( B ) Box plots show frequencies ( % ) of each subset defined by cluster in samples of non - inflamed controls , UC or CD after stimulation with PMA / ionomycin ± BTP2 for 4h ex vivo . Statistical significances were calculated by one - paired Wilcoxon matched - pairs signed - rank , * p < 0 . 05 . Boxes range from 25th to 75th percentiles . Whisker plots show min ( smallest ) max ( largest ) values . in box denotes median obtained from 1 experiment ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) .

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(B) Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4h ex vivo. Statistical significances were calculated by one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).


( C ) viSNE plots of one exemplary CD . Identification of subsets ( left ) expression of markers after PMA / ionomycin stimulation in presence or absence of BTP2 ( blue ; low , red : high ) .

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(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cell markers after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).


( D ) Heatmaps indicating median fold change expression of cytokines surface markers in CD45 + CD3 - LPMCs activated with PMA / ionomycin ± BTP2 for 4h in vitro . Values are normalized to stimulated , non - BTP2 treated samples .

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(D) Heatmaps indicating the median fold change expression of cytokines and surface markers in CD45+CD3- LPMCs activated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.


( A - B ) Adult - derived intestinal epithelial spheroids of wild - type C57BL / 6 mice were cultured for 4 days in Wnt - free medium to induce differentiation . Initial viability was determined on day 0 . DMSO control ( vehicle ) or 1 μM BTP2 were added to culture medium viability was measured every 24h by resazurin viability microscopy . Scale bar depicts 100 µm . ( C ) Transepithelial electrical resistance ( TEER ) was measured on differentiated murine colon monolayers in presence or absence of 1 µM BTP2 .

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(A-B) Adult stem cell-derived intestinal epithelial spheroids of wild-type C57BL/6 mice were cultured for 4 days in Wnt-free medium to induce differentiation. Initial viability was determined on day 0. DMSO control (vehicle) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy. Scale bar depicts 100 µm. (C) Transepithelial electrical resistance (TEER) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2.


( D ) Human colonic spheroids were generated from human epithelial crypts obtained from colon resectates of 2 CD cultured for 6 days in Wnt - free medium to induce differentiation into colonic organoids . Initial viability was determined on day 0 . DMSO control or 1 μM BTP2 were added to culture medium viability was measured on days 2 , 4 6 .

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(D) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt-free medium to induce differentiation into colonic organoids. Initial viability was determined on day 0. DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2, 4 and 6.


( E ) TEER of differentiated human colon monolayers was analyzed in presence or absence of 1 µM BTP2 .

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(E) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2.


( F ) Relative mRNA expression of markers for enterocytes ( ALPI ) , goblet ( MUC2 ) , entero - endocrine ( CHGA ) was quantified by RT - PCR in differentiated human colon monolayers after addition of 1 µM BTP2 .

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(F) Relative mRNA expression of markers for enterocytes (ALPI), goblet cells (MUC2), and entero-endocrine cells (CHGA) was quantified by RT-PCR in differentiated human colon monolayers after addition of 1 µM BTP2.


( A - C ) Acute administration of CRAC channel blocker CM4620 inhibits CRAC currents ( Icrac ) . ( A ) Time course of peak amplitudes of ICRAC before after application of 3 μM CM4620 to HEK293 transfected with ORAI1 STIM1 . ( B ) Current - voltage ( I - V ) relationship of ICRAC before after CM4620 treatment . I - Vs were obtained at time points indicated by arrowheads in ( A ) . ( C ) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before after CM4620 administration ( arrowheads ) using formula 1 - ( ICM4620 / ICtrl ) . Data are from 8 per conditions shown as mean ± .

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(A-C) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents (Icrac). (A) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1. (B) Current-voltage (I-V) relationship of ICRAC before and after CM4620 treatment. I-Vs were obtained at the time points indicated by the arrowheads in (A). (C) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration (arrowheads) using the formula 1- (ICM4620 / ICtrl). Data are from 8 cells per conditions and shown as the mean ± SEM.


( E ) Relative loss of mice treated with vehicle or CM4620 ; start of treatment is indicated by arrow . Data are mean ± of 9 mice per cohort , statistical by unpaired student ' s t - , * p < 0 . 05 .

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(E) Relative weight loss of mice treated with vehicle or CM4620; the start of treatment is indicated by the white arrow. Data are the mean ± SEM of 9 mice per cohort, statistical analysis by unpaired student's t-test , *p<0.05.


( F ) Concentrations of CM4620 in serum of mice at day 49 , data are mean ± . Statistical by unpaired student ' s t - , * * * p < 0 . 001 .

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(F) Concentrations of CM4620 in the serum of mice at day 49, data are the mean ± SEM. Statistical analysis by unpaired student's t-test, ***p<0.001.


( G ) Representative H & E of distal colon of Rag1 - / - mice treated with vehicle or CM4620 . Colitis scores of 9 mice per cohort . Each symbol represents one mouse .

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(G) Representative H&E staining of the distal colon of Rag1-/- mice treated with vehicle or CM4620. Colitis scores of 9 mice per cohort. Each symbol represents one mouse.


( H ) Number of CD11b + Gr - 1 + neutrophils CD4 + T ( normalized to grams of tissue ) in colon lamina propria ( CLP ) of mice on day 49 quantified by flow cytometry . Each dot represents one mouse . Data are mean ± of 9 mice per cohort .

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(H) Number of CD11b+Gr-1+ neutrophils and CD4+ T cells (normalized to grams of tissue) in the colon lamina propria (CLP) of mice on day 49 quantified by flow cytometry. Each dot represents one mouse. Data are the mean ± SEM of 9 mice per cohort.


( I ) Frequencies of RORγt + Th17 Foxp3 + Treg in CLP of mice treated with CM4620 or vehicle . Each dot represents one mouse . Data are mean ± of 6 control 5 CM4620 treated mice .

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(I) Frequencies of RORγt+ Th17 cells and Foxp3+ Treg cells in the CLP of mice treated with CM4620 or vehicle. Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.


( J ) Frequencies of IFN - γ , TNF - α IL - 2 producing CD4 + T isolated from CLP , restimulated ex vivo with PMA ionomycin for 4 h ( without addition of CM4620 during stimulation in vitro ) analyzed by flow cytometry . Bar graphs represent percentages of IFN - γ + , TNF - α + , IL - 2 + in CD4 + ( top row ) mean ( MFI ) of IFN - γ , TNF - α , IL - 2 on CD4 + ( bottom row ) . Each dot represents one mouse . Data are mean ± of 6 control 5 CM4620 treated mice .

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(J) Frequencies of IFN-γ, TNF-α and IL-2 producing CD4+ T cells isolated from the CLP, restimulated ex vivo with PMA and ionomycin for 4 h (without addition of CM4620 during stimulation in vitro) and analyzed by flow cytometry. Bar graphs represent the percentages of IFN-γ+, TNF-α+, IL-2+ cells in CD4+ cells (top row) and mean fluorescent intensity (MFI) of IFN-γ, TNF-α, IL-2 on CD4+ cells (bottom row). Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.


A Scheme of lentiviral vector with inducible FlagOmomyc ( top ) immunoblotting ( bottom ) of FlagOmomyc 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 BT168GFPcells upon Dox treatment for 0 - 96 h ( n = 3 ; mean ± SD ) . Viable were counted using a haemocytometer .

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 upon Dox treatment . C : histograms showing of capable of re - forming a neurosphere seven days after dissociation ( n = 3 ; mean ± SD ) . D : representative of BT168FO 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 , differentiation markers ( CCND1 , PTEN , SOX2 , NOTCH1 , NESTIN , MYC ) in BT168FO after 48 h Dox treatment , compared to uninduced ( n = 3 ; mean ± SD ) . Expression in non - induced 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 of BT168FO after 3 days with or without Dox ( n = 3 ; mean ± SD ) . 10 fields were counted per .

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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 presence of serum treated with doxycycline for up to 7 days . upper panel displays immunofluorescence of NESTIN , GFAP , ß - tubulin III , SOX2 , FlagOmomyc expression . FlagOmomyc blunted SOX2 expression decreased GFAP NESTIN , while inducing onset of ß - tubulin III . lower panel shows of positive for each marker evaluated ( n = 4 ; mean ± SD ) . 16 fields for each 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 ) Omomyc expressing BT168FO . A - D ) Flag , Ki67 , CD31 , DCX ( doublecortin ) immunostaining of adjacent sections in control Omomycxenografts . E - G ) Differentiation phenotype investigated by GFAP , ß - tubulin III , OLIG2 . H ) Immunostaining for anti - human nuclei . Representative are shown . Scale bar = 100μm .

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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 Omomyc around TSSs of MYC promoter - target genes in BT168FO ( A ) , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated . Each row shows ± 5 kb region centred on TSSs . Colour scaled intensities are in units of tags per 50bp . plots adjacent to each depict MYC Omomyc binding patterns at genes in two gene clusters denoted by arrows , in 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 Omomyc around TSSs of MYC promoter - target genes in U87FO ( B ) , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated . Each row shows ± 5 kb region centred on TSSs . Colour scaled intensities are in units of tags per 50bp . plots adjacent to each depict MYC Omomyc binding patterns at genes in two gene clusters denoted by arrows , in 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 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 of Pol II around TSSs of MYC promoter - target genes in BT168FO , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated . Each row shows ± 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 , DUSP10 genes in BT168FO treated or not with Dox for 24 h . y - axis shows Pol II binding signals as tags / 500bp / million reads . x - axis displays genomic positions ; introns as lines , exons as boxes . Arrowheads denote direction of transcription . Blue green bars denote 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 ( FPKM from - seq data ) in BT168FO of 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 between fold enrichments ( specific antibody / IgG signal ) derived from qChIPs of Pol II - Ser2p 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 Polymerase II traveling distribution from Pol II ChIP - seqs , in presence or absence of Dox .

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 in untreated with doxycycline ( black ) . In doxycycline treated promoter occupancy by MYC is lower , no longer increases together with transcript . scatter plot displays MYCChIP - seq reads at promoters ( - 1000 , + 100 regions with respect to TSS ) in BT168FO - untreated ( black ) or upon 48 h Dox treatment ( blue ) - versus transcript ( FPKM from - 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 , plotted against expression in untreated ( 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 table first row shows number of genes significantly up or down regulated at 24 h of Dox treatment , subdivided in promoter - , intragenic - , intergenic - targets ( ± 50 kbp from TSS ) , . following rows depict - for each gene class - mean ( average ) values of FPKM in treated or not with Dox for 24 h corresponding Δ FPKM 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 treated with Dox for 24 h vs . untreated . Transcript , expressed as FPKM , represent averages of three independent experiments .

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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 - seq expression values of select genes in BT168FO GSCs along a 48 h time course of Omomyc induction ( n = 3 independent biological replicates ) . block on left represents DUSP ( dual specificity phosphatase ) family genes . middle block contains transcription factors ( TFs ) : upper thirteen ( ASCL1 - SALL1 ) are GSC - specific remaining ones are oncogenic . right block contains genes , including well - known MYC targets , with functions in proliferation , neurodifferentiation , gliomagenesis . first column of each block represents average expression ( log2 FPKM ) in untreated ( 0 h ) in colour scale illustrated by lower bar : violet indicates low blue high expression . other columns depict relative expression versus untreated ( average log2 FC ) at different times ( 8 - 48h ) of Dox treatment , according to scale shown by upper bar : red indicating low green high expression . table in upper left summarizes GSEA score of 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 ) of - seq data from BT168FO . Left : genes downregulated by Omomyc in BT168FO identify set of genes targeted by GSC TFs [ 40 ] . Middle : Omomyc downregulated genes identify a gene set upregulated by MYC in cancer [ 66 ] . Right : genes upregulated by Omomyc identify genes downregulated in small 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 GAPDH loading control expression in BT168FO BT275FO after 2 days with ( + ) or without ( - ) Dox . Representative 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 of selected miRs in U87FO grown 48 h in presence ( + ) or absence ( - ) of Dox ( n = 3 ; mean ± SD ) . Expression were normalized using U6RNA as control . Expression in non - induced 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 , GAPDHimmunoblots in same as in panel C . Representative 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 , GAPDHimmunoblots in uninduced U87FO ( - ) in transfected with LNA oligos targeting miR - 200a - 200b - 429 ( LNA 200 ) miR - 146b ( LNA 146b ) , or with a non - targeting LNA ( C ) , induced with Dox for 48 h ( + Dox ) . Representative 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 proliferation of uninduced or Dox induced U87FO , transfected with either a non - targeting LNA ( LNA C ) , or LNA oligos targeting miR - 200a - 200b - 429 cluster ( LNA 200 ) , miR - 146b ( LNA 146b ) , or both ( LNAmix ) analysed at 24 , 48 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 of U87FO transfected with non - targeting control LNA oligos ( LNA C ) or with LNA oligos targeting miR - 200a - 200b - 429 ( LNA 200 ) ; were either untreated ( - ) or treated with Dox for 24 h ( + Dox ) ; ( n = 3 ; mean ± SD ) .

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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).


A . Relative antiviral activity of IFNα ( circles ) or IFNλ ( triangles ) . AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations ( ng / ml ) induction of indicated ISGs was assessed by qPCR ( data shown is representative of four independent experiments , n = 3 - 4 ) .

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A. Relative antiviral activity of IFNα (circles) or IFNλ (triangles). AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations (ng/ml) and induction of indicated ISGs was assessed by qPCR (data shown is representative of four independent experiments, n=3-4).


B , C . Mice were pretreated with equivalent doses of IFNα ( 1 . 45μg / 50μl ) or IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( squares , 50μl PBS ) 24hrs prior to infection with PR8 ; loss survival was assessed throughout infection ( B ) , viral load ( C ) assessed at 4dpi ( data shown is representative of two independent experiments , n = 8 - 10 ( B ) , n = 3 ( C ) ) .

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B, C. Mice were pretreated with equivalent doses of IFNα (1.45μg/50μl) or IFNλ (2.6μg/50μl) or Veh Ctrl (squares, 50μl PBS) 24hrs prior to infection with PR8; weight loss and survival was assessed throughout infection (B), and viral load (C) assessed at 4dpi (data shown is representative of two independent experiments, n=8-10 (B), n=3 (C)).


D , E . Mice were infected with PR8 treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 5 post infection ; survival loss was monitored ( D , data pooled from 4 independent experiments , n = 12 - 29 ) ( data representative of two independent experiments , n = 3 - 5 ) .

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D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; survival and weight loss was monitored (D, data pooled from 4 independent experiments, n=12-29) (data representative of two independent experiments, n=3-5).


D , E . Mice were infected with PR8 treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 5 post infection ; viral load assessed at 4dpi ( E ) ( data representative of two independent experiments , n = 3 - 5 ) .

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D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; viral load assessed at 4dpi (E) (data representative of two independent experiments, n=3-5).


A , B . Mice were infected with PR8 treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine ( A ) ( data shown is representative of two independent experiments , n = 2 - 6 ) .

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A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay (A) (data shown is representative of two independent experiments, n=2-6).


A , B . Mice were infected with PR8 treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry of pDCs Inflammatory Monocytes in lung was performed ( B ) ( data shown is representative of two independent experiments , n = 2 - 6 ) .

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A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed (B) (data shown is representative of two independent experiments, n=2-6).


C , D . Lung sections from control infected mice treated as indicated , were stained by TUNEL for apoptotic at 6dpi . of TUNEL + in whole lung slides by Icy Spot Detector ( ICY - R3M2Y2 ) ( C ) ( data shown is pooled from three independent experiments , n = 3 - 8 ) . Red arrowheads indicate TUNEL signal ( D ) . Scale bar , 200 μM ( data shown is representative of two independent experiments , n = 3 - 4 ) .

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C, D. Lung sections from control and infected mice treated as indicated, were stained by TUNEL for apoptotic cells at 6dpi. Quantification of TUNEL+ cells in whole lung slides by Icy Spot Detector (ICY-R3M2Y2) (C) (data shown is pooled from three independent experiments, n=3-8). Red arrowheads indicate TUNEL signal (D). Scale bar, 200 μM (data shown is representative of two independent experiments, n=3-4).


A , B . IL - 6 , IP - 10 MCP - 1 concentrations were measured by multiplex cytokine in AEC culture supernatants ( A ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) or LPS ( AEC only ) ( data shown is representative of two independent experiments , n = 3 - 6 ) .

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A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants (A) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) or LPS (AEC only) (data shown is representative of two independent experiments, n=3-6).


A , B . IL - 6 , IP - 10 MCP - 1 concentrations were measured by multiplex cytokine in Macrophage , pDC cDC culture supernatants ( B ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) ( data shown is representative of two independent experiments , n = 3 - 6 ) .

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A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in Macrophage, pDC and cDC culture supernatants (B) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) (data shown is representative of two independent experiments, n=3-6).


C . BAL samples taken from mice treated with IFNα , IFNλ or Veh Ctrl at specified time points ( data shown is representative of two independent experiments , n = 5 - 6 ) .

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C. BAL samples taken from mice treated with IFNα, IFNλ or Veh Ctrl at specified time points (data shown is representative of two independent experiments, n=5-6).


Mice were treated with IFNα ( 1 . 45μg / 50μl ) , IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( 50μl PBS ) , whole lungs were taken at 18hrs post treatment for global by Illumina . SingleColor . Mouse WG - 6 _ V2 _ 0 _ R0 _ 1127 microarrays . Samples ( n = 5 ) were normalized to median of vehicle control group filtered for a fold change of 1 . 5 , yielding 553 genes differently regulated between treatments ( One way ANOVA , P < 0 . 01 , Benjamini - Hochberg multiple correction ) , of which 429 genes are upregulated . K means clustering revealed six gene clusters , one of which encompassed genes primarily induced by IFNα4 ( A ) , while remaining clusters contained genes upregulated by both IFNα4 IFNλ2 ( B ) . two clusters of genes were analysed by Ingenuity Pathway ( IPA ) ( C , D ) .

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Mice were treated with IFNα (1.45μg/50μl), IFNλ (2.6μg/50μl) or Veh Ctrl (50μl PBS), and whole lungs were taken at 18hrs post treatment for global analysis by Illumina.SingleColor.Mouse WG-6_V2_0_R0_1127 microarrays. Samples (n=5) were normalized to the median of the vehicle control group and filtered for a fold change of 1.5, yielding 553 genes differently regulated between treatments (One way ANOVA, P<0.01, Benjamini-Hochberg multiple test correction), of which 429 genes are upregulated. K means clustering revealed six gene clusters, one of which encompassed genes primarily induced by IFNα4 (A), while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 (B). The two clusters of genes were analysed by Ingenuity Pathway Analysis (IPA) (C,D).


A . HumanAEC cultures were stimulated for 4hrs with IFNα ( circles ) or IFNλ ( triangles ) at specified concentrations then assessed for stated ISG induction by qPCR ( data is representative of 2 independent experiments , n = 3 ) .

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A. HumanAEC cultures were stimulated for 4hrs with IFNα (circles) or IFNλ (triangles) at specified concentrations then assessed for stated ISG induction by qPCR (data is representative of 2 independent experiments, n=3).


B , C . ISG induction in humanPBMCs was assessed at 4 24hrs post IFNα ( 21 U / ml ) or IFNλ ( 1 . 2 ng / ml ) stimulation ( B ) ( data shown is pooled from 6 independent donors ) .

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B, C. ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα (21 U/ml) or IFNλ (1.2 ng/ml) stimulation (B) (data shown is pooled from 6 independent donors).


B , C . PBMC proinflammatory cytokine secretion was measured by multiplex cytokine at 4 24hrs post stimulation with IFNα or IFNλ ( C ) ( data shown is pooled from 6 independent donors ) .

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B, C. PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ (C) (data shown is pooled from 6 independent donors).


( A ) injured DRG axons showing growth cone regeneration ( upper panels ) regenerative failure ( lower panels ) . Arrows as indicated .

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(A) Laser injured DRG axons showing growth cone regeneration (upper panels) and regenerative failure (lower panels). Arrows as indicated.


( B ) Upper graph shows of regenerating axons of DIV 1 - 2 adult DRG neurons in presence of p110 inhibitors , 2 h after injury . Lower graph shows time taken to regenerate a new growth cone . Inhibitors were added 1hr before axons were injured , maintained throughout : LY294002 , 20 μM . A66 5 μM . XL - 147 5 μM . TGX221 500 nM . IC - 87114 10μM . Idelalisib 500 nM . Upper graph data are shown as mean + / - . P values indicate statistical significance analysed by Fishers exact ( upper graph ) or Kruskal - Wallis ( lower graph ) .

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(B) Upper graph shows percentage of regenerating axons of DIV 1-2 adult DRG neurons in the presence of p110 inhibitors, 2 h after injury. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: LY294002, 20 μM. A66 5 μM. XL-147 5 μM. TGX221 500 nM. IC-87114 10μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance analysed by Fishers exact (upper graph) or Kruskal-Wallis test (lower graph).


( C ) Examples of static vs . growing uninjured axons treated with p110α or δ inhibitors as indicated . Both arrows in bottom right image indicate growing axons .

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(C) Examples of static vs. growing uninjured axons treated with p110α or δ inhibitors as indicated. Both arrows in bottom right image indicate growing axons.


( D ) of uninjured , growing axons in presence of p110 inhibitors . Inhibitors were added as for ( B ) . Lower graph shows grown in 2 h . Data are shown as mean + / - . P values indicate significance measured by Fishers exact ( upper graph ) or ANOVA with Tukey ' s post - hoc ( lower graph ) .

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(D) Percentage of uninjured, growing axons in the presence of p110 inhibitors. Inhibitors were added as for (B). Lower graph shows length grown in 2 h. Data are shown as the mean +/- SEM. P values indicate significance measured by Fishers exact test (upper graph) or ANOVA with Tukey's post-hoc analysis (lower graph).


( E ) Adult DRG neurons in microfluidic compartmental chambers . bodies on left side , axons extending through microchannels on right . Lower panels are schematics .

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(E) Adult DRG neurons in microfluidic compartmental chambers. Cell bodies on the left side, axons extending through microchannels on the right. Lower panels are schematics.


( F ) Upper graph shows of regenerating axons in microfluidic chambers . p110α or δ inhibitors were applied to either soma or axons . Lower graph shows time taken to regenerate a new growth cone . Inhibitors were added 1hr before axons were injured , maintained throughout : A66 5 μM . Idelalisib 500 nM . Upper graph data are shown as mean + / - . P values indicate statistical significance as measured by Fishers exact ( upper graph ) or by Kruskal - Wallis ( lower graph ) .

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(F) Upper graph shows percentage of regenerating axons in microfluidic chambers. p110α or δ inhibitors were applied to either soma or axons. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: A66 5 μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance as measured by Fishers exact test (upper graph) or by Kruskal-Wallis test (lower graph).


( A ) DIV 3 cortical neurons immunolabelled for PIP3 . Spectrum shows . ( B ) DIV 8 cortical neuron transfected with GFP immunolabelled for PIP3 . Spectrum shows . ( C ) DIV 16 cortical neuron transfected GFP immunolabelled for PIP3 . Spectrum shows . ( D ) Somatic PIP3 in soma at increasing days in vitro . Data are shown as mean + / - . P values are significance measured by ANOVA with Tukey ' s post - hoc . N = 3 experiments , 60 neurons . ( E ) Growth cone PIP3 at increasing days in vitro . Data are shown as mean + / - . P values indicate significance measured by ANOVA with Tukey ' s post - hoc . N = 3 experiments , 60 growth cones .

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(A) DIV 3 cortical neurons immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (B) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (C) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (D) Somatic PIP3 quantification in the soma at increasing days in vitro. Data are shown as the mean +/- SEM. P values are significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (E) Growth cone PIP3 quantification at increasing days in vitro. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.


( A ) PIP3 immunofluorescence in soma of DIV 16 cortical neurons expressing p110 isoforms GFP . Spectrum shows . ( B ) PIP3 immunofluorescence at axonal growth cone of DIV 16 neurons expressing p110 isoforms GFP . Spectrum shows . ( C ) of PIP3 immunofluorescence in soma . Data are shown as mean + / - . P values indicate significance as measured by ANOVA with Tukey ' s post - hoc . N = 3 experiments , 60 neurons . ( D ) Graph showing PIP3 of immunofluorescence at axon growth cone . Data are shown as mean + / - . P values indicate significance measured by ANOVA with Tukey ' s post - hoc . N = 3 experiments , 60 growth cones .

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(A) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (B) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (C) Quantification of PIP3 immunofluorescence in the soma. Data are shown as the mean +/- SEM. P values indicate significance as measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (D) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.


( A ) DIV 4 cortical neurons expressing p110 isoforms GFP . Arrow marks axon tip . ( B ) of axon . n = 3 experiments , 45 neurons per group . ( C ) of dendrite . n = 3 experiments , 45 neurons per group . ( D ) of axon : dendrite . n = 3 experiments , 45 neurons per group .

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(A) DIV 4 cortical neurons expressing p110 isoforms and GFP. Arrow marks the axon tip. (B) Quantification of axon length. n = 3 experiments, 45 neurons per group. (C) Quantification of dendrite length. n = 3 experiments, 45 neurons per group. (D) Quantification of the axon:dendrite length ratio. n = 3 experiments, 45 neurons per group.


( E ) DIV 14 cortical neurons expressing p110 isoforms GFP , immunolabelled for pS6 . ( F ) Sholl of branches . n = 60 neurons per group . Data are shown as mean + / - . P values indicate significance measured by repeated measure ANOVA with Bonferonni ' s post - hoc . ( G ) of total neurite . n = 3 experiments , 60 neurons per group . ( H ) of soma . n = 3 experiments , 60 neurons per group . ( I ) of pS6 immunofluorescence . n = 3 experiments , 60 neurons per group .

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(E) DIV 14 cortical neurons expressing p110 isoforms and GFP, immunolabelled for pS6. (F) Sholl analysis of branches. n = 60 neurons per group. Data are shown as the mean +/- SEM. P values indicate significance measured by repeated measure ANOVA with Bonferonni's post-hoc test. (G) Quantification of the total neurite length. n = 3 experiments, 60 neurons per group. (H) Quantification of soma area. n = 3 experiments, 60 neurons per group. (I) Quantification of the pS6 immunofluorescence. n = 3 experiments, 60 neurons per group.


( A ) Axotomised DIV 14 - 16 rat cortical neurons expressing p110δ GFP or GFP alone . Axons were cut > 1000 μm from soma imaged for 14 h . Black arrows mark cut site , arrows marks axon tip . See also Movies EV7 EV8 . ( B ) of regenerating axons 14 h after axotomy . Numbers on bars are injured axons per group , from 4 experiments . ( C ) of axon regeneration 14 h post - axotomy . n = 9 axons for GFP , 9 for p110α , 25 for p110αH1047R 27 for p110δ from 4 experiments . ( D ) of time to start of regeneration . n = 9 axons for GFP , 9 for p110α , 25 for p110αH1047R 27 for p110δ from 4 experiments .

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(A) Axotomised DIV 14-16 rat cortical neurons expressing p110δ and GFP or GFP alone. Axons were cut >1000 μm from the soma imaged for 14 h. Black arrows mark the cut site, white arrows marks the axon tip. See also Movies EV7 and EV8. (B) Percentage of regenerating axons 14 h after laser axotomy. Numbers on bars are injured axons per group, from 4 experiments. (C) Quantification of axon regeneration length 14 h post-axotomy. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments. (D) Quantification of time to start of regeneration. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments.


( E ) Axotomised human embryonic neurons p110δ GFP or GFP alone . Black arrow marks cut site , arrows mark axon tip . ( F ) of regenerating axons of human hESC neurons , Numbers on bars are injured axons per group , from 5 experiments . ( G ) of axon regeneration . n = 22 axons for GFP , 44 for p110δ , from 5 experiments . Data are shown as mean + / - . Data were analysed by two Student ' s T - . ( H ) of time to start of regeneration . n = 22 axons for GFP , 44 for p110δ , from 5 experiments . Data are shown as mean + / - . Data were analysed by two Student ' s T - .

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(E) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone. Black arrow marks the cut site, white arrows mark the axon tip. (F) Percentage of regenerating axons of human hESC neurons, Numbers on bars are injured axons per group, from 5 experiments. (G) Quantification of axon regeneration length. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test. (H) Quantification of time to start of regeneration. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test.


( A ) of regenerating axons after axotomy of DIV 14 - 17 rat cortical neurons expressing either GFP ( control ) or GFP p110δ in presence of indicated inhibitors , 14h after injury . Numbers on bars are injured axons per group .

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(A) Percentage of regenerating axons after laser axotomy of DIV 14-17 rat cortical neurons expressing either GFP (control) or GFP and p110δ in the presence of the indicated inhibitors, 14h after laser injury. Numbers on bars are injured axons per group.


( B ) Single confocal section through a DIV 16 neuron expressing p110δ GFP ( upper panels ) . Green colour is GFP , red is overexpressed p110δ , detected with anti - p110δ . Lower panels show p110δ GFP in a distal axon section , at growth cone . Arrow indicates axon .

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(B) Single confocal section through a DIV 16 neuron expressing p110δ and GFP (upper panels). Green colour is GFP, red is overexpressed p110δ, detected with anti-p110δ. Lower panels show p110δ and GFP in a distal axon section, and at the growth cone. Arrow indicates the axon.


( C ) Active ARF total ARF6 ( red ) in axons of DIV 16 rat cortical neurons expressing GFP or GFP p110δ , as indicated . Arrows indicate axons . ( D ) of mean axonal ARF activity total ( mean ) ARF6 in DIV 16 neurons expressing GFP or GFP p110δ n = 60 axons for each condition .

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(C) Active ARF and total ARF6 (red) in axons of DIV 16 rat cortical neurons expressing GFP or GFP and p110δ, as indicated. Arrows indicate axons. (D) Quantification of mean axonal ARF activity and total (mean) ARF6 in DIV 16 neurons expressing GFP or GFP and p110δ n=60 axons for each condition.


( E ) Kymographs showing dynamics of α9 integrin - GFP in distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . ( F ) of α9 integrin - GFP dynamics in distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( G ) of total α9 integrin - GFP number in distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n = 43 for control , 47 for co - transfected with p110δ . n numbers are axon sections analysed for each condition .

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(E) Kymographs showing dynamics of α9 integrin-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. (F) Quantification of α9 integrin-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition. (G) Quantification of total α9 integrin-GFP number in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 47 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.


( H ) Kymographs showing dynamics of Rab11 - GFP in distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . ( I ) of Rab11 - GFP dynamics in distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( J ) of total Rab11 - GFP in distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n = 43 for control , 53 for co - transfected with p110δ . n numbers are axon sections analysed for each condition .

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(H) Kymographs showing dynamics of Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. (I) Quantification of Rab11-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition. (J) Quantification of total Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 53 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.


C ) Retinal sections from AAV - injected mice , immunolabelled for phospho - S6 . Blue colour is DAPI to indicate nuclei . Inset show colours at same scale as full image .

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C) Retinal sections from AAV-injected mice, immunolabelled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image.


( D ) of phospho - S6 positive in RGC layer 2 after delivery of AAV - Cre - GFP or AAV - GFP .

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(D) Percentage of phospho-S6 positive cells in the RGC layer 2 weeks after delivery of AAV-Cre-GFP or AAV-GFP.


( E ) Retinal whole mounts of AAV - injected mice immunolabelled for RGC marker Brn3A to indicate survival .

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(E) Retinal whole mounts of AAV-injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival.


( F ) of RGC survival 28 days after optic nerve crush ( of Brn3A positive RGC neurons ) .

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(F) Analysis of RGC survival 28 days after optic nerve crush (counts of Brn3A positive RGC neurons).


G ) Representative of CTB - labelled RGC axons 4 after optic nerve crush in wild type ( c57bl / 6 ) , Rosa26 - p110αH1047R , Rosa26 - p110δ transgenic mice injected with AAV2 . Cre . GFP . Dashed indicates crush site .

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G) Representative images of CTB-labelled RGC axons 4 weeks after optic nerve crush in wild type (c57bl/6), Rosa26-p110αH1047R, and Rosa26-p110δ transgenic mice injected with AAV2.Cre.GFP. Dashed line indicates crush site.


( H ) Regenerating axons at different distances distal to lesion sites .

(H) Regenerating axons at different distances distal to lesion sites.


( A ) Retinal sections from mice injected with AAV2 as indicated , immunolabeled for phospho - S6 . Blue colour is DAPI to indicate nuclei . Inset show colours at same scale as full image . ( B ) of phospho S6 positive in retinal ganglion layer of mice injected with AAV2 . shScramble . GFP or AAV2 . shPTEN . GFP . ( C ) Number of pS6 - postive RGCs of mice injected with AAV2 . GFP or AAV2 . p110δ .

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(A) Retinal sections from mice injected with AAV2 viruses as indicated, immunolabeled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image. (B) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2.shScramble.GFP or AAV2.shPTEN.GFP. (C) Number of pS6-postive RGCs of mice injected with AAV2.GFP or AAV2.p110δ.


( D ) Representative of retinal whole mounts from AAV - injected mice stained for RGC marker Brn3A to indicate RGC survival . ( E ) RGC survival 28 days after optic nerve crush .

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(D) Representative images of retinal whole mounts from AAV-injected mice stained for the RGC marker Brn3A to indicate RGC survival. (E) RGC survival 28 days after optic nerve crush.


( F ) CTB - labelled RGC axons 4 after optic nerve crush . Boxes i . to iv . show regenerating axons . Dashed indicates crush site . arrows mark end of axons . Graph shows of regenerating axons at different distances distal to lesion sites .

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(F) CTB-labelled RGC axons 4 weeks after optic nerve crush. Boxes i. to iv. show regenerating axons. Dashed line indicates crush site. White arrows mark the end of axons. Graph shows quantification of regenerating axons at different distances distal to the lesion sites.


Giemsa of a femoral section from 8 - day - old mice showing endochondral ossification defects . Scale bar : 100 μm .

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Giemsa staining of a femoral section from 8-day-old mice showing endochondral ossification defects. Scale bar: 100 μm.


Representative femur sections from 8 - day - old wild - type Dnmt2 − / − mice . of perivascular ( GLS , green ) is strongly reduced in Dnmt2 − / − bone marrow . Nuclei are counterstained with DAPI . Scale bar : 100 μm .

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Representative femur sections from 8-day-old wild-type and Dnmt2−/− mice. The staining of perivascular cells (GLS, green) is strongly reduced in Dnmt2−/− bone marrow. Nuclei are counterstained with DAPI. Scale bar: 100 μm.


Quantitative of capillary numbers . GLS - positive capillaries were counted in wild - type ( wt ) Dnmt2 − / − ( D2 - ) mice , in three independent high - power fields ( HPF ) per mouse ( n = 7 ) .

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Quantitative analysis of capillary numbers. GLS-positive capillaries were counted in wild-type (wt) and Dnmt2−/− (D2-) mice, in three independent high-power fields (HPF) per mouse (n = 7).


of numbers in haematopoietic tissues of 8 - day - old mice ( n = 8 ) .

Quantification of cell numbers in haematopoietic tissues of 8-day-old mice (n = 8).


Representative FACS plots of wild - type Dnmt2 − / − BM stained for Lin − Sca1 + cKit + .

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Representative FACS plots of wild-type and Dnmt2−/− BM stained for Lin− Sca1+ and cKit+ cells.


of bone marrow LSK in wild - type ( three independent experiments , each with pooled BM samples from 4 to 6 mice ) Dnmt2 − / − bone marrow ( three independent experiments , each with pooled BM samples of 5 - 9 mice ) .

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Quantification of bone marrow LSK cells in wild-type (three independent experiments, each with pooled BM samples from 4 to 6 mice) and Dnmt2−/−bone marrow (three independent experiments, each with pooled BM samples of 5-9 mice).


Restoration of LSK population in adult Dnmt2 − / − mice . Frequencies of LSK were normalized to frequencies of LSK from bone marrow of wild - type at indicated ( 2 - 3 independent experiments , each with pooled bone marrow samples from 3 to 9 mice ) .

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Restoration of the LSK cell population in adult Dnmt2−/− mice. Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild-type littermates at the indicated age (2-3 independent experiments, each with pooled bone marrow samples from 3 to 9 mice).


Increased LSK CD34 − reduced LSK CD34 + populations in 8 - day - 1 - year - old Dnmt2 − / − mice compared to wild - type ( two independent experiments , each with pooled bone marrow samples from 3 - 9 mice ) .

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Increased LSK CD34− and reduced LSK CD34+cell populations in 8-day- and 1-year-old Dnmt2−/− mice compared to wild-type (two independent experiments, each with pooled bone marrow samples from 3-9 mice).


qPCR of Dnmt2 mRNA expression in haematopoietic tissues , normalized to GAPDH .

qPCR analysis of Dnmt2 mRNA expression in haematopoietic tissues, normalized to GAPDH.


Western blot of Dnmt2 expression in haematopoietic tissues .

Western blot analysis of Dnmt2 protein expression in haematopoietic tissues.


Proliferation of bone marrow suspension . bone marrow suspension isolated from femurs of 8 - day - old mice were grown together with attached stromal . Dnmt2 − / − population doubling of three biological replicates , each plated three times , were calculated relative to wild - type culture .

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Proliferation analysis of primary bone marrow suspension cells. Primary bone marrow suspension cells isolated from the femurs of 8-day-old mice were grown together with the attached stromal cells. Dnmt2−/− population doubling levels of three biological replicates, each plated three times, were calculated relative to wild-type culture.


Reduced replating abilities of Dnmt2 − / − bone marrow in a myeloid colony formation . For ( 1 ° ) colony formation , 1 . 25 × 104 total bone marrow were plated in semisolid medium allowing myeloid differentiation . For secondary ( 2 ° ) colony formation , were harvested 0 . 1 % of colonies were replated into semisolid medium . Results are from two independent experiments analyzing 9 wild - type 13 Dnmt2 − / − mice in total .

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Reduced replating abilities of primary Dnmt2−/−bone marrow cells in a myeloid colony formation assay. For primary (1°) colony formation, 1.25 × 104 total bone marrow cells were plated in semisolid medium allowing myeloid differentiation. For secondary (2°) colony formation, cells were harvested and 0.1% of primary colonies were replated into semisolid medium. Results are from two independent experiments analyzing 9 wild-type and 13 Dnmt2−/−mice in total.


Reduced secondary CFU - preB colony formation of Dnmt2 − / − . Numbers of CFU - preB - colonies are indicated per 5 × 104 wild - type or Dnmt2 − / − total bone marrow . 10 % of colonies were replated into secondary CFU - preB assays .

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Reduced secondary CFU-preB colony formation of Dnmt2−/− cells. Numbers of CFU-preB-cell colonies are indicated per 5 × 104 wild-type or Dnmt2−/− total bone marrow cells. 10% of primary colonies were replated into secondary CFU-preB assays.


Stable engraftment ( upper panel ) multi - lineage reconstitution ( lower panel ) of wild - type Dnmt2 − / − bone marrow transplants . CD11b : macrophages ; Ly6G : granulocytes ; CD45R : B ; CD3 : T .

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Stable engraftment (upper panel) and multi-lineage reconstitution (lower panel) of wild-type and Dnmt2−/− bone marrow transplants. CD11b: macrophages; Ly6G: granulocytes; CD45R: B cells; CD3: T cells.


Flow cytometry of bone marrow mesenchymal .

Flow cytometry analysis of bone marrow primary mesenchymal cells.


of fibroblast colony - forming units ( CFU - F ) .

Quantification of fibroblast colony-forming units (CFU-F).


of alkaline phosphatase colony - forming units ( CFU - ALP ) .

Quantification of alkaline phosphatase colony-forming units (CFU-ALP).


Dnmt2 expression after 3 days of mesenchymal differentiation .

Dnmt2 expression after 3 days of mesenchymal cell differentiation.


Alizarin Red , indicating enhanced osteoblast differentiation of stromal from Dnmt2 − / − mice . Scale bar : 50 μm . was performed with three biological replicates .

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Alizarin Red staining, indicating enhanced osteoblast differentiation of primary stromal cells from Dnmt2−/− mice. Scale bar: 50 μm. Quantification was performed with three biological replicates.


qPCR of osteoblast markers Osterix Osteocalcin .

qPCR analysis of the osteoblast markers Osterix and Osteocalcin.


Oil Red O / hematoxylin , showing reduced adipocyte differentiation of stromal from Dnmt2 − / − mice . Scale bar : 50 μm . was performed with three biological replicates .

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Oil Red O/hematoxylin staining, showing reduced adipocyte differentiation of primary stromal cells from Dnmt2−/− mice. Scale bar: 50 μm. Quantification was performed with three biological replicates.


qPCR of adipocyte markers CEBp alpha Glut4 .

qPCR analysis of the adipocyte markers CEBp alpha and Glut4.


Bisulfite sequencing maps of 454 reads showing average methylation status of cytosines ( boxes ) within tRNAs from bone marrow ( BM ) , mesenchymal stromal ( MSC ) , in vitro differentiated adipocytes osteoblasts . Methylation are indicated on a color scale from green ( unmethylated ) to red ( completely methylated ) .

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Bisulfite sequencing maps of 454 reads showing the average methylation status of individual cytosines (boxes) within tRNAs from bone marrow (BM), mesenchymal stromal cells (MSC), and in vitro differentiated adipocytes and osteoblasts. Methylation levels are indicated on a color scale from green (unmethylated) to red (completely methylated).


Representative Northern blot showing tRNA ( Gly ) tRNA ( Asp ) fragmentation in bone marrow from 8 - day - old adult mice . tRNA ( Ser ) was included as a Dnmt2 non - substrate tRNA , 5S rRNA was included as a loading control .

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Representative Northern blot showing tRNA(Gly) and tRNA(Asp) fragmentation in bone marrow from 8-day-old and adult mice. tRNA(Ser) was included as a Dnmt2 non-substrate tRNA, and 5S rRNA was included as a loading control.


tRNA fragment coverage for Dnmt2 substrates tRNA ( Gly ) tRNA ( Asp ) , non - substrate tRNA ( Ser ) in bone marrow , as determined by deep sequencing . Wild - type coverage is represented in red , Dnmt2 − / − coverage is indicated in blue . plots illustrate specific increase of 5 ′ - halves from tRNA - Gly 3 ′ - halves from tRNA - Asp fragments in Dnmt2 mutants .

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tRNA fragment coverage for the Dnmt2 substrates tRNA(Gly) and tRNA(Asp), and the non-substrate tRNA(Ser) in bone marrow, as determined by deep sequencing. Wild-type coverage is represented in red, and Dnmt2−/− coverage is indicated in blue. The plots illustrate the specific increase of 5′-halves from tRNA-Gly and 3′-halves from tRNA-Asp fragments in Dnmt2 mutants.


of synthesis by dynamic SILAC . top 10 % of that are translated slower in Dnmt2 − / − versus wild - type are indicated in orange . top 10 % of that are translated faster in Dnmt2 − / − versus wild - type are shown in blue . A change of > twofold is indicated by black lines .

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Analysis of protein synthesis by dynamic SILAC. The top 10% of proteins that are translated slower in Dnmt2−/− versus wild-type cells are indicated in orange. The top 10% of proteins that are translated faster in Dnmt2−/− versus wild-type cells are shown in blue. A change of >  twofold is indicated by the black lines.


Dimethyl - labeling of 8 - day - old mouse bone marrow tissue . that were down - regulated ( < twofold change ) in Dnmt2 - deficient are indicated in red , up - regulated ( > twofold change ) are shown in green . Nestin periostin are marked with arrows .

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Dimethyl-labeling analysis of 8-day-old mouse bone marrow tissue. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. Nestin and periostin are marked with arrows.


Correlation between mRNA expression for differentially expressed . that were down - regulated ( < twofold change ) in Dnmt2 - deficient are indicated in red , up - regulated ( > twofold change ) are shown in green . Pearson correlation coefficient is − 0 . 03 .

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Correlation between protein and mRNA expression for differentially expressed proteins. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. The Pearson correlation coefficient is −0.03.


Representative of nestin periostin in femur sections from 8 - day - old wild - type Dnmt2 − / − mice . anti - periostin is comparable in periosteum of wild - type Dnmt2 − / − mice , but is reduced in bone marrow ( compare highlighted areas ) . Scale bar : 100 μm . Nuclei are counterstained with DAPI ( blue ) .

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Representative staining of nestin and periostin in femur sections from 8-day-old wild-type and Dnmt2−/− mice. The anti-periostin staining is comparable in the periosteum of wild-type and Dnmt2−/− mice, but is reduced in the bone marrow cells (compare the highlighted areas). Scale bar: 100 μm. Nuclei are counterstained with DAPI (blue).


Quantitative differences in of nestin - positive trabecular zone ( upper panel ) between wild - type ( 30 measurements , three mice ) Dnmt2 − / − ( 21 measurements , two mice ) in bone periostin - positive marrow areas ( lower panel ) between wild - type ( 26 measurements , three mice ) Dnmt2 − / − ( 45 measurements , three mice ) in femur sections from 8 - day - old mice . Data are presented as mean ± SD .

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Quantitative differences in the length of the nestin-positive trabecular zone (upper panel) between wild-type (30 measurements, three mice) and Dnmt2−/− (21 measurements, two mice) and in bone periostin-positive marrow areas (lower panel) between wild-type (26 measurements, three mice) and Dnmt2−/− (45 measurements, three mice) in femur sections from 8-day-old mice. Data are presented as mean ± SD.


Frequency of acid substitutions in dSILAC dataset . Differences between Asp > Glu Glu > Asp transitions versus Asn > Lys His > Gln were found to be highly significant ( P < 0 . 01 , Fisher ' s exact ) .

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Frequency of amino acid substitutions in the dSILAC dataset. Differences between Asp > Glu and Glu > Asp transitions versus Asn > Lys and His > Gln were found to be highly significant (P < 0.01, Fisher's exact test).


B . lines generated from WT , null , mutant p53 esophagi were assessed by Western blot for p53 expression . Relative densitometry of Rab11 - FIP1 results are shown at bottom .

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B. Cell lines generated from WT, null, and mutant p53 esophagi were assessed by Western blot analysis for p53 protein expression. Relative intensity densitometry of Rab11-FIP1 results are shown at the bottom.


C . Organoids generated from WT , mutant , null p53 lines . Scale bar : 100μm . D . of portion of organoids exhibiting a solid , intermediate or stratified ( > 37 organoids from 2 - 3 lines per group ) .

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C. Organoids generated from WT, mutant, and null p53 cell lines. Scale bar: 100μm. D. Quantification of the portion of organoids exhibiting a solid, intermediate or stratified morphology (> 37 organoids from 2-3 cell lines per group).


E - G . were subcutaneously injected in lower flanks of nude mice ( n = 4 flanks / . WT p53 2 lines , Mutant p53 - 3 lines , p53 null - 3 lines ) . E . number of tumors formed in each group was quantified . F . growth was measured at indicated time points post - implantation . Graph represents total per group . G . Average following dissection ( n = 2 biological replicates for WT p53 lines , n = 3 biological replicates for mutant p53 lines , n = 3 biological replicates for p53 null lines , n = 4 technical replicates injected per , calculated as average per ) . Error bars represent ± S . E . M . * p = 0 . 02 Mutant p53 vs WT p53 , * p = 0 . 02 Mutant p53 vs Null p53 ( One - way Anova ) .

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E-G. Cells were subcutaneously injected in the lower flanks of nude mice (n= 4 flanks/cell line. WT p53 2 cell lines, Mutant p53-3 cell lines, p53 null-3 cell lines). E. The number of tumors formed in each group was quantified. F. Tumor growth was measured at the indicated time points post-implantation. Graph represents total tumor size per group. G. Average tumor weight following dissection (n=2 biological replicates for WT p53 cell lines, n=3 biological replicates for mutant p53 cell lines, n=3 biological replicates for p53 null cell lines, n=4 technical replicates injected per cell line, calculated as average tumor weight per cell line). Error bars represent ±S.E.M. *p=0.02 Mutant p53 vs WT p53, *p=0.02 Mutant p53 vs Null p53 (One-way Anova).


B . Relative fold change in expression level of endocytic recycling genes in invasive vs . non - invasive , in mutant vs . WT p53 on microarray - seq , respectively .

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B. Relative fold change in expression level of endocytic recycling genes in invasive vs. non-invasive cells, and in mutant vs. WT p53 cells based on microarray and RNA-seq analysis, respectively.


C . qPCR of recycling - related gene expression in mutant p53 relative to WT p53 ( red black bars , respectively ) . Graphs represent mean ± ( n = 3 biological replicates for Rab11 - FIP1 , Rab25 , Myo5b Clic3 ; n = 2 biological replicates for Rab5 ) . Rab11 - FIP1 * p = 0 . 0109 , Rab25 * * p = 0 . 0021 , Myo5b * * * p = 0 . 0001 , Clic3 * * p = 0 . 0051 , Rab5 no significant differences ( n . s . ) . ( Unpaired t - ) .

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C. qPCR analysis of recycling-related gene expression in mutant p53 relative to WT p53 cells (red and black bars, respectively). Graphs represent mean ± SEM (n=3 biological replicates for Rab11-FIP1, Rab25, Myo5b and Clic3; n=2 biological replicates for Rab5). Rab11-FIP1 *p=0.0109, Rab25 **p=0.0021, Myo5b ***p=0.0001, Clic3 **p=0.0051, Rab5 no significant differences (n.s.). (Unpaired t-test).


A . Western blot confirming knockdown of Rab11 - FIP1 by shRNAs . Relative by densitometry of Rab11 - FIP1 results are shown at bottom .

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A. Western blot confirming knockdown of Rab11-FIP1 by shRNAs. Relative intensity by densitometry of Rab11-FIP1 results are shown at the bottom.


3D organoids were generated from WT mutant p53 expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 sh2 ) . B . Diameter of organoids ( n = 80 - 130 organoids ) . Bar graph represents mean diameter ± . * * * * p < 0 . 0001 WT or mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0006 WT non - targeting control vs Rab11 - FIP1 shRNA2 , * * * * p < 0 . 0001 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) . * * * * p < 0 . 0001 WT vs mutant p53 non - targeting control ( Unpaired t - ) .

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3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). B. Diameter of organoids (n= 80-130 organoids). Bar graph represents mean diameter ± SEM. ****p<0.0001 WT or mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0006 WT non-targeting control vs Rab11-FIP1 shRNA2, ****p<0.0001 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova). ****p<0.0001 WT vs mutant p53 non-targeting control (Unpaired t-test).


3D organoids were generated from WT mutant p53 expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 sh2 ) . C . of WT p53 ( top panel ) mutant ( bottom panel ) organoids exhibiting a solid , intermediate , or stratified ( n > 50 organoids generated from three different passages quantified ) . Bar graph represents mean diameter ± . n . s . WT p53 non - targeting control vs Rab11 - FIP1 shRNAs , * p = 0 . 046 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0002 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) .

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3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). C. Percent of WT p53 (top panel) and mutant (bottom panel) organoids exhibiting a solid, intermediate, or stratified morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova).


3D organoids were generated from WT mutant p53 expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 sh2 ) . D . of WT p53 mutant organoids exhibiting a basaloid - like ( n > 50 organoids generated from three different passages quantified ) . Bar graph represents mean diameter ± . n . s . WT p53 non - targeting control vs Rab11 - FIP1 shRNAs , * p = 0 . 046 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0002 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) .

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3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). D. Percent of WT p53 and mutant organoids exhibiting a basaloid-like morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova).


3D organoids were generated from WT mutant p53 expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 sh2 ) . E . WT p53 organoids stained for H & E ( left ) , Rab11 - FIP1 ( middle ) , basal marker p63 ( right ) by . 40X . Scale bar = 100 μm . F . Mutant p53 organoids stained for H & E ( left ) , Rab11 - FIP1 ( middle ) , basal marker p63 ( right ) by . 40X . Scale bar = 100 μm .

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3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). E. WT p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm. F. Mutant p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm.


A . EPC2 - hTERT - EGFR - p53R175H grown in OTC stained for RCP . Arrow indicates invasive region . ( scale bar = 100 μm ) .

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A. EPC2-hTERT-EGFR-p53R175H cells grown in OTC stained for RCP. Arrow indicates invasive region. (scale bar = 100 μm).


B . Transwell Boyden Chamber invasion of WT mutant p53 expressing non - targeting control ( NT ) Rab11 - FIP1 shRNAs ( sh1 sh2 ) ( n = 9 technical replicates from 3 different passages ) . Error bars represent ± S . E . M . n . s . WT p53 NT vs sh1 , * * * p = 0 . 0005 WT p53 NT vs sh2 , n . s . Mutant p53 NT vs sh1 , * * p = 0 . 0026 Mutant p53 NT vs sh2 , ( One - way Anova ) * p = 0 . 0173 WT p53 NT vs Mutant p53 NT , ( unpaired t - ) .

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B. Transwell Boyden Chamber invasion assay of WT and mutant p53 cells expressing non-targeting control (NT) and Rab11-FIP1 shRNAs (sh1 and sh2) (n=9 technical replicates from 3 different passages). Error bars represent ±S.E.M. n.s. WT p53 NT vs sh1, ***p=0.0005 WT p53 NT vs sh2, n.s. Mutant p53 NT vs sh1,** p=0.0026 Mutant p53 NT vs sh2, (One-way Anova) *p=0.0173 WT p53 NT vs Mutant p53 NT, (unpaired t-test).


C - D . WT mutant p53 expressing non - target control Rab11 - FIP1 shRNAs were grown in OTC . C . H & E of OTC with WT p53 ( top ) mutant p53 ( bottom ) with invasive regions . Scale bar = 50 μm . D . Graph shows relative invasive from OTC of WT p53 non - target control Rab11 - FIP1 shRNAs . n = 5 technical replicates in WT p53 NT , n = 6 technical replicates in WT p53 sh1 , n = 6 technical replicates in WT p53 sh2 . Error bars represent ± S . E . M . * * * * p < 0 . 0001 WT p53 NT vs sh1 , n . s . WT p53 NT vs sh2 ( One - way Anova ) .

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C-D. WT and mutant p53 cells expressing non-target control and Rab11-FIP1 shRNAs were grown in OTC. C. H&E staining of OTC with WT p53 (top) and mutant p53 (bottom) with invasive regions. Scale bar = 50 μm. D. Graph shows relative invasive area from OTC of WT p53 non-target control and Rab11-FIP1 shRNAs. n=5 technical replicates in WT p53 NT, n=6 technical replicates in WT p53 sh1, n=6 technical replicates in WT p53 sh2. Error bars represent ±S.E.M. **** p<0.0001 WT p53 NT vs sh1, n.s. WT p53 NT vs sh2(One-way Anova).


A . Immunofluorescent for Rab11 - FIP1 in TE1 TE2 ESCC lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm B . Immunofluorescent for f - actin in TE1 TE2 ESCC lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm C . Immunofluorescent for E - cadherin , in TE1 TE2 ESCC lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm

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A. Immunofluorescent staining for Rab11-FIP1 in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm B. Immunofluorescent staining for f-actin in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm C. Immunofluorescent staining for E-cadherin, in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm


D . qPCR of epithelial mesenchymal gene expression in TE1 ( left ) TE2 ( right ) expressing Rab11 - FIP1 shRNA relative to control ( dotted ) . Graph error bars represent mean ( n = 3 technical replicates from three different passages ) . n . s . TE2 SNAI2 , * p < 0 . 05 , ( Unpaired t - ) .

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D. qPCR analysis of epithelial and mesenchymal gene expression in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). n.s. TE2 SNAI2, *p<0.05,(Unpaired t-test).


E . qPCR of epithelial mesenchymal gene expression following ZEB1 depletion in TE1 ( left ) TE2 ( right ) expressing Rab11 - FIP1 shRNA relative to control ( dotted ) . Graph error bars represent mean ( n = 3 technical replicates from three different passages ) . * * p < 0 . 01 , * p < 0 . 05 , n . s . TE1 CDH1 , VIM . SNAI1 , SNAI2 , TE2 VIM , SNAI1 ( Unpaired t - ) .

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E. qPCR analysis of epithelial and mesenchymal gene expression following ZEB1 depletion in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). **p<0.01, * p<0.05, n.s. TE1 CDH1, VIM. SNAI1, SNAI2, TE2 VIM, SNAI1 (Unpaired t-test).


( A ) Survival curves of C57BL / 6J naïve adrenalectomized ( Adx ) mice treated for 7 days with 25 mM ZnSO4 in drinking water challenged , i . p . , with 30 µg ( naïve ) or 2 µg ( Adx ) TNF , solved in sterile PBS , per 20 g . No deaths occurred later than 72 h after TNF injection . Results represent combined data of 3 experiments .

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(A) Survival curves of C57BL/6J naïve and adrenalectomized (Adx) mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 30 µg (naïve) or 2 µg (Adx) TNF, solved in sterile PBS, per 20 g body weight. No deaths occurred later than 72 h after TNF injection. Results represent combined data of 3 experiments.


( B ) Corticosterone in serum of C57BL / 6J naïve Adx mice after 7 days of 25 mM ZnSO4 supplementation to drinking water ( N = 6 per group ) .

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(B) Corticosterone levels in the serum of C57BL/6J naïve and Adx mice after 7 days of 25 mM ZnSO4 supplementation to the drinking water (N=6 per group).