells, transfected with either a non-targeting LNA (LNA C), or LNA oligos targeting the miR-200a-200b-429 cluster (LNA 200), miR-146b (LNA 146b), or both (LNAmix) and analysed at 24, 48 and 72h (n = 3; mean ± SD).


G Migration assay of U87FO cells transfected with non - targeting control LNA oligos ( LNA C ) or with LNA oligos targeting miR - 200a - 200b - 429 ( LNA 200 ) ; cells were either untreated ( - ) or treated with Dox for 24 h ( + Dox ) ; ( n = 3 ; mean ± SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Migration assay of U87FO cells transfected with non-targeting control LNA oligos (LNA C) or with LNA oligos targeting miR-200a-200b-429 (LNA 200); cells were either untreated (-) or treated with Dox for 24 h (+Dox); (n = 3; mean ± SD).


Curated heatmaps of top 76 up and 72 down regulated genes . Highlighted genes were chosen based on biological interest . The coloured bar represents the expression level from low ( green ) to high ( red ) .

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

Curated heatmaps of top 76 up and 72 down regulated genes. Highlighted genes were chosen based on biological interest. The coloured bar represents the expression level from low (green) to high (red).


Real - time qPCR analysis of fetal organoids cultured for one month showing decrease in relative expression of indicated neonatal markers A Ass1 , B Blimp - 1 , C FcRn and D Lct and increase of the adult markers E Sis , F Treh and G Arg2 ( n = 3 individual wells from single organoid culture ( see material and methods ) ; experiment was repeated in four to eight independent organoid cultures with similar results ) .

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

Real-time qPCR analysis of fetal organoids cultured for one month showing decrease in relative expression of indicated neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct and increase of the adult markers E Sis, F Treh and G Arg2 (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four to eight independent organoid cultures with similar results).


Enzyme activity assay of fetal organoids for H Lactase , I sucrase , J maltase , K trehalase and L arginase . Activity is given in µM glucose / µg protein · min - 1 ( experiment was generated from single organoid culture ( see material and methods ) and repeated in 3 independent organoid cultures with similar results ) .

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

Enzyme activity assay of fetal organoids for H Lactase, I sucrase, J maltase, K trehalase and L arginase. Activity is given in µM glucose/µg protein·min-1 (experiment was generated from single organoid culture (see material and methods) and repeated in 3 independent organoid cultures with similar results).


Relative expression detected by Real - time qPCR in fetal organoids ( ) and adult organoids ( ) . Neonatal markers A Ass1 , B Blimp - 1 , C FcRn and D Lct in fetal organoids cultured for one month compare to levels adult organoids . Mature markers E Sis , F Treh and G Arg2 increase in fetal organoids to adult levels ( n = 3 individual wells from single organoid culture ( see material and methods ) ; experiment was repeated in four independent organoid cultures with similar results ) .

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

Relative expression detected by Real-time qPCR in fetal organoids ( ) and adult organoids ( ). Neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct in fetal organoids cultured for one month compare to levels adult organoids. Mature markers E Sis, F Treh and G Arg2 increase in fetal organoids to adult levels (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four independent organoid cultures with similar results).


Enzyme activity assay of fetal ( ) and adult ( ) organoids . H Lactase activity is higher in fetal organoids decreasing to adult levels at day 28 , while I sucrase , J maltase , K trehalase and L arginase increase to adult levels . Activity is given in µM glucose / µg protein · min - 1 ( n = 3 independent organoid cultures ) .

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

Enzyme activity assay of fetal ( ) and adult ( ) organoids. H Lactase activity is higher in fetal organoids decreasing to adult levels at day 28, while I sucrase, J maltase, K trehalase and L arginase increase to adult levels. Activity is given in µM glucose/µg protein·min-1 (n=3 independent organoid cultures).


Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ) , as revealed by real - time qPCR . Decrease in relative expression of neonatal marker A Blimp - 1 and increase in expression of adult marker B Sis occur earlier in dexamethasone treated

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

Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. Decrease in relative expression of neonatal marker A Blimp-1 and increase in expression of adult marker B Sis occur earlier in dexamethasone treated organoids


Dexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of the culture . Activity is given in µM glucose / µg protein · min - 1 ( n = 3 independent organoid cultures ) .

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

Dexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).


Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ) , as revealed by real - time qPCR . expression of adult marker E Treh and F Arg2 remains unchanged ( n = 3 individual wells from single organoid

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

Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. expression of adult marker E Treh and F Arg2 remains unchanged (n=3 individual wells from single organoid culture


Dexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture . Activity is given in µM glucose / µg protein · min - 1 ( n = 3 independent organoid cultures ) .

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

Dexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).


Microscopic images of fetal organoids at day 3 , 13 , 20 and 30 of culture . Scale bars : 500 µm .

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

Microscopic images of fetal organoids at day 3, 13, 20 and 30 of culture. Scale bars: 500 µm.


Percentage of fetal spheroids decreases during culture ( n = 3 individual wells from single organoid culture ( see material and methods ) ; experiment was repeated in ten independent organoid cultures with similar results ) .

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

Percentage of fetal spheroids decreases during culture (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in ten independent organoid cultures with similar results).


Immmunohistochemistry of fetal organoids for C Ass1 D Sis and E Arg2 . Quantification of 3 slides per staining , 7 to 10 organoids / spheroids per slide . Scale bars : 50µm .

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

Immmunohistochemistry of fetal organoids for C Ass1 D Sis and E Arg2. Quantification of 3 slides per staining, 7 to 10 organoids / spheroids per slide. Scale bars: 50µm.


A . Viability of primary human PMNs following treatment with purified recombinant bi - component leukocidins using the viability dye CellTiter . Bars indicate Mean ± SEM , with n = 5 donors .

A. Viability of primary human PMNs following treatment with purified recombinant bi-component leukocidins using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 5 donors.


B . Half maximal effective concentrations ( EC50 ) of native and heterologous toxin pairings on primary human PMNs using the viability dye CellTiter . E = LukE , D = LukD , A = HlgA , B = HlgB , C = HlgC , S = LukS - PV , F = LukF - PV . Bars indicate Mean ± SEM , with n = 6 donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Half maximal effective concentrations (EC50) of native and heterologous toxin pairings on primary human PMNs using the viability dye CellTiter. E = LukE, D = LukD, A = HlgA, B = HlgB, C = HlgC, S = LukS-PV, F = LukF-PV. Bars indicate Mean ± SEM, with n = 6 donors.


D . Viability of primary human PMNs following incubation with WT and mutant toxin pairings using the viability dye CellTiter . Insert : 2 μg per lane of purified recombinant toxins visualized by Coomassie Blue staining on a 12 % SDS - PAGE gel . E = LukE , D = LukD , Emut1 = LukEmut1 , Dmut = LukDmut . Bars indicate Mean ± SEM , with n = 4 donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability of primary human PMNs following incubation with WT and mutant toxin pairings using the viability dye CellTiter. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie Blue staining on a 12% SDS-PAGE gel. E = LukE, D = LukD, Emut1 = LukEmut1, Dmut = LukDmut. Bars indicate Mean ± SEM, with n = 4 donors.


E . Viability of primary humanPMNs following simultaneous incubation with a lethal dose of WT LukED ( 78 . 12 nM per subunit ) and a dose response of WT or dominant - negative ( DN ) toxin subunits using the viability dye LukE . E = LukE , D = LukD , Emut1 = LukEmut1 , Emut2 = LukEmut2 , Emut1Dmut = LukEmut1LukDmut , Emut2Dmut = LukEmut2LukDmut . Bars indicate Mean ± SEM , with n = 6 donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability of primary humanPMNs following simultaneous incubation with a lethal dose of WT LukED (78.12 nM per subunit) and a dose response of WT or dominant-negative (DN) toxin subunits using the viability dye LukE. E = LukE, D = LukD, Emut1 = LukEmut1, Emut2 = LukEmut2, Emut1Dmut = LukEmut1LukDmut, Emut2Dmut = LukEmut2LukDmut . Bars indicate Mean ± SEM, with n = 6 donors.


C . Viability of primary human PMNs following incubation with mutant toxin pairings using the viability dye CellTiter . Bars indicate Mean ± SEM , with n = 3 donors .

O O O O O O O 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. Viability of primary human PMNs following incubation with mutant toxin pairings using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 3 donors.


D - F . Viability of primary humanPMNs following incubation with WT or mutant toxin pairings . Insert : 2 μg per lane of purified recombinant toxins visualized by Coomassie blue staining using a 12 % SDS - PAGE gel . Amut1 = HlgAmut1 , Bmut = HlgBmut , Cmut1 = HlgCmut1 , Smut1 = LukS - PVmut1 , Fmut = LukF - PVmut . Bars indicate Mean ± SEM , with n = 4 donors ( D ) , n = 4 donors ( E ) , and n = 5 donors ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. Viability of primary humanPMNs following incubation with WT or mutant toxin pairings. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie blue staining using a 12% SDS-PAGE gel. Amut1 = HlgAmut1, Bmut = HlgBmut, Cmut1 = HlgCmut1, Smut1 = LukS-PVmut1, Fmut = LukF-PVmut. Bars indicate Mean ± SEM, with n = 4 donors (D), n = 4 donors (E), and n = 5 donors (F).


A - D . Viability of primary human PMNs following incubation with a LD90 of LukED ( 78 . 12 nM ) , HlgAB ( 78 . 12 nM ) , HlgCB ( 39 . 1 nM ) , or LukSF - PV ( 19 . 53 nM ) and the indicated dominant - negative toxins using the viability dye CellTiter . Open white symbols indicate no treatment . Filled black symbols indicate treatment with dominant - negative toxins . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 5 donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. Viability of primary human PMNs following incubation with a LD90 of LukED (78.12 nM), HlgAB (78.12 nM), HlgCB (39.1 nM), or LukSF-PV (19.53 nM) and the indicated dominant-negative toxins using the viability dye CellTiter. Open white symbols indicate no treatment. Filled black symbols indicate treatment with dominant-negative toxins. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 5 donors.


E . Viability of primary human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA - MRSA USA300 strain LAC using the viability dye CellTiter . Bars indicate Mean ± SEM , with n = 6 donors .

O O O O O O O O O O O O O O O O O O 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. Viability of primary human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA-MRSA USA300 strain LAC using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 6 donors.


F . Viability of primary human PMNs following incubation with 1 . 25 % filtered culture supernatants and a dose response of the indicated dominant - negative ( DN ) toxins using the viability dye CellTiter . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 3 donors .

F. Viability of primary human PMNs following incubation with 1.25% filtered culture supernatants and a dose response of the indicated dominant-negative (DN) toxins using the viability dye CellTiter. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.


G . Viability of primary human PMNs during a 3 - hour ex vivo infection with the S . aureus Newman toxinless strain overproducing the indicated toxins using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Bars indicate Mean ± SEM , with n = 3 donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability of primary human PMNs during a 3-hour ex vivo infection with the S. aureus Newman toxinless strain overproducing the indicated toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3 donors.


H . Viability of primary human PMNs during a 4 - hr ex vivo infection with a MOI of 50 of S . aureus Newman toxinless strain overproducing the indicated toxins in the presence of a dose response of the DN toxins using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 3 donors .

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

H. Viability of primary human PMNs during a 4-hr ex vivo infection with a MOI of 50 of S. aureus Newman toxinless strain overproducing the indicated toxins in the presence of a dose response of the DN toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.


B . Exoproteome analysis of LukAB mutants produced by S . aureus strain Newman in a toxinless background . Samples were electrophoresed on a 12 % SDS - PAGE gel and visualized by staining with Coomassie blue . A representative of two experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O 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. Exoproteome analysis of LukAB mutants produced by S. aureus strain Newman in a toxinless background. Samples were electrophoresed on a 12% SDS-PAGE gel and visualized by staining with Coomassie blue. A representative of two experiments is shown.


C . Exoproteome and immunoblot analyses of wildtype S . aureus strain Newman transformed with empty plasmid ( p - ) or with plasmids containing the mutated lukAB loci . A representative of two independent experiments is shown .

O O O O O O O O O O O O O O O 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. Exoproteome and immunoblot analyses of wildtype S. aureus strain Newman transformed with empty plasmid (p-) or with plasmids containing the mutated lukAB loci. A representative of two independent experiments is shown.


D . Viability of primary human PMNs during a 2 - hour ex vivo infection with the indicated S . aureus strains using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Bars indicate Mean ± SEM , with n = 3 . Statistical significance is indicated as follows : MOI of 100 : * * p = 0 . 0026 for p - vs LukABmut1 and * * * p = 0 . 0003 for p - vs LukABmut2 ; MOI of 50 : * * * * p < 0 . 0001 for p - vs LukABmut1 and * * * * p < 0 . 0001 for p - vs LukABmut2 ; MOI of 25 : * * * p = 0 . 0007 for p - vs LukABmut1 and * * * * p < 0 . 0001 for p - vs LukABmut2 using two - way ANOVA with Tukey ' s post hoc test correction for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability of primary human PMNs during a 2-hour ex vivo infection with the indicated S. aureus strains using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3. Statistical significance is indicated as follows: MOI of 100: **p = 0.0026 for p- vs LukABmut1 and ***p = 0.0003 for p- vs LukABmut2; MOI of 50: ****p <0.0001 for p- vs LukABmut1 and ****p <0.0001 for p- vs LukABmut2; MOI of 25: ***p = 0.0007 for p- vs LukABmut1 and ****p < 0.0001 for p- vs LukABmut2 using two-way ANOVA with Tukey's post hoc test correction for multiple comparisons.


B . Competition assay using GFP - LukE ( 312 . 5 nM ) and a dose response of unlabeled WT or dominant - negative ( DN ) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs . E = LukE , Emut1 = LukEmut1 , A = HlgA , Amut1 = HlgAmut1 , S = LukS - PV , C = HlgC . Data is presented as total fluorescence emitted from PMNs . A representative of three independent experiments each done with hPMNs isolated from different donors is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Competition assay using GFP-LukE (312.5 nM) and a dose response of unlabeled WT or dominant-negative (DN) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs. E = LukE, Emut1 = LukEmut1, A = HlgA, Amut1 = HlgAmut1, S = LukS-PV, C = HlgC. Data is presented as total fluorescence emitted from PMNs. A representative of three independent experiments each done with hPMNs isolated from different donors is shown.


D . Recruitment assay using GFP - LukD ( 156 . 3 nM ) and a dose response of unlabeled dominant - negative ( DN ) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs . Emut1 = LukEmut1 , Amut1 = HlgAmut1 . Data is presented as total fluorescence emitted from GFP - positive PMNs . A representative of three independent experiments each done with hPMNs isolated from different donors is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Recruitment assay using GFP-LukD (156.3 nM) and a dose response of unlabeled dominant-negative (DN) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs. Emut1 = LukEmut1, Amut1 = HlgAmut1. Data is presented as total fluorescence emitted from GFP-positive PMNs. A representative of three independent experiments each done with hPMNs isolated from different donors is shown.


E . Immunoblot using anti - His antisera to detect dimer and oligomer formation by purified polyhistidine ( 6xHis ) - tagged WT or dominant - negative LukED toxins in the absence or presence of 2 - methyl - 2 , 4 - pentanediol ( MPD , 40 % v / v ) . Emut1 = LukEmut1 , Dmut = LukDmut . A representative of two independent experiments is shown .

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

E. Immunoblot using anti-His antisera to detect dimer and oligomer formation by purified polyhistidine (6xHis)-tagged WT or dominant-negative LukED toxins in the absence or presence of 2-methyl-2,4-pentanediol (MPD, 40% v/v). Emut1 = LukEmut1, Dmut = LukDmut. A representative of two independent experiments is shown.


A . Survival of Swiss - Webster mice following intravenous administration of purified recombinant toxins : LukED ( 10 μg per subunit ) , HlgAB ( 5 μg per subunit ) , HlgCB ( 35 μg per subunit ) and LukSF - PV / PVL ( 35 μg per subunit ) . Buffer : n = 2 mice ; LukED : n = 3 mice ; HlgAB : n = 3 mice ; HlgCB : n = 2 mice ; PVL : n = 3 . A representative of two independent experiments is shown .

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

A. Survival of Swiss-Webster mice following intravenous administration of purified recombinant toxins: LukED (10 μg per subunit), HlgAB (5 μg per subunit), HlgCB (35 μg per subunit) and LukSF-PV/PVL (35 μg per subunit). Buffer: n = 2 mice; LukED: n = 3 mice; HlgAB: n = 3 mice; HlgCB: n = 2 mice; PVL: n = 3. A representative of two independent experiments is shown.


B . Survival of Swiss - Webster mice following prophylactic intravenous administration of dominant - negative mutants , followed by intravenous challenge with a lethal dose of WT LukED . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . n = 6 mice per treatment . * * * p = 0 . 0005 for Buffer vs Emut1Dmut , Buffer vs Amut1Bmut , and Buffer vs Cmut1Bmut . p = 0 . 1050 ( not significant , ns ) for Buffer vs Smut1Fmut . Statistics performed using Log - rank ( Mantel - Cox ) test . A representative of two independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival of Swiss-Webster mice following prophylactic intravenous administration of dominant-negative mutants, followed by intravenous challenge with a lethal dose of WT LukED. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. n = 6 mice per treatment. ***p = 0.0005 for Buffer vs Emut1Dmut, Buffer vs Amut1Bmut, and Buffer vs Cmut1Bmut. p = 0.1050 (not significant, ns) for Buffer vs Smut1Fmut. Statistics performed using Log-rank (Mantel-Cox) test. A representative of two independent experiments is shown.


C . Survival of Swiss - Webster mice following prophylactic intravenous administration of dominant - negative mutants , followed by intravenous challenge with a lethal dose of WT HlgAB . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . n = 6 mice per treatment . * * * p = 0 . 0004 for Buffer vs Emut1Dmut and Buffer vs Amut1Bmut . * * p = 0 . 0015 for Buffer vs Cmut1Bmut . p = 0 . 7955 ( not significant , ns ) for Buffer vs Smut1Fmut . Statistics performed using Log - rank ( Mantel - Cox ) test . A representative of two independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival of Swiss-Webster mice following prophylactic intravenous administration of dominant-negative mutants, followed by intravenous challenge with a lethal dose of WT HlgAB. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. n = 6 mice per treatment. ***p = 0.0004 for Buffer vs Emut1Dmut and Buffer vs Amut1Bmut. **p = 0.0015 for Buffer vs Cmut1Bmut. p = 0.7955 (not significant, ns) for Buffer vs Smut1Fmut. Statistics performed using Log-rank (Mantel-Cox) test. A representative of two independent experiments is shown.


D . Bacterial burden 96 hours post infection in the liver of mice infected systemically with ~ 1 . 5 x 107 CFU of S . aureus strain Newman and intravenously treated with buffer or LukEmut1LukDmut ( Emut1Dmut , 10 μg per subunit ) at t = 0 , 24 , and 48 h . p . i . Bars indicate Mean ± SEM , with n = 20 mice ( Buffer ) and n = 19 mice ( Emut1Dmut2 ) combined from two independent experiments . * p = 0 . 0303 by unpaired , parametric , two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bacterial burden 96 hours post infection in the liver of mice infected systemically with ~1.5 x 107 CFU of S. aureus strain Newman and intravenously treated with buffer or LukEmut1LukDmut (Emut1Dmut, 10 μg per subunit) at t = 0, 24, and 48 h.p.i. Bars indicate Mean ± SEM, with n = 20 mice (Buffer) and n = 19 mice (Emut1Dmut2) combined from two independent experiments. *p = 0.0303 by unpaired, parametric, two-tailed Student's t-test.


B FACS analysis of p - S6 in WT mouse splenic CD8 + T cells stimulated for 8 min with α - mouse CD3ε ( 145 - 2C11 ) after pretreatment with DSF ( 5 µM ) for 2 h ( n = 3 ) . Data information : , data are representative of 3 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; ns , not significant [ unpaired t tests for measurements between the two groups in ( B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FACS analysis of p-S6 in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with DSF (5 µM) for 2 h (n = 3). Data information: , data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (B)


C Representative FACS of p - S6 in WT mouse splenic CD8 + T cells stimulated for 8 min with α - mouse CD3ε ( 145 - 2C11 ) after pretreatment with the indicated concentrations of DSF for 2 h ( n = 2 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; ns , not significant [ unpaired t tests for measurements between the two groups in ( C )

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

C Representative FACS of p-S6 in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with the indicated concentrations of DSF for 2 h (n = 2). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (C)


D Western blot analysis of the phosphorylation level of S6 and p70S6K in WT naïve CD8 + T cells stimulated by α - mouse CD3ε ( 145 - 2C11 ) ( 1 µgml - 1 ) in the presence of α - mouse CD28 ( 1 µgml - 1 ) for the indicated times after pretreatment with DSF ( 5 µM ) for 2 h . The immunoblot shows normalized expression of p - S6 ( S235 / 236 ) to S6 , p - p70S6K to p70S6K , p - Zap70 to Zap70 , as well as that of p - LCK ( Y394 ) and P - Lck ( Y505 ) to Lck . Data information : , data are representative of 3 independent experiments , , Image J analysis in ( D )

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

D Western blot analysis of the phosphorylation level of S6 and p70S6K in WT naïve CD8+ T cells stimulated by α-mouse CD3ε(145-2C11) (1 µgml-1) in the presence of α-mouse CD28 (1 µgml-1) for the indicated times after pretreatment with DSF (5 µM) for 2 h. The immunoblot shows normalized expression of p-S6(S235/236) to S6, p-p70S6K to p70S6K, p-Zap70 to Zap70, as well as that of p-LCK(Y394) and P-Lck(Y505) to Lck. Data information: , data are representative of 3 independent experiments, , Image J analysis in (D)


E FACS analysis of p - CD3ζ ( PY142 ) in mouse naïve CD8 + T cells treated with DSF or stimulated with α - mouse CD3ε ( 145 - 2C11 ) in the presence of α - mouse CD28 ( 1 µgml - 1 ) for 2 h ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; ns , not significant [ unpaired t tests for measurements between the two groups in ( E )

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

E FACS analysis of p-CD3ζ(PY142) in mouse naïve CD8+ T cells treated with DSF or stimulated with α-mouse CD3ε(145-2C11) in the presence of α-mouse CD28 (1 µgml-1) for 2 h (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (E)


F Western blot analysis of the indicated proteins in Jurkat T cells treated with the indicated concentrations of DSF for 2 h . The immunoblot analysis shows normalized expression of p - CD3ζ to CD3ζ as well as that of p - Zap70 ( Tyr319 ) / Syk ( Tyr352 ) , p - LCK ( Y394 ) , and p - LAT ( Tyr191 ) to β - actin . Data information : data are representative of 3 independent experiments , Image J analysis in ( F )

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

F Western blot analysis of the indicated proteins in Jurkat T cells treated with the indicated concentrations of DSF for 2 h. The immunoblot analysis shows normalized expression of p-CD3ζ to CD3ζ as well as that of p-Zap70(Tyr319)/Syk(Tyr352), p-LCK(Y394), and p-LAT(Tyr191) to β-actin. Data information: data are representative of 3 independent experiments, Image J analysis in (F)


G , H Luciferin assay of NFAT activity ( G ) and Nfatc1 mRNA expression ( H ) in J76 - NFATRE - luc and TCR052 cells stimulated with α - human CD3 ( OKT3 ) ( 1 µgml - 1 ) in the presence of α - human CD28 ( 1 µgml - 1 ) for 4 h or treated with DSF ( 5 µM ) for 2 h . The levels of NFAT activity were normalized with TCR052 cells treated with DMSO ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; two - way analysis of variance ( ANOVA ) in ( G , H ) ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H Luciferin assay of NFAT activity (G) and Nfatc1 mRNA expression (H) in J76-NFATRE-luc and TCR052 cells stimulated with α-human CD3 (OKT3) (1 µgml-1) in the presence of α-human CD28 (1 µgml-1) for 4 h or treated with DSF (5 µM) for 2 h. The levels of NFAT activity were normalized with TCR052 cells treated with DMSO (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; two-way analysis of variance (ANOVA) in (G, H)].


A , B MST analysis determined the Kd of DSF towards His - LCK ( WT ) ( 86 . 812 nm ) ( A ) or His - LCK ( C20 / 23S ) ( 22 . 797 µM ) ( B ) labeled with RED - tris - NTA 2nd Generation dye . Concentration is reported in nanomolar . Data information : , data are representative of 3 ( A , B , independent

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

A, B MST analysis determined the Kd of DSF towards His-LCK(WT) (86.812 nm) (A) or His-LCK(C20/23S) (22.797 µM) (B) labeled with RED-tris-NTA 2nd Generation dye. Concentration is reported in nanomolar. Data information: , data are representative of 3 (A, B, independent experiments


C , D MS / MS spectra of the Cys20 / 23 - containing human LCK peptide WMENIDVCENCHYPIVPLDGK ( aa 13 - 33 ; 2532 . 87 Da ) ( LC retention time was 49 . 16 min ; a triplet - charged precursor ion m / z 844 . 7163 ( mass , 2531 . 1284 Da ; ΔM of − 0 . 5 ppm ; 10logP = 200 ) was observed ) ( C ) , or of the corresponding LCK peptide after LCK incubation with DSF , which was modified on Cys23 ( red ) by the diethyldithiocarbamate moiety of DSF ( an increase of 147 . 0176 Da ; LC retention time was 56 . 31 min ; a triplet charged precursor ion m / z 893 . 7295 ( mass , 2678 . 1460 Da ; ΔM of 7 . 7 ppm ; − 10logP = 116 . 99 ) was observed ( D ) . Data information : , data are representative of 2 independent

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

C, D MS/MS spectra of the Cys20/23-containing human LCK peptide WMENIDVCENCHYPIVPLDGK (aa 13-33; 2532.87 Da) (LC retention time was 49.16 min; a triplet-charged precursor ion m/z 844.7163 (mass, 2531.1284 Da; ΔM of −0.5 ppm; 10logP = 200) was observed) (C), or of the corresponding LCK peptide after LCK incubation with DSF, which was modified on Cys23 (red) by the diethyldithiocarbamate moiety of DSF (an increase of 147.0176 Da; LC retention time was 56.31 min; a triplet charged precursor ion m/z 893.7295 (mass, 2678.1460 Da; ΔM of 7.7 ppm; −10logP = 116.99) was observed (D). Data information: , data are representative of 2 independent experiments


E Recombinant human LCK proteins left untreated ( Ctrl ) or incubated with ATP and increasing concentrations of DSF for 15 min . Enzyme - linked immunosorbent assay ( ELISA ) of LCK kinase activity was analyzed by using α - p - LCK ( Y394 ) antibody . The results show the average values of the optical density at 450 nm ( OD450 ) from five replicates ( n = 5 ) . Data information : data are representative of 2 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; ns , not significant [ one - way ANOVA in ( E )

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Recombinant human LCK proteins left untreated (Ctrl) or incubated with ATP and increasing concentrations of DSF for 15 min. Enzyme-linked immunosorbent assay (ELISA) of LCK kinase activity was analyzed by using α-p-LCK (Y394) antibody. The results show the average values of the optical density at 450 nm (OD450) from five replicates (n = 5). Data information: data are representative of 2 independent experiments, mean ± SD. **P<0. 01; ***P<0.001; ns, not significant [one-way ANOVA in (E)


F Confocal analysis of p - Lck ( Y394 ) ( red ) in naïve CD8 + T cells treated with DSF ( 5 µM ) or stimulated with α - mouse CD3 and α - mouse CD28 for 2 h ( left ) . The total fluorescence intensity ( IntDen ) was quantified ( right ; n = 20 cells ) . Scale bars , 3 µm . Data information : data are representative of F , independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; ns , not significant [ one - way ANOVA in ( F ) ] .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Confocal analysis of p-Lck(Y394) (red) in naïve CD8+ T cells treated with DSF (5 µM) or stimulated with α-mouse CD3 and α-mouse CD28 for 2 h (left). The total fluorescence intensity (IntDen) was quantified (right; n= 20 cells). Scale bars, 3 µm. Data information: data are representative of F, independent experiments, mean ± SD. **P<0. 01; ***P<0.001; ns, not significant [one-way ANOVA in (F)].


G Western blot analysis of the active LCK enrichment [ assessed by p - Lck ( Y394 ) ] in Triton X - 100 - insoluble fractions after DSF ( 5 µM ) treatment in naïve CD8 + T cells . Data information : data are representative of independent experiments ,

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

G Western blot analysis of the active LCK enrichment [assessed by p-Lck (Y394)] in Triton X-100-insoluble fractions after DSF (5 µM) treatment in naïve CD8+ T cells. Data information: data are representative of independent experiments,


H IVP assay of His - tagged CD3εCD peptides was phosphorylated by LCK in the presence of DSF . CD3ε phosphorylation was analyzed by immunoblotting using α - pTyr ( PY100 ) . Data information : , data are representative of 2 , H ) independent

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

H IVP assay of His-tagged CD3εCD peptides was phosphorylated by LCK in the presence of DSF. CD3ε phosphorylation was analyzed by immunoblotting using α-pTyr(PY100). Data information: , data are representative of 2 , H) independent experiments


I Western blot analysis of p - CD3ζ , LCK and p - Zap70 in LCK - deficient cells were infected with the lentivirus collected from human HEK293T cells transfected with control plasmid ( Control ) ( 1 µg ) , LCK ( WT ) ( 1 µg ) , or LCK ( C20 / 23S ) ( 1 µg ) with psPAX2 ( 1 µg ) and pMD2 . G ( 1 µg ) for 48 h followed by treatment with DSF ( 5 µM ) for 2 h stimulated with α - human CD3 and α - human CD28 for 5 min . Data information : data are representative of I ) independent

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

I Western blot analysis of p-CD3ζ, LCK and p-Zap70 in LCK-deficient cells were infected with the lentivirus collected from human HEK293T cells transfected with control plasmid (Control) (1 µg), LCK(WT) (1 µg), or LCK(C20/23S) (1 µg) with psPAX2 (1 µg) and pMD2.G (1 µg) for 48 h followed by treatment with DSF (5 µM) for 2 h stimulated with α-human CD3 and α-human CD28 for 5 min. Data information: data are representative of I) independent experiments


C Scatter plot comparing global gene - expression profiles of DMSO and DSF ( n = 3 ) . Data information : data are representative of 3 independent

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

C Scatter plot comparing global gene-expression profiles of DMSO and DSF (n = 3). Data information: data are representative of 3 independent experiments


D A heatmap of upregulated genes associated with TCR and T cell activation - related genes in DSF relative to that in DMSO . One column indicates one sample in each condition ( n = 3 ) . Data information : data are representative of 3 independent

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

D A heatmap of upregulated genes associated with TCR and T cell activation-related genes in DSF relative to that in DMSO. One column indicates one sample in each condition (n = 3). Data information: data are representative of 3 independent experiments


E ELISA analysis of the production of IL - 2 in primary OT - I T cell supernatants treated with DMSO , DSF ( 5 µM ) or OVA peptide ( 5 µgml - 1 ) for 4 h ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . ns , not significant [ unpaired t tests for the measurements between the two groups in ( E )

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

E ELISA analysis of the production of IL-2 in primary OT-I T cell supernatants treated with DMSO, DSF (5 µM) or OVA peptide (5 µgml-1) for 4 h (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [unpaired t tests for the measurements between the two groups in (E)


F CFSE dilution of proliferation of primary OT - I T cells treated with DMSO or DSF ( 5 µM ) for 2 h ( n = 3 ) . Data information : , data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . ns , not significant [ unpaired t tests for the measurements between the two groups in ( F )

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

F CFSE dilution of proliferation of primary OT-I T cells treated with DMSO or DSF (5 µM) for 2 h (n = 3). Data information: , data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [unpaired t tests for the measurements between the two groups in (F)


G FACS analysis of IFNγ and TNFα production in primary OT - I T cells primed by N4 ( SIINFEKL , 0 . 01 μgml - 1 ) and A4 ( SAINFEKL , 0 . 01 μgml - 1 ) for 2 h followed by treatment with DSF ( 5 µM ) for 2 h ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . two - way ANOVA in ( G )

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

G FACS analysis of IFNγ and TNFα production in primary OT-I T cells primed by N4 (SIINFEKL, 0.01 μgml-1) and A4 (SAINFEKL, 0.01 μgml-1) for 2 h followed by treatment with DSF (5 µM) for 2 h (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. two-way ANOVA in (G)


H , I Extracellular acidification rate ( ECAR ) ( H ) and oxygen consumption rate ( OCR ) ( I ) in primary OT - I T cells ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . ns , not significant [ unpaired t tests for the measurements between the two groups in ( H ) , two - way ANOVA in ( I ) ] .

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

H, I Extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I) in primary OT-I T cells (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [unpaired t tests for the measurements between the two groups in (H), two-way ANOVA in (I)].


A , Tumor growth [ ( A ) , ( Vehicle , n = 5 ; DSF , n = 4 , one tumor had disappeared ) ] ( n = 10 mice per group ) ] in DSF ( suspended in 0 . 5 % NaCMC ) - and Vehicle ( equal volume of 0 . 5 % NaCMC ) - treated mice after B16F10 melanoma inoculation . Data information : , data are representative of 3 independent experiments , ns , not significant two - way ANOVA in ( A )

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

A, Tumor growth [(A), (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared)] (n = 10 mice per group)] in DSF (suspended in 0.5% NaCMC)- and Vehicle (equal volume of 0.5% NaCMC)-treated mice after B16F10 melanoma inoculation. Data information: , data are representative of 3 independent experiments, ns, not significant two-way ANOVA in (A)


B Images and tumor weight of subcutaneous mouse B16F10 tumors extracted from mice at day 21 ( Vehicle , n = 5 ; DSF , n = 4 , one tumor had disappeared ) . Data information : , data are representative of 3 independent experiments , ns , not significant [ multiple t tests between the two groups in ( B )

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

B Images and tumor weight of subcutaneous mouse B16F10 tumors extracted from mice at day 21 (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: , data are representative of 3 independent experiments, ns, not significant [multiple t tests between the two groups in (B)


survival curves [ ( C ) , ( n = 10 mice per group ) ] in DSF ( suspended in 0 . 5 % NaCMC ) - and Vehicle ( equal volume of 0 . 5 % NaCMC ) - treated mice after B16F10 melanoma inoculation . Data information : data are representative of 3 independent experiments , mean ± SD . * ns , not significant [ , log - rank ( Mantel - Cox ) test in ( C )

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survival curves [(C), (n = 10 mice per group)] in DSF (suspended in 0.5% NaCMC)- and Vehicle (equal volume of 0.5% NaCMC)-treated mice after B16F10 melanoma inoculation. Data information: data are representative of 3 independent experiments, mean ± SD. * ns, not significant [ , log-rank (Mantel-Cox) test in (C)


D - Phenotypic analysis of tumor - infiltrating T cells at day 21 after melanoma inoculation ( Vehicle , n = 5 ; DSF , n = 4 , one tumor had disappeared ) . Data information : , data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . ns , not significant [ multiple t tests between the two groups in ( D )

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D - Phenotypic analysis of tumor-infiltrating T cells at day 21 after melanoma inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: , data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [multiple t tests between the two groups in (D)


immunofluorescence ( E ) analysis of tumor - infiltrating T cells at day 21 after melanoma inoculation ( Vehicle , n = 5 ; DSF , n = 4 , one tumor had disappeared ) . Scale bars , 20 µm . Statistics data are from 40 × field of view .

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immunofluorescence (E) analysis of tumor-infiltrating T cells at day 21 after melanoma inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Scale bars, 20 µm. Statistics data are from 40× field of view.


F Phenotypic analysis of tumor - infiltrating T cells at day 21 after melanoma inoculation ( Vehicle , n = 5 ; DSF , n = 4 , one tumor had disappeared ) . Data information : data are representative of 3 independent experiments , mean ± SD .

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F Phenotypic analysis of tumor-infiltrating T cells at day 21 after melanoma inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: data are representative of 3 independent experiments, mean ± SD.


G Phenotypic analysis of IFNγ and TNFα production in T cells isolated from draining lymph nodes at day 19 after melanoma inoculation ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . ns , not significant [ multiple t tests between the two groups in ( G )

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G Phenotypic analysis of IFNγ and TNFα production in T cells isolated from draining lymph nodes at day 19 after melanoma inoculation (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [multiple t tests between the two groups in (G)


H , I Tumor growth [ ( H ) , ( n = 5 mice per group ) ] and survival curves [ ( I ) , ( n = 5 mice per group ) ] in Vehicle - and DSF - treated mice injected s . c . with B16F10 - melanoma cells followed by intraperitoneal ( i . p . ) injection with PD - 1 antibody on days 0 and 4 and then every third day ( n = 5 ) . Data information : data are representative of 3 independent experiments , mean ± SD . ns , not significant two - way ANOVA in ( H ) , log - rank ( Mantel - Cox ) test in ( I ) ] .

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H, I Tumor growth [(H), (n = 5 mice per group)] and survival curves [(I), (n = 5 mice per group)] in Vehicle- and DSF-treated mice injected s.c. with B16F10-melanoma cells followed by intraperitoneal (i.p.) injection with PD-1 antibody on days 0 and 4 and then every third day (n = 5). Data information: data are representative of 3 independent experiments, mean ± SD. ns, not significant two-way ANOVA in (H), log-rank (Mantel-Cox) test in (I)].


A Tumor growth in Vehicle - and DSF - treated mice injected s . c . with B16F10 melanoma cells followed by intraperitoneal ( i . p . ) injection with CD8 ( 2A3 ) antibody on days − 1 , 0 , and 2 and then every second day ( n = 5 ) . Data information : data are representative of 3 independent experiments , mean ± SD . ns , not significant [ two - way ANOVA in ( A )

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

A Tumor growth in Vehicle- and DSF-treated mice injected s.c. with B16F10 melanoma cells followed by intraperitoneal (i.p.) injection with CD8(2A3) antibody on days −1, 0, and 2 and then every second day (n = 5). Data information: data are representative of 3 independent experiments, mean ± SD. ns, not significant [two-way ANOVA in (A)


B FACS analysis of cytotoxicity of B16F10 - OVA cells after coculture with DMSO - or DSF - treated primary OT - I T cells ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 . ns , not significant [ two - way

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

B FACS analysis of cytotoxicity of B16F10-OVA cells after coculture with DMSO- or DSF-treated primary OT-I T cells (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ****P<0.0001. ns, not significant [two-way ANOVA


C , D Tumor growth [ ( C ) , ( n = 5 mice per group ) ] and survival curves [ ( D ) , ( n = 10 mice per group ) ] of B16F10 - OVA tumor - bearing mice after adoptive transfer of Vehicle - or DSF - treated primary OT - I T cells . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 . ns , not significant [ two - way ANOVA in ( C ) , log - rank ( Mantel - Cox ) test in ( D ) , mean ± SD ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D Tumor growth [(C), (n = 5 mice per group)] and survival curves [(D), (n = 10 mice per group)] of B16F10-OVA tumor-bearing mice after adoptive transfer of Vehicle- or DSF-treated primary OT-I T cells. Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ****P<0.0001. ns, not significant [two-way ANOVA in (C), log-rank (Mantel-Cox) test in (D), mean ± SD].


A . NLK but not kinase - negative NLK ( Flag - NLK - KM ; K155M ) caused mobility shift of EGFP - YAP . EGFP - YAP was transfected with empty vector , Flag - NLK - WT , or Flag - NLK - KM into HEK293T cells . Mobility shift was shown by immunoblotting .

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A. NLK but not kinase-negative NLK (Flag-NLK-KM; K155M) caused mobility shift of EGFP-YAP. EGFP-YAP was transfected with empty vector, Flag-NLK-WT, or Flag-NLK-KM into HEK293T cells. Mobility shift was shown by immunoblotting.


B . NLK phosphorylates YAP in vitro . Flag - NLK - WT , or Flag - NLK - KM was immunoprecipitated with anti - Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST - YAP in the presence of [ 32P - ATP ] . Phosphorylation of GST - YAP and autophosphorylation of NLK were examined by autoradiography ( top panel ) , and Coomassie Blue staining showed similar levels of GST - YAP and Flag - NLK ( bottom panel ) .

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B. NLK phosphorylates YAP in vitro. Flag-NLK-WT, or Flag-NLK-KM was immunoprecipitated with anti-Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST-YAP in the presence of [32P-ATP]. Phosphorylation of GST-YAP and autophosphorylation of NLK were examined by autoradiography (top panel), and Coomassie Blue staining showed similar levels of GST-YAP and Flag-NLK (bottom panel).


C . NLK interacts with YAP at the endogenous level . Immunoprecipitates from HEK293T cell lysates with indicated antibody were blotted with anti - YAP or anti - NLK antibody .

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C. NLK interacts with YAP at the endogenous level. Immunoprecipitates from HEK293T cell lysates with indicated antibody were blotted with anti-YAP or anti-NLK antibody.


B . NLK - WT but not NLK - KM reduces Ser127 phosphorylation of endogenous YAP . Cell lysates from HEK293 cells transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in the figure ( left panel ) . The ratio of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± sem . * * P < 0 . 01 . Student t - test was used for statistical analysis .

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B. NLK-WT but not NLK-KM reduces Ser127 phosphorylation of endogenous YAP. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. **P < 0.01. Student t-test was used for statistical analysis.


C . Deficiency of NLK leads to increased YAP phosphorylation at Ser127 . Lysates from wild type and two independent NLK knockout HEK293 cells were immunoblotted with antibodies indicated in the figure ( left panel ) . The ratio of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± sem . * P < 0 . 05 and * * P < 0 . 01 . Student t - test was used for statistical analysis .

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C. Deficiency of NLK leads to increased YAP phosphorylation at Ser127. Lysates from wild type and two independent NLK knockout HEK293 cells were immunoblotted with antibodies indicated in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. *P < 0.05 and **P < 0.01. Student t-test was used for statistical analysis.


D . Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128 . Cell lysates from HEK293 cells transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in the figure .

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D. Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure.


E . Substitution of Ser127 with alanine enhances phosphorylation at Ser128 , and vice versa . Cell lysates from HEK293T cells transfected with empty vector ( - ) , EGFP - YAP , EGFP - YAP - S127A , or EGFP - YAP - S128A were immunoblotted with antibodies shown in the figure .

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E. Substitution of Ser127 with alanine enhances phosphorylation at Ser128, and vice versa. Cell lysates from HEK293T cells transfected with empty vector (-), EGFP-YAP, EGFP-YAP-S127A, or EGFP-YAP-S128A were immunoblotted with antibodies shown in the figure.


A . Ectopic expression of NLK attenuates interaction between YAP and 14 - 3 - 3 . Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti - HA antibody and immunoblotted with anti - GFP antibody ( top two panels ) . Whole cell lysates ( WCL ) were immunoblotted with antibodies shown in the figure ( bottom three panels ) .

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A. Ectopic expression of NLK attenuates interaction between YAP and 14-3-3. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-HA antibody and immunoblotted with anti-GFP antibody (top two panels). Whole cell lysates (WCL) were immunoblotted with antibodies shown in the figure (bottom three panels).


B . Deficiency of NLK leads to increased interaction between YAP and 14 - 3 - 3 . Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti - YAP antibody and immunoblotted with anti - 14 - 3 - 3 antibody ( top two panels ) . WCL were immunoblotted with antibodies indicated in the figure ( bottom three panels ) .

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B. Deficiency of NLK leads to increased interaction between YAP and 14-3-3. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-YAP antibody and immunoblotted with anti-14-3-3 antibody (top two panels). WCL were immunoblotted with antibodies indicated in the figure (bottom three panels).


C . Phosphorylation status of Ser127 or Ser128 determines interaction between 14 - 3 - 3 and YAP . Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti - Flag antibody and immunoblotted with anti - HA antibodies ( top two panels ) . Lysates were immunoblotted with anti - HA or anti - Flag antibodies ( bottom two panels ) . " n . s . " arrow indicates a non - specific band also visible in the non - transfected lane .

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C. Phosphorylation status of Ser127 or Ser128 determines interaction between 14-3-3 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibodies (top two panels). Lysates were immunoblotted with anti-HA or anti-Flag antibodies (bottom two panels). "n.s." arrow indicates a non-specific band also visible in the non-transfected lane.


D . Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP . Immunofluorescence analysis was performed in HeLa cells transfected with EGFP - YAP , EGFP - YAP - S127A , EGFP - YAP - S128A , or EGFP - YAP - S128D at high cell density . Figure shows a representative of multiple areas . Nuclei were stained with Hoechst 33342 . Scale bars : 20 μm .

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D. Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP. Immunofluorescence analysis was performed in HeLa cells transfected with EGFP-YAP, EGFP-YAP-S127A, EGFP-YAP-S128A, or EGFP-YAP-S128D at high cell density. Figure shows a representative of multiple areas. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm.


E . NLK knockout leads to increased cytoplasmic localization of endogenous YAP . Immunoblot analysis shows significant reduction of NLK level in NLK knockout HEK293 cell pools ( top panel ) .

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E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. Immunoblot analysis shows significant reduction of NLK level in NLK knockout HEK293 cell pools (top panel).


E . NLK knockout leads to increased cytoplasmic localization of endogenous YAP . To determine subcellular localization of YAP indirect immunofluorescence analysis was performed in NLK knockout HEK293 cell pools ( bottom panel ) . Figure shows a representative of multiple areas . Nuclei were stained with DAPI . Scale bars : 20 μm .

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E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. To determine subcellular localization of YAP indirect immunofluorescence analysis was performed in NLK knockout HEK293 cell pools (bottom panel). Figure shows a representative of multiple areas. Nuclei were stained with DAPI. Scale bars: 20 μm.


A . Localization of endogenous YAP and NLK in low ( LD ) or high density ( HD ) MCF10A ( upper panel ) or NIH3T3 cells ( bottom panel ) was examined by indirect immunofluorescence . Nuclei were stained with DAPI . Scale bars : 20 μm .

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A. Localization of endogenous YAP and NLK in low (LD) or high density (HD) MCF10A (upper panel) or NIH3T3 cells (bottom panel) was examined by indirect immunofluorescence. Nuclei were stained with DAPI. Scale bars: 20 μm.


B . Level of NLK decreases in a cell density - dependent manner . Lysates from low ( LD ) and high density ( HD ) of MCF10A , HEK293T , and NIH3T3 cells were immunoblotted with antibodies indicated in the figure .

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B. Level of NLK decreases in a cell density-dependent manner. Lysates from low (LD) and high density (HD) of MCF10A, HEK293T, and NIH3T3 cells were immunoblotted with antibodies indicated in the figure.


C . Level of endogenous NLK mRNA is unaffected , whereas the expression of YAP target genes was reduced , at high cell density . Quantitative real - time PCR analyses for expression of AMOTL2 , CTGF , CYR61 , and NLK in low ( LD ) and high density ( HD ) NIH3T3 cells were performed . Quantification of AMOTL2 , CTGF , CYR61 , and NLK mRNA was normalized with the level of Gapdh . Data represent average values from a representative of multiple experiments performed in triplicate . Error bars indicate standard deviations of triplicate measurements . Data are presented as mean ± sem . * P < 0 . 05 and * * * P < 0 . 005 . Student t - test was used for statistical analysis .

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C. Level of endogenous NLK mRNA is unaffected, whereas the expression of YAP target genes was reduced, at high cell density. Quantitative real-time PCR analyses for expression of AMOTL2, CTGF, CYR61, and NLK in low (LD) and high density (HD) NIH3T3 cells were performed. Quantification of AMOTL2, CTGF, CYR61, and NLK mRNA was normalized with the level of Gapdh. Data represent average values from a representative of multiple experiments performed in triplicate. Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05 and ***P < 0.005. Student t-test was used for statistical analysis.


A . Knockout of NLK reduces the interaction between endogenous YAP and TEAD . Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti - TEAD1 antibody and immunoblotted with anti - TEAD1 antibody or anti - YAP antibody ( top panel ) . Whole cell lysates ( WCL ) were immunoblotted with antibodies indicated in the figure .

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A. Knockout of NLK reduces the interaction between endogenous YAP and TEAD. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-TEAD1 antibody and immunoblotted with anti-TEAD1 antibody or anti-YAP antibody (top panel). Whole cell lysates (WCL) were immunoblotted with antibodies indicated in the figure.


B . Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 and YAP . Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti - Flag antibody and immunoblotted with indicated antibodies in the figure .

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B. Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies in the figure.