D . Control ( sh Control ; top ) GIV - GEM depleted ( shGIV ; bottom ) HeLa were co - transfected with Gαi1 - YFP , Gβ1 - CFP Gγ2 ( untagged ) live were analyzed by FRET imaging at steady - state , after being serum starved in 0 . 2 % FBS overnight then after stimulation with 50 nM EGF . Representative freeze - frame FRET are shown . FRET image panels display intensities of acceptor emission due to efficient energy transfer in each pixel . FRET scale is shown in inset . Golgi PM regions of interest are indicated with arrows . Scale bar = 10 µm . See also Figure EV2A - B for free - frame for additional time points in control HeLa .

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

D. Control (sh Control; top) and GIV-GEM depleted (shGIV; bottom) HeLa cells were co-transfected with Gαi1-YFP, Gβ1-CFP and Gγ2 (untagged) and live cells were analyzed by FRET imaging at steady-state, after being serum starved in 0.2% FBS overnight and then after stimulation with 50 nM EGF. Representative freeze-frame FRET images are shown. FRET image panels display intensities of acceptor emission due to efficient energy transfer in each pixel. FRET scale is shown in the inset. Golgi and PM regions of interest are indicated with arrows. Scale bar = 10 µm. See also Figure EV2A-B for free-frame images for additional time points in control HeLa cells.


F . HeLa starved with 0 . 2 % FBS overnight or stimulated subsequently with 50 nM EGF were fixed stained for active Gαi ( green ; anti - Gαi : GTP mAb ) Man II ( red ) analyzed by confocal microscopy . Activation of Gαi was detected exclusively after EGF stimulation . When detected , active Gαi colocalizes with Man II ( yellow pixels in merge panel ) . See also Figure EV2C - D for additional time points stimulus . Scale bar = 7 . 5 µm .

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

F. HeLa cells starved with 0.2% FBS overnight or stimulated subsequently with 50 nM EGF were fixed and stained for active Gαi (green; anti-Gαi:GTP mAb) and Man II (red) and analyzed by confocal microscopy. Activation of Gαi was detected exclusively after EGF stimulation. When detected, active Gαi colocalizes with Man II (yellow pixels in merge panel). See also Figure EV2C-D for additional time points and stimulus. Scale bar = 7.5 µm.


G . Model fit for fold change of active tGTPase ( denoted as tG * ) . Experiment data is fold change of ∆ FRET / CFP in D is shown as mean ± S . E . M ( n = 8 ; 3 - 5 from 2 independent biological experiments ) . Continuous lines display model simulation results after parameter

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

G. Model fit for the fold change of active tGTPase (denoted as tG*). Experiment data is the fold change of ∆FRET/CFP in D and is shown as mean ± S.E.M (n=8; 3-5 cells from 2 independent biological experiments). Continuous lines display the model simulation results after parameter fitting


I . Immunoblot shows bound Arf1 ( active ; top ) total Arf1 ( input lysates ; bottom ) from equal aliquots of lysates of control ( sh Control ) GIV - depleted ( shGIV ) HeLa . were stimulated with EGF for indicated time points prior to lysis . Bar graphs in Figure EV2E display fold change in Arf1 activity normalized to t0 min . ' Low ' ' high ' indicate exposures .

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

I. Immunoblot shows bound Arf1 (active; top) and total Arf1 (input lysates; bottom) from equal aliquots of lysates of control (sh Control) and GIV-depleted (shGIV) HeLa cells. Cells were stimulated with EGF for the indicated time points prior to lysis. Bar graphs in Figure EV2E display the fold change in Arf1 activity normalized to t0 min. 'Low' and 'high' indicate exposures.


D - E . secretion ( D ) or number ( E ) as a function of stimulus in coupled uncoupled switches . stimulus = 0 , 0 . 00046 , 0 . 0046 , 0 . 046 , 0 . 115 , 0 . 23 0 . 46 correspond to varying doses of EGF in simulations , ranging from 0 , 0 . 1 nM , 1 nM , 10 nM , 25 nM , 50 nM , 100 nM , respectively . error bar denotes S . D on 1000 repeated independent simulations of ODEs when noise is in stimulus connections .

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D-E. The secretion (D) or the cell number (E) as a function of stimulus in coupled and uncoupled switches. The stimulus =0, 0.00046, 0.0046, 0.046, 0.115, 0.23 and 0.46 correspond to varying doses of EGF in simulations, ranging from 0, 0.1 nM, 1 nM, 10 nM, 25 nM, 50 nM, and 100 nM, respectively. The error bar denotes the S.D based on 1000 repeated independent simulations of ODEs when noise is in the stimulus and connections.


F . bar plot depicts numbers achieved by with either coupled or uncoupled switches , at different of stimulus . For first two bars , height error bars are mean S . D . of number when stimulus = 0 . 046 in E , respectively . For last two bars , height error bars are mean S . D . of number when stimulus = 0 in E , respectively .

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F. The bar plot depicts cell numbers achieved by cells with either coupled or uncoupled switches, at different levels of stimulus. For the first two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0.046 in E, respectively. For the last two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0 in E, respectively.


B . Immunoblots showing intracellular ( left ) secreted ( in media ; right ) GFP - MMP9 at 24 h after stimulation with varying doses of EGF . Tubulin , used as a loading control , confirms presence of a similar number of plated in .

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B. Immunoblots showing intracellular (left) and secreted (in the media; right) GFP-MMP9 at 24 h after stimulation with varying doses of EGF. Tubulin, used as a loading control, confirms the presence of a similar number of plated cells in the assay.


C . Left : Graph displays experimentally determined secretion of GFP - MMP9 in response to varying doses of EGF in control ( shControl ) GIV - depleted ( shGIV ) HeLa ( as in B ) , quantified by band densitometry . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . p values were determined by two - sided unpaired t - . Right : Schematic diagram of dose responses ( mG * secretion ) for single switch coupled switches . Coupled switches stretch range of proportionate responses . Single mG switch results in misaligned responses . DoRA , dose response alignment .

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

C. Left: Graph displays experimentally determined secretion of GFP-MMP9 in response to varying doses of EGF in control (shControl) and GIV-depleted (shGIV) HeLa cells (as in B), and quantified by band densitometry. Results are expressed as mean ± S.E.M; n = 3 biological replicates. p values were determined by two-sided unpaired t-test. Right: Schematic diagram of dose responses (mG* and secretion) for the single switch and coupled switches. Coupled switches stretch the range of proportionate responses. Single mG switch results in misaligned responses. DoRA, dose response alignment.


G - H . Control ( parental ) GIV - depleted ( GIV KO ) HeLa grown in different concentrations of serum ( FBS % ) were treated or not with varying concentrations of BFA ( µM ) as indicated . graphs in 3D ( G ) depict formazan absorbance expressed as a measure of viability from HeLa in various conditions tested . Bar graphs ( H ) depict number in serum - free growth conditions that are supported exclusively by autocrine secrete - - sense loop ( without BFA ; BFA = 0 . 0 µM ) or when such loop is interrupted ( BFA = 0 . 1 µM ) . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . Statistical significance was determined by one way ANOVA .

O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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. Control (parental) and GIV-depleted (GIV KO) HeLa cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as indicated. Line graphs in 3D (G) depict the formazan absorbance expressed as a measure of cell viability from the HeLa cells in various conditions tested. Bar graphs (H) depict the cell number in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA.


I - K . Control ( parental ) GIV - depleted ( GIV KO ) MDA MB - 231 grown in different concentrations of serum ( FBS % ) were treated or not with varying concentrations of BFA ( µM ) as in G - H . graphs in 3D ( I ) depict formazan absorbance expressed as a measure of viability from MDA MB - 231 in various conditions tested . Bar graphs ( J ) depict viability of MDA MB - 231 in serum - free growth conditions that are supported exclusively by autocrine secrete - - sense loop ( without BFA ; BFA = 0 . 0 µM ) or when such loop is interrupted ( BFA = 0 . 1 µM ) . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . Statistical significance was determined by one way ANOVA . Immunoblots ( K ) of equal aliquots of whole lysates confirm depletion of GIV compared to tubulin ( loading control ) . See also Appendix Figure S5D - H for dot plots early late apoptotic fractions . Results are expressed as mean ± S . E . M ; n = 3 biological replicates .

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

I-K. Control (parental) and GIV-depleted (GIV KO) MDA MB-231 cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as in G-H. Line graphs in 3D (I) depict the formazan absorbance expressed as a measure of cell viability from the MDA MB-231 cells in various conditions tested. Bar graphs (J) depict the viability of the MDA MB-231 cells in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA. Immunoblots (K) of equal aliquots of whole cell lysates confirm the depletion of GIV compared to tubulin (loading control). See also Appendix Figure S5D-H for dot plots and early and late apoptotic fractions. Results are expressed as mean ± S.E.M; n = 3 biological replicates.


( A ) SA - β - gal of U2 - OS , harvested at indicated times post irradiation ( IR ) ; h , hours ; D , days ; UT , untreated . n = 3 . Scale bar : 50 μm . Bar Graph : Significance was determined by Brown Forsythe ANOVA with Dunnett ' s 3T , using n = 3 biological replicates . SD shown . * * * p < 0 . 001 , n . s . = not significant .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SA-β-gal staining of U2-OS cells, harvested at indicated times post irradiation (IR); h, hours; D, days; UT, untreated. n = 3. Scale bar: 50 μm. Bar Graph: Significance was determined by Brown Forsythe ANOVA with Dunnett's 3T test, using n = 3 biological replicates. SD shown. *** p < 0.001, n.s. = not significant.


( B ) fractions from U2 - OS were harvested at indicated time points post IR ( 20 Gy ) as in ( A ) . Top panel : NF - κB binding was analyzed by EMSA . Lower panels : Western blot of indicated NF - κB subunits PARP1 as loading control . two phases of

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

(B) Nuclear fractions from U2-OS cells were harvested at indicated time points post IR (20 Gy) as in (A). Top panel: NF-κB DNA binding was analyzed by EMSA. Lower panels: Western blot of indicated NF-κB subunits and PARP1 as loading control. The two phases of nuclear translocation


( C ) Gene expression of two NF - κB phases following damage . - Seq ( n = 5 biological replicates per group ) of U2 - OS , either untreated or analyzed 1 . 5 hours or 7 days after irradiation ( 20 Gy ) , as indicated . shows significantly regulated genes ( log2 value > 0 . 5 p value < 0 . 05 ) . Black arrow points to NFKBIA . Significance determined by ANOVA with Bonferroni correction for multiple testing . See also Dataset EV1 .

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

(C) Gene expression analysis of two NF-κB phases following damage. RNA-Seq analysis (n = 5 biological replicates per group) of U2-OS cells, either untreated or analyzed 1.5 hours or 7 days after irradiation (20 Gy), as indicated. The heatmap shows significantly regulated genes (log2 value > 0.5 and p value < 0.05). Black arrow points to NFKBIA. Significance determined by ANOVA with Bonferroni correction for multiple testing. See also Dataset EV1.


( D ) mice 12 - 16 old ( n = 6 mice per condition ( with three technical repeats per mouse ) , were irradiated ( 5 Gy ) sacrificed after 1 hour ( first phase ) , 7 days ( second phase ) or left untreated . Skin or renal tissue was analyzed by RT - qPCR , shown as a mean + / - SD . Significance was confirmed by ANOVA with Tukey multiple comparisons . SD shown : * = p < 0 . 05 , * * = p < 0 . 01 .

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(D) Female mice 12-16 weeks old (n = 6 mice per condition (with three technical repeats per mouse), were irradiated (5 Gy) and sacrificed after 1 hour (first phase), 7 days (second phase) or left untreated. Skin or renal tissue RNA was analyzed by RT-qPCR, shown as a mean +/- SD. Significance was confirmed by ANOVA with Tukey multiple comparisons test. SD shown: * = p < 0.05, ** = p < 0.01.


( E ) In situ hybridization using an anti - sense IκBα / Nfkbia riboprobe on longitudinal skin sections from mice ( 5 - 8 of ) treated as in ( D ) for time indicated . Representative sections shown from n = 6 mice per condition . Bu , bulge region ( dashed lines ) , HS , hair shaft , IFE , interfollicular epidermis . Scale bar : 50 μm . ( F ) Quantitation of ( E ) from n = 6 mice per condition 3 - 4 sections per mouse . Bulge regions positive for Nfkbia were counted presented as of total . One way ANOVA with Tukey multiple comparisons was performed . SD shown . * * = p < 0 . 01 .

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

(E) In situ hybridization using an anti-sense IκBα/Nfkbia riboprobe on longitudinal skin sections from male and female mice (5-8 weeks of age) treated as in (D) for the time indicated. Representative sections shown from n = 6 mice per condition. Bu, bulge region (dashed lines), HS, hair shaft, IFE, interfollicular epidermis. Scale bar: 50 μm. (F) Quantitation of (E) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for Nfkbia were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. ** = p < 0.01.


( G ) Skin sections as in ( E ) analyzed by immunofluorescence with IκBα ( red ) or IL - 6 antibody ( green ) DAPI ( blue ) . Dotted lines delineate hair follicles , Bu , bulge region , HS , hair shaft , IFE , interfollicular epidermis , SG , sebaceous . Scale bar : 50 μm . ( H ) Quantitation of ( G ) from n = 6 mice per condition 3 - 4 sections per mouse . Bulge regions positive for IκBα or IL - 6 were counted presented as of total . One way ANOVA with Tukey multiple comparisons was performed . SD shown . * = p < 0 . 05 , * * * = p < 0 . 001 .

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

(G) Skin sections as in (E) analyzed by immunofluorescence with IκBα (red) or IL-6 antibody (green) and nuclear DAPI staining (blue). Dotted lines delineate hair follicles, Bu, bulge region, HS, hair shaft, IFE, interfollicular epidermis, SG, sebaceous gland. Scale bar: 50 μm. (H) Quantitation of (G) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for IκBα or IL-6 were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. * = p < 0.05, *** = p < 0.001.


( A ) Left panel : IκBα western blot of U2 - OS . Lane 1 , untreated ( ut ) ; Lanes 2 - 4 , single IR ( 20 Gy ) at indicated time points prior to harvest ; Lane 5 , IR at 5 days prior to harvest , plus 1 . 5 hours prior to harvest . Right panel : Samples treated as in left panel , analyzed by RT - qPCR . Significance from n = 3 replicates per condition determined by ANOVA with Tukey multiple comparisons . SD shown . * * * = p < 0 . 001 .

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

(A) Left panel: IκBα western blot of U2-OS cells. Lane 1, untreated (ut); Lanes 2-4, single IR (20 Gy) at indicated time points prior to harvest; Lane 5, IR at 5 days prior to harvest, plus 1.5 hours prior to harvest. Right panel: Samples treated as in the left panel, analyzed by RT-qPCR. Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test. SD shown. *** = p < 0.001.


( B ) NFKBIA knockdown by dox - inducible shRNA in U2 - OS . were pre - treated with Dox to induce knockdown . For knockdown efficiency see Fig . S2F . were irradiated ( 20 Gy ) or not , as indicated described above . NF - κB activity was analyzed by EMSA . ns , non - specific band . A representative gel from n = 3 biological replicates is shown .

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(B) NFKBIA knockdown by dox-inducible shRNA in U2-OS cells. Cells were pre-treated with Dox to induce knockdown. For knockdown efficiency see Fig. S2F. Cells were irradiated (20 Gy) or not, as indicated and described above. NF-κB activity was analyzed by EMSA. ns, non-specific band. A representative gel from n = 3 biological replicates is shown.


( E ) GSEA was performed comparing IκBα IEC ‐ KO gene list ( n = 4 villin - Cre x floxed IκBα mice n = 4 littermate controls ( GSE 139251 ) Mikuda et al , 2020 ( ) , with a gene signature on a list of SASP factors Coppe et al , 2010 ( ) . NES : normalized enrichment score . FDR : false discovery rate .

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(E) GSEA was performed comparing the IκBα IEC‐KO gene list (n = 4 villin-Cre x floxed IκBα mice and n = 4 littermate controls (GSE 139251) Mikuda et al, 2020(), with a gene signature based on a list of SASP factors Coppe et al, 2010(). NES: normalized enrichment score. FDR: false discovery rate.


( F ) RT - qPCR of indicated genes was performed using extracted from duodenum of 5 8 old control villin - Cre ( n = 6 ) or villin - Cre x floxed IκBα mice ( n = 6 ) both . Expression is shown as fold change between , paired t - . SD shown . * p < 0 . 05 * * = P < 0 . 01 .

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

(F) RT-qPCR analysis of indicated genes was performed using RNA extracted from the duodenum of 5 and 8 weeks old control villin-Cre (n = 6) or villin-Cre x floxed IκBα mice (n = 6) both male and female. Expression is shown as fold change between littermates, paired t-test. SD shown. * p < 0.05 ** = P < 0.01.


( A ) fractions from either untreated or irradiated ( 20 Gy ) U2 - OS , 1 . 5 hours or 7 days prior to harvest , were analyzed by EMSA with supershift . For supershifting , lysates were incubated with antibodies or water , as indicated . Arrows point to shift location of a given subunit , due to antibody binding . ns , non - specific band . antibodies were previously tested for supershift compatibility . Representative gel shown from n = 3 replicates .

O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nuclear fractions from either untreated or irradiated (20 Gy) U2-OS cells, 1.5 hours or 7 days prior to harvest, were analyzed by EMSA with supershift analysis. For supershifting, lysates were incubated with antibodies or water, as indicated. Arrows point to shift location of a given subunit, due to antibody binding. ns, non-specific band. All antibodies were previously tested for supershift compatibility. Representative gel shown from n = 3 replicates.


( B ) Expression of SASP targets of NF - κB obtained from - seq ( Dataset EV1B ) , was quantitated using RT - qPCR from shRNA ( RELA ) stably expressing U2 - OS . For knockdown efficiency see Appendix Fig S4A . were treated with doxycycline ( Dox ) to induce knockdown of p65 . represents targets normalized to untreated Scrambled control . Expression is shown as log2 change with p values < 0 . 05 . Statistical performed using ANOVA with Bonferroni correction for multiple testing . First - phase second - phase samples were irradiated ( 20 Gy ) 1 . 5h 7 Days prior to harvest , respectively . on n = 3 biological replicates .

O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of SASP targets of NF-κB obtained from RNA-seq analysis (Dataset EV1B), was quantitated using RT-qPCR from the shRNA (RELA) stably expressing U2-OS cells. For knockdown efficiency see Appendix Fig S4A. Cells were treated with doxycycline (Dox) to induce knockdown of p65. Heatmap represents targets normalized to the untreated Scrambled control. Expression is shown as log2 change with p values < 0.05. Statistical analysis performed using ANOVA with Bonferroni correction for multiple testing. First-phase and second-phase samples were irradiated (20 Gy) 1.5h and 7 Days prior to harvest, respectively. Analysis based on n = 3 biological replicates.


( C ) duplication was measured at time points indicated in U2 - OS bearing either scrambled control or Dox - inducible shRNA against RELA ( n = 3 biological replicates ) . Treatment with Dox was initiated at two days prior to IR ( 20 Gy ) or after IR . Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons . SD shown . * = P < 0 . 05 , * * * = p < 0 . 001 .

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

(C) Cell duplication was measured at time points indicated in U2-OS cells bearing either scrambled control or Dox-inducible shRNA against RELA (n = 3 biological replicates). Treatment with Dox was initiated at two days prior to IR (20 Gy) or after IR. Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = P < 0.05, *** = p < 0.001.


( A ) ( Nuc . ) ( Cyto . ) fractions of U2 - OS were analyzed at times indicated after IR exposure by SDS - PAGE western blotting . PARP1 LDHA are fractionation controls phospho - specific p65 signals are indicated . Representative gels from n = 3 biological replicates are shown . Right panel : quantitation of p65 phosphorylated p65 species ( n = 3 ) . Fold changes compared to untreated samples ( ut ) time points post IR are indicated on Y X axes , respectively .

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

(A) Nuclear (Nuc.) and cytoplasmic (Cyto.) fractions of U2-OS cells were analyzed at times indicated after IR exposure by SDS-PAGE western blotting. PARP1 and LDHA are fractionation controls and phospho-specific p65 signals are indicated. Representative gels from n = 3 biological replicates are shown. Right panel: quantitation of nuclear p65 and phosphorylated p65 species (n = 3). Fold changes compared to untreated samples (ut) and time points post IR are indicated on Y and X axes, respectively.


( B ) U2 - OS treated with dox to deplete endogenous p65 , were irradiated ( 20 Gy ) transfected with plasmids encoding p65 , p65 - S276A or p65 - S468A , as indicated . ( N ) ( C ) lysates were analyzed by SDS - PAGE at day seven post IR . Representative gel from n = 3 biological replicates is shown . PARP1 αTubulin serve as fractionation loading controls .

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

(B) U2-OS cells treated with dox to deplete endogenous p65, were irradiated (20 Gy) and transfected with plasmids encoding p65, p65-S276A or p65-S468A, as indicated. Nuclear (N) and cytoplasmic (C) lysates were analyzed by SDS-PAGE at day seven post IR. Representative gel from n = 3 biological replicates is shown. PARP1 and αTubulin serve as fractionation and loading controls.


( C ) ChIP performed with U2 - OS irradiated ( 20 Gy ) 1 . 5h or 7 days prior to , or left untreated ( UT ) . Normalized relative enrichment of NFKBIA is shown ( relative to input two non - recruiting reference regions ) . Left panel : p65 antibody . n = 5 biological replicates with 3 technical repeats per biological replicate . Right : Antibody against p65 pSer468 . n = 2 biological replicates with three technical repeats per biological replicate . Statistical significance was determined by ANOVA with Tukey multiple comparisons . SD shown . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 .

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

(C) ChIP performed with U2-OS cells irradiated (20 Gy) 1.5h or 7 days prior to assay, or left untreated (UT). Normalized relative enrichment of NFKBIA is shown (relative to input and two non-recruiting reference regions). Left panel: p65 antibody. n = 5 biological replicates with 3 technical repeats per biological replicate. Right: Antibody against p65 pSer468. n = 2 biological replicates with three technical repeats per biological replicate. Statistical significance was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = p < 0.05, ** = p < 0.01, *** = p < 0.001


( D ) Kinase proteome array was performed with fractions of U2 - OS that were either left untreated ( UT ) or irradiated ( 20 Gy ) at time points indicated prior to harvest . Kinase binding to was quantitated ( n = 2 ) only significant results ( p < 0 . 05 ) are shown . Significance was determined by ANOVA with Tukey multiple comparisons .

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

(D) Kinase proteome array was performed with nuclear fractions of U2-OS cells that were either left untreated (UT) or irradiated (20 Gy) at time points indicated prior to harvest. Kinase binding to membrane was quantitated (n = 2) and only significant results (p < 0.05) are shown. Significance was determined by ANOVA with Tukey multiple comparisons test.


( E ) U2 - OS left untreated ( ut ) or irradiated ( 20 Gy ) 7 days before harvesting , as indicated . Two days prior to harvest were transfected with siRNA against GSK3β or scrambled control ( Scr ) . Whole lysates were analyzed by SDS - PAGE western blotting . A representative example from n = 3 experiments is shown .

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

(E) U2-OS cells left untreated (ut) or irradiated (20 Gy) 7 days before harvesting, as indicated. Two days prior to harvest cells were transfected with siRNA against GSK3β or scrambled control (Scr). Whole cell lysates were analyzed by SDS-PAGE and western blotting. A representative example from n = 3 experiments is shown.


( F ) U2 - OS were left untreated or exposed overnight to 10 mM LiCl , with or without prior irradiation , as indicated , analyzed as in ( E ) . Representative data from n = 3 experiments are shown .

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

(F) U2-OS cells were left untreated or exposed overnight to 10 mM LiCl, with or without prior irradiation, as indicated, and analyzed as in (E). Representative data from n = 3 experiments are shown.


( G ) RT - qPCR of U2 - OS irradiated ( 20 Gy ) either 1 . 5h or 7 days prior to harvest . CHIR denotes treatment with GSK3 inhibitor CHIR - 99021 ( 10nM overnight ) . DMSO overnight treatment was used as control . Quantitation was performed statistical significance obtained from n = 3 replicates , using ANOVA with Tukey multiple comparisons . SD shown . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR of U2-OS cells irradiated (20 Gy) either 1.5h or 7 days prior to harvest. CHIR denotes treatment with GSK3 inhibitor CHIR-99021 (10nM overnight). DMSO overnight treatment was used as control. Quantitation was performed and statistical significance obtained from n = 3 replicates, using ANOVA with Tukey multiple comparisons test. SD shown. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.


( B ) SDS - PAGE western blot of whole lysates of U2 - OS irradiated ( 20 Gy ) at time points indicated prior to harvest . ut , untreated . LDHA . Representative gel shown from n = 4 biological replicates .

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

(B) SDS-PAGE western blot of whole cell lysates of U2-OS cells irradiated (20 Gy) at time points indicated prior to harvest. ut, untreated. LDHA. Representative gel shown from n = 4 biological replicates.


( C ) U2 - OS transfected with TRAF6 siRNA or scrambled control siRNA TRAF6 IKKβ analyzed by western blotting ( top panels ) . NF - κB activity was determined by EMSA in untreated ( ut ) after IR at indicated time points ( lower panel ) . ns , unspecific band . Representative gels are shown from n = 2 biological replicates .

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

(C) U2-OS cells transfected with TRAF6 siRNA or scrambled control siRNA and TRAF6 and IKKβ levels analyzed by western blotting (top panels). NF-κB activity was determined by EMSA in untreated cells (ut) and after IR at indicated time points (lower panel). ns, unspecific band. Representative gels are shown from n = 2 biological replicates.


( D ) U2 - OS were exposed to IR at indicated time points prior to harvest analyzed by western blotting for of IκBα , NIK , IL - 1α PARP1 . Treatment with proteasomal inhibitor MG132 ( 10 μM ) started 4 hours prior to harvest . NIK serves as positive control for efficient proteasome inhibition . Quantitation from n = 4 biological replicates SD is shown for IκBα ( right panel ) . Brown - Forsynthe ANOVA : p = 0 . 0005 .

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

(D) U2-OS cells were exposed to IR at indicated time points prior to harvest and analyzed by western blotting for levels of IκBα, NIK, IL-1α and PARP1. Treatment with proteasomal inhibitor MG132 (10 μM) started 4 hours prior to harvest. NIK serves as positive control for efficient proteasome inhibition. Quantitation from n = 4 biological replicates and SD is shown for IκBα (right panel). Brown-Forsynthe ANOVA test: p = 0.0005.


( E ) U2 - OS CRISPR IKBKB knockout control lines were irradiated ( 20 Gy ) harvested at indicated time points . NF - κB activity was analyzed by EMSA ( lower panel ) . ns indicates an non - specific band . Upper panel , western blot of actin IKKβ . Representative gels shown from n = 3 replicates .

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

(E) U2-OS CRISPR IKBKB knockout and control cell lines were irradiated (20 Gy) and harvested at indicated time points. NF-κB activity was analyzed by EMSA (lower panel). ns indicates an non-specific band. Upper panel, western blot analysis of actin and IKKβ. Representative gels shown from n = 3 replicates.


( F ) Irradiated U2 - O2 control IKBKB knockout ( as above ) were analyzed by RT - qPCR at time points indicated for expression of NF - κB target genes identified by - Seq ( Dataset EV1 ) . Significantly regulated targets , cutoff set at p = 0 . 05 ( as determined by ANOVA with Tukey multiple comparison ) , are shown .

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

(F) Irradiated U2-O2 control and IKBKB knockout cells (as above) were analyzed by RT-qPCR at the time points indicated for expression of NF-κB target genes identified by RNA-Seq (Dataset EV1). Significantly regulated targets, cutoff set at p = 0.05 (as determined by ANOVA with Tukey multiple comparison), are shown.


Display of differentially expressed genes in NeuNT tumors ( WT , TNCKO ) as volcano plot representing associated genes in WT tumors . N = 3 tumors per genotype . ( A ) Log2 Fold Change ( X - axis ) negative log10 ( P . Value ) ( Y - axis ) is displayed , blue indicating p = 0 . 05 ( Y - axis ) , points above , p < 0 . 05 . dashed red lines indicate log 2 Fold Change cutoff of - 0 . 25 0 . 25 , respectively ( X - axis ) . Green dots represent genes that display both statistical significance ( p < 0 . 05 ) fold changes - 0 . 25 0 . 25 , respectively . Selected genes regulated by TNC are marked in red ( type I interferon response ) or pink ( Cxcl12 ) dot .

O O O B-EXP_ASSAY O O B-DISEASE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O

Display of differentially expressed genes in NeuNT tumors (WT, TNCKO) as volcano plot representing all associated genes in WT tumors. N = 3 tumors per genotype. (A) Log2 Fold Change (X-axis) and the negative log10 (P.Value) (Y-axis) is displayed, the blue line indicating p = 0.05 (Y-axis), points above the line, p < 0.05. The dashed red lines indicate the log 2 Fold Change cutoff of -0.25 and 0.25, respectively (X-axis). Green dots represent genes that display both statistical significance (p < 0.05) and fold changes -0.25 and 0.25, respectively. Selected genes regulated by TNC are marked in red (type I interferon response) or pink (Cxcl12) dot.


( A ) Representative ( N = 5 ) of tissue from TNC high ( WT / shC ) TNC low ( KO / shTNC ) tumors ( N = 5 per condition ) with indicated antibodies . Arrows point at respective immune subtype . Scale bar , 50 µm .

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

(A) Representative staining images (N = 5) of tissue from TNC high (WT/shC) and TNC low (KO/shTNC) tumors (N = 5 per condition) with the indicated antibodies. Arrows point at the respective immune subtype. Scale bar, 50 µm.


( D , E ) Apoptosis measurement upon for cl . casp3 ( D ) signal per field ( E ) in TNC high TNC low tumors ( N = 7 per condition ) , 2 sections per , 5 random fields . * * * p = 0 . 0006 , Mann - Whitney , Mean ± . Scale bar , 50 µm .

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

(D, E) Apoptosis measurement upon tumor staining for cl. casp3 (D) and signal quantification per field (E) in TNC high and TNC low tumors (N = 7 per condition), 2 sections per tumor, 5 random fields. *** p = 0.0006, Mann-Whitney test, Mean ± SEM. Scale bar, 50 µm.


( I , J ) Detection ( I ) ( J ) of CD8 TIL in lung metastasis of NeuNT WT TNCKO mice ( N = 3 mice , n = 7 12 lung metastases , respectively ) . Scale bar , 100 µm . * * p = 0 . 0097 , Mann Whitney . Mean ± .

O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-DISEASE I-DISEASE O B-DISEASE O B-GENEPROD B-ORGANISM O O O O B-ORGANISM O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O

(I, J) Detection (I) and quantification (J) of CD8 TIL in lung metastasis of NeuNT WT and TNCKO mice (N = 3 mice, n = 7 and 12 lung metastases, respectively). Scale bar, 100 µm. ** p = 0.0097, Mann Whitney test. Mean ± SEM.


( E ) of Cxcl12 ( qRT - PCR ) in upon pretreatment with indicated inhibitors for 45 minutes ( GW , BOP , DMSO PBS as control respectively ) or 60 minutes ( Sunitinib ) 6 hours ( Cli95 ) ( 6 hours DMSO as control ) before incubation with TNC for 24 hours . N = 6 independent experiments , * * p = 0 . 0022 , Mann Whitney . Mean ± .

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

(E) Analysis of Cxcl12 (qRT-PCR) in tumor cells upon pretreatment with the indicated inhibitors for 45 minutes (GW, BOP, DMSO and PBS as control respectively) or 60 minutes (Sunitinib) and 6 hours (Cli95) (6 hours DMSO as control) before incubation with TNC for 24 hours. N = 6 independent experiments, ** p = 0.0022, Mann Whitney test. Mean ± SEM.


CXCL12 binding to TNC as determined by negative EM imaging ( G ) , followed by ( H ) . ( G ) scale bars , 100 nm ( left ) , 50 nm ( right ) , arrowheads point at gold labeled CXCL12 asterisk points at fibrinogen globe of murine TNC monomer . ( H ) Representation of TNC monomer , oligomerization domain ( triangle ) to form hexamers as seen in ( G ) , FNIII repeats ( grey boxes , contant domains , boxes , alternative domains ) , fibrinogen like domain ( circle ) . Representative result ( 3 independent experiments ) .

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

CXCL12 binding to TNC as determined by negative EM imaging (G), followed by quantification (H). (G) scale bars, 100 nm (left), 50 nm (right), the arrowheads point at gold labeled CXCL12 and the asterisk points at the fibrinogen globe of the murine TNC monomer. (H) Representation of TNC monomer, oligomerization domain (triangle) to form hexamers as seen in (G), FNIII repeats (grey boxes, contant domains, white boxes, alternative domains), fibrinogen like domain (circle). Representative result (3 independent experiments).


Attraction retention were determined towards coatings with collagen I ( COL ) , fibronectin ( FN ) or TNC in presence of CXCL10 , CCL21 , CXCL12 ( B , C ) 3 independent experiments in duplicates , Mann - Whitney , ns > 0 . 05 ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 005 . Mean ± .

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Attraction and retention were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CXCL10, CCL21, CXCL12 (B, C) 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM.


( B , C ) Imaging signal upon for cl . casp3 DAPI in PBS AMD treated tumors , N = 5 tumors per condition , 2 sections per , 5 random fields , * * p = 0 . 0079 , Mann - Whitney . Mean ± . Scale bar , 50µm , arrows point at cl . Casp3 .

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

(B, C) Imaging and signal quantification upon staining for cl. casp3 and DAPI in PBS and AMD treated tumors, N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50µm, arrows point at cl. Casp3.


( D , E ) CD8 TIL abundance localization in PBS AMD treated tumors , arrow points at CD8 TIL , scale bar , 50µm ( D ) . ( E ) of CD8 T per randomly chosen fields . N = 5 tumors per condition , 2 sections per , 5 random fields , * * p = 0 . 0079 , Mann - Whitney . Mean ± . ( F ) Gzmb in PBS AMD treated tumors . N = 10 tumors , * * * * p < 0 . 0001 , unpaired t - . Mean ± . (

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

(D, E) CD8 TIL abundance and localization in PBS and AMD treated tumors, arrow points at CD8 TIL, scale bar, 50µm (D). (E) Quantification of CD8 T cells per randomly chosen fields. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. (F) Gzmb levels in PBS and AMD treated tumors. N = 10 tumors, **** p < 0.0001, unpaired t-test. Mean ± SEM. (


( I , J ) Abundance of F4 / 80 + macrophages in PBS AMD treated tumors , ( I ) for indicated molecules , ( arrow , macrophages in stroma ; asterisk , macrophages in nest ) , ( J ) of F4 / 80 + inside nest . N = 5 tumors per condition , 2 sections per , 5 random fields , * * p = 0 . 0079 , Mann - Whitney . Mean ± . Scale bar , 50 µm .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-CELL_TYPE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-CELL_TYPE O B-TISSUE O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I, J) Abundance and quantification of F4/80+ macrophages in PBS and AMD treated tumors, (I) staining for the indicated molecules, (arrow, macrophages in the stroma; asterisk, macrophages in the tumor nest), (J) quantification of F4/80+ cells inside the tumor nest. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50 µm.


( I - L ) qRT - PCR assessment of Neu ( I ) , Cd8a ( J ) , Ifnγ ( K ) Gzmb ( L ) by qRT - PCR . PBS ( N = 16 ) AMD ( N = 19 ) treated mice , * , p = 0 . 0364 , Mann whitney ( I ) , * * , p = 0 . 0086 unpaired t - ( J ) , * * , p = 0 . 0055 , unpaired t - ( K ) , * * * , p = 0 . 0003 unpaired t - ( L ) , respectively . central band represents median , ends of box first third quartiles , whiskers reach from each quartile to minimum or maximum .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L) qRT-PCR assessment of Neu (I), Cd8a (J), Ifnγ (K) and Gzmb (L) by qRT-PCR. PBS (N = 16) and AMD (N = 19) treated mice, *, p = 0.0364, Mann whitney test (I), **, p = 0.0086 unpaired t-test (J), **, p = 0.0055, unpaired t-test (K), ***, p = 0.0003 unpaired t-test (L), respectively. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum.


( A - C ) Adjacent human breast cancer tissue microarray ( TMA ) sections were stained with indicated antibodies , scale bar , 50 μm , arrows point at CD8 TIL ( A ) , CD8 TIL were counted per field ( B ) , or in stroma nest , respectively ( C ) , N = 103 TNC + tumors , N = 116 TNC - tumors . . ( B ) * * p = 0 . 0055 , Fisher ' s exact . central band represents median , ends of box first third quartiles , whiskers reach from each quartile to minimum or maximum . ( C ) * * p = 0 , 0055 , Mann - Whitney .

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

(A-C) Adjacent human breast cancer tissue microarray (TMA) sections were stained with the indicated antibodies, scale bar, 50 μm, arrows point at CD8 TIL (A) and, CD8 TIL were counted per field (B), or in the stroma and nest, respectively (C), N = 103 TNC+ tumors, N = 116 TNC- tumors.. (B) ** p = 0.0055, Fisher's exact test. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum. (C) ** p = 0,0055, Mann-Whitney test.


( D - G ) Kaplan Meier to address correlation of TNC expression ( D ) , total numbers of CD8 TIL ( E ) , combined high ( above median ) CD8 TIL high or low ( above / below median ) TNC ( F ) stromal CD8 ( G ) with metastasis - free survival ( MFS ) of breast cancer . Stromal CD8 - or CD8 + is defined as below or above median stromal CD8 TIL per . Log - rank .

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

(D-G) Kaplan Meier analysis to address correlation of TNC expression (D), total numbers of CD8 TIL (E), combined high (above median) CD8 TIL and high or low (above/below median) TNC (F) and stromal CD8 cells (G) with metastasis-free survival (MFS) of breast cancer patients. Stromal CD8- or CD8+ is defined as below or above median stromal CD8 TIL per area. Log-rank test.


( H , I ) Correlation of TNC CD8a with overall survival ( OS ) using survival on breast cancer cohort GSE 19783 - GPL6580 . Hazard ( HR ) p values are indicated in Appendix Fig . S5N . Log - rank .

O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O B-DISEASE I-DISEASE 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) Correlation of TNC and CD8a levels with overall survival (OS) using survival analysis on breast cancer patient cohort GSE 19783-GPL6580. Hazard ratio (HR) and p values are indicated in Appendix Fig. S5N. Log-rank test.


( A ) Representative of CXCL12 pattern in TMA . N = 219 tumors . Upper panel , scale bar , 400 μm ; lower panel , scale bar , 100 μm . Arrows point at punctate in stroma .

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(A) Representative IHC images of CXCL12 intensity and staining pattern in the TMA. N = 219 tumors. Upper panel, scale bar, 400 μm; lower panel, scale bar, 100 μm. Arrows point at punctate staining in the stroma.


( D ) Kaplan Meier survival of breast cancer upon stratification into tumors with a CXCL12 Hscore below or above median , p = 0 . 002 . Mann - Whitney . ( E ) Survival of breast cancer in GSE 19783 - GPL6580 cohort upon stratification into tumors with high or low expression of TNC CXCL12 . Log - rank . HR p values are indicated ,

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(D) Kaplan Meier survival analysis of breast cancer patients upon stratification into tumors with a CXCL12 Hscore below or above the median, p = 0.002. Mann-Whitney test. (E) Survival analysis of breast cancer patients in the GSE 19783-GPL6580 cohort upon stratification into tumors with high or low expression of TNC and CXCL12. Log-rank test. HR and p values are indicated,


A . Ribbon representation of SARS - CoV - 2 N - NTD molecules in one asymmetric unit . four molecules are highlighted with different color

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A. Ribbon representation of SARS-CoV-2 N-NTD molecules in one asymmetric unit. The four molecules are highlighted with different color respectively


Naïve CD4 + T were isolated from spleen LNs of WT , Stim2fl / fl Cd4Cre ( S2 ) , Orai1fl / fl Cd4Cre ( O1 ) Stim1fl / fl Cd4Cre ( S1 ) mice differentiated into Th1 , Th17 iTreg . ( A ) Fura - 2 loaded were stimulated with 0 . 3 μM ionomycin to induce SOCE . Representative Ca2 + traces ( left ) peak Ca2 + ( right ) . Data are from mean ± of 4 mice per group .

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

Naïve CD4+ T cells were isolated from the spleen and LNs of WT, Stim2fl/fl Cd4Cre (S2), Orai1fl/fl Cd4Cre (O1) and Stim1fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17 and iTreg cells. (A) Fura-2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca2+ traces (left) and peak Ca2+ levels (right). Data are from the mean ± SEM of 4 mice per group.


( B ) Induction of colitis in Rag1 - / - mice by adoptive transfer of naive CD4 + T from wildtype , Stim2fl / fl Cd4Cre , Orai1fl / fl Cd4Cre Stim1fl / fl Cd4Cre mice . curves of recipient Rag1 - / - mice following injection of naive CD4 + T . Data are from two independent experiments with 8 recipient mice per group .

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(B) Induction of colitis in Rag1-/- mice by adoptive transfer of naive CD4+ T cells from wildtype, Stim2fl/fl Cd4Cre, Orai1fl/fl Cd4Cre and Stim1fl/fl Cd4Cre mice. Weight curves of recipient Rag1-/- mice following injection of naive CD4+ T cells. Data are from two independent experiments with 8 recipient mice per group.


( C ) H & E stains of colon tissues histological inflammation scores of Rag1 - / - mice 8 after T transfer . Data are from one representative experiment with 5 mice per group .

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(C) H&E stains of colon tissues and histological inflammation scores of Rag1-/- mice 8 weeks after T cell transfer. Data are from one representative experiment with 5 mice per group.


( D - E ) Representative flow cytometry plots frequencies of donor CD4 + T producing IFNγ , IL - 17A , TNF or expressing Foxp3 that were isolated from mesenteric LNs of recipient Rag1 - / - mice . Data are from 2 independent experiments with 3 - 8 mice per group .

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

(D-E) Representative flow cytometry plots and frequencies of donor CD4+ T cells producing IFNγ, IL-17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1-/- mice. Data are from 2 independent experiments with 3-8 mice per group.


( A , B ) Ca2 + influx in human lamina propria CD4 + CD8 + T from 3 IBD samples analyzed by flow cytometry . ( A ) Experimental design . ( B ) Ca2 + influx rates in T pre - incubated for 4 h in presence of 15 - 1000 nM BTP2 , which was present until data acquisition . SOCE was induced by stimulation with sarco - endoplasmic reticulum Ca2 + ATPase ( SERCA ) inhibitor thapsigargin ( TG ) in Ca2 + free buffer followed by addition of 2 mM Ca2 + . Bar graphs show mean ± of Ca2 + influx rates after Ca2 + readdition from 1 experiment ( n = 3 IBD ) .

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

(A,B) Ca2+ influx in human lamina propria CD4+ and CD8+ T cells from 3 IBD patient samples analyzed by flow cytometry. (A) Experimental design. (B) Ca2+ influx rates in T cells pre-incubated for 4 h in the presence of 15-1000 nM BTP2, which was present until data acquisition. SOCE was induced by stimulation with the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin (TG) in Ca2+ free buffer followed by the addition of 2 mM Ca2+. Bar graphs show the mean ± SEM of Ca2+ influx rates after Ca2+ readdition from 1 experiment (n=3 IBD patients).


( D ) viSNE plots of CD45 + CD3 + LPMCs stimulated with 20 ng / ml PMA 1 µg / ml ionomycin for 4 hours in presence of 15 - 1000 nM BTP2 . viSNE plots are colored according to expression level of IL - 2 ( blue : low , red : high ) are representative of one CD .

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(D) viSNE plots of CD45+CD3+ LPMCs stimulated with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 hours in the presence of 15-1000 nM BTP2. viSNE plots are colored according to the expression level of IL-2 (blue: low, red: high) and are representative of one CD patient.


( E ) Dose - response curves of frequencies of cytokine producing CD4 + CD8 + T after PMA / ionomycin stimulation for 4h in presence of increasing doses of BTP2 . frequencies of cytokine producing were normalized to control samples treated with PMA / ionomycin alone . Bar graphs show mean ± of 5 CD samples .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-DISEASE O O

(E) Dose-response curves of the frequencies of cytokine producing CD4+ and CD8+ T cells after PMA/ionomycin stimulation for 4h in the presence of increasing doses of BTP2. The frequencies of cytokine producing cells were normalized to control samples treated with PMA/ionomycin alone. Bar graphs show the mean ± SEM of 5 CD patient samples.


( F ) Heatmaps representing median fold change of cytokine surface marker expression in CD45 + CD3 + LPMCs stimulated for 4h with PMA / ionomycin in presence of BTP2 ; data are normalized to PMA / ionomycin treatment alone .

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(F) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45+CD3+ LPMCs stimulated for 4h with PMA/ionomycin in the presence of BTP2; data are normalized to PMA/ionomycin treatment alone.


( G ) Cytokine production by CD19 + HLA - DR + B CD11c + dendritic in presence of increasing doses ( 15 - 1 , 000 nM ) of BTP2 . Left : Dose - response curves showing frequencies of cytokine - producing after 4h stimulation with PMA / ionomycin ex vivo . Data are normalized to control samples stimulated with PMA / ionomycin in absence of BTP2 . Error bars represent obtained from 1 experiment ( n = 5 CD ) . Right : heatmaps showing median fold change of cytokines surface markers in CD19 + HLA - DR + B CD11c + dendritic treated as described for left panel .

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

(G) Cytokine production by CD19+HLA-DR+ B cells and CD11c+ dendritic cells in the presence of increasing doses (15-1,000 nM) of BTP2. Left: Dose-response curves showing frequencies of cytokine-producing cells after 4h stimulation with PMA/ionomycin ex vivo. Data are normalized to control samples stimulated with PMA/ionomycin in the absence of BTP2. Error bars represent SEM obtained from 1 experiment (n=5 CD patients). Right: heatmaps showing the median fold change of cytokines and surface markers in CD19+HLA-DR+ B cells and CD11c+ dendritic cells treated as described for the left panel.


( A ) Experimental setup for mass cytometric assays . Non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 . ( B ) FlowSOM plot of merged FCS files from LPMCs of non - inflamed controls , UC or CD after stimulation with PMA / ionomycin ± 1 μM BTP2 for 4h ex vivo . Colors of t - SNE plot represent 20 clusters of distinct CD45 + LPMC lineages . shows expression of 21 markers used for defining clusters .

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

(A) Experimental setup for mass cytometric assays. Non-inflamed: n = 4, CD: n =6, UC: n =6. (B) FlowSOM plot of merged FCS files from LPMCs of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4h ex vivo. Colors of the t-SNE plot represent 20 clusters of distinct CD45+ LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters.


( C ) FlowSOM map of merged FCS files from CD45 + of non - inflamed controls , UC CD that were left unstimulated or treated with PMA / ionomycin ± 1 μM BTP2 . Colors indicate altered clusters in CD vs . controls , UC vs . controls CD vs . UC . ( D ) viSNE plots of one exemplary non - inflamed control , UC CD colored by marker expression ( blue : low , red : high ) .

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

(C) FlowSOM map of merged FCS files from CD45+ cells of non-inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. (D) viSNE plots of one exemplary non-inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high).


( E ) Quantified frequencies ( % ) of each subset defined by cluster . Statistical significance was calculated using edgeR statistical framework with negative binomial GLM a false discovery rate adjusted to 10 % using Benjiamini - Hochberg procedure ( * padj < 0 . 05 , * * padj < 0 . 01 , * * * padj < 0 . 001 ) . Boxes extend from 25th to 75th percentiles . Whisker plots show min ( smallest ) max ( largest ) values . in box denotes median from 1 experiment ( non - inflamed n = 4 , CD : n = 6 , UC : n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-DISEASE O O O O O B-DISEASE O O O O O O

(E) Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure (*padj< 0.05, **padj < 0.01, ***padj< 0.001). Boxes extend from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median from 1 experiment (non-inflamed n = 4, CD: n =6, UC: n =6 patients).


( F ) Heatmaps representing median fold change of cytokine surface marker expression in CD45 + LPMCs that were isolated from non - inflamed , UC CD samples activated for 4h with PMA / ionomycin ex vivo . Statistical significance was calculated using an unpaired t with FDR adjustment to 1 % using Benjamini , Krieger Yekutieli procedure ; *

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

(F) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45+ LPMCs that were isolated from non-inflamed, UC and CD samples and activated for 4h with PMA/ionomycin ex vivo. Statistical significance was calculated using an unpaired t test with FDR adjustment to 1% using the Benjamini, Krieger and Yekutieli procedure; *


( A ) FlowSOM plot of merged FCS files from samples treated with PMA / ionomycin ± 1 µM BTP2 ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) . Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 + LPMCs . Heatmaps show expression of 24 markers used for cluster determination . table shows mean ± of frequencies ( % ) for each subset defined in non - inflamed controls , UC CD after 4h stimulation with PMA / ionomycin in vitro . Statistical significance was calculated using edgeR statistical framework with negative binomial GLM a false discovery rate adjusted to 10 % using Benjiamini - Hochberg procedure .

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

(A) FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3+ LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-inflamed controls, UC and CD patients after 4h stimulation with PMA/ionomycin in vitro. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure.


( B ) Box plots showing frequencies ( % ) of each subset defined by cluster in non - inflamed , UC CD samples following stimulation with PMA / ionomycin ± BTP2 for 4h in vitro . Statistical significances were calculated using a 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 ) .

O O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O B-DISEASE O O O O O O

(B) Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non-inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4h in vitro. Statistical significances were calculated using a 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 cytokines 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 cytokines 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 molecules in CD45 + CD3 + LPMCs stimulated 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 molecules in CD45+CD3+ LPMCs stimulated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.


( A ) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA / ionomycin ± 1 µM BTP2 ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) . Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 - LPMCs . clusters showing expression of 34 markers used for cluster determination . table shows mean ± of frequencies ( % ) for each subset defined in non - Inflamed , UC CD samples after stimulation with PMA / ionomycin for 4h . Statistical significance was calculated using edgeR statistical framework with negative binomial GLM a false discovery rate adjusted to 10 % using Benjiamini - Hochberg

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(A) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3- LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-Inflamed, UC and CD samples after stimulation with PMA/ionomycin for 4h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure


( 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 ) .

(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 .

(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 .

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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 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 .

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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 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.