( f ) Volcano plot showing TMZ - induced metabolic changes ( 1 mM , 48 hours ) in A549 cells in serum starvation Shown are differences in log2 fold - change between TMZ treatment and serum starvation alone ( x - axis ) , and their statistical significance ( y - axis , adjusted p - value , t - test and Benjamini - Hochberg correction ) .

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(f) Volcano plot showing TMZ-induced metabolic changes (1 mM, 48 hours) in A549 cells in serum starvation Shown are differences in log2 fold-change between TMZ treatment and serum starvation alone (x-axis), and their statistical significance (y-axis, adjusted p-value, t-test and Benjamini-Hochberg correction).


( h ) Comparison of relative changes ( log2 fold - change ) in the abundance of 1791 putatively annotated metabolites upon TMZ treatment ( 1 mM , y - axis ) against untreated quiescence - induced A549 cells ( x - axis ) . Fold - changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations ( Spearman , p < 0 . 01 ) with cell death at 96 hours are highlighted in blue and yellow , respectively .

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(h) Comparison of relative changes (log2 fold-change) in the abundance of 1791 putatively annotated metabolites upon TMZ treatment (1 mM, y-axis) against untreated quiescence-induced A549 cells (x-axis). Fold-changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations (Spearman, p < 0.01) with cell death at 96 hours are highlighted in blue and yellow, respectively.


( i ) Fractions of early apoptotic cells in A549 or HCT116 cancer cell cultures in quiescence - inducing conditions ( serum starvation , left panels ) or full growth medium ( 5 % dFBS , right panels ) , treated with TMZ ( 0 . 4 or 1 mM ) , 5 - fluorouracil ( 5 - FU , 0 . 5 mM , positive control ) or medium ( untreated ) . Early apoptotic cells were detected using ApoTracker Green under co - staining with Hoechst 33342 ( nuclei ) and propidium iodide ( dead cells ) . Cell staining was continuously monitored in 3 - hours intervals for 96 hours using automated fluorescence microscopy in a TECAN Spark Cyto . Data points and shaded areas indicate mean ± SD over three biological replicates . Approximately 3400 and 7500 cells / well were seeded of A549 and HCT116 cells , respectively .

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(i) Fractions of early apoptotic cells in A549 or HCT116 cancer cell cultures in quiescence-inducing conditions (serum starvation, left panels) or full growth medium (5% dFBS, right panels), treated with TMZ (0.4 or 1 mM), 5-fluorouracil (5-FU, 0.5 mM, positive control) or medium (untreated). Early apoptotic cells were detected using ApoTracker Green under co-staining with Hoechst 33342 (nuclei) and propidium iodide (dead cells). Cell staining was continuously monitored in 3-hours intervals for 96 hours using automated fluorescence microscopy in a TECAN Spark Cyto. Data points and shaded areas indicate mean ± SD over three biological replicates. Approximately 3400 and 7500 cells/well were seeded of A549 and HCT116 cells, respectively.


B . Single - cell lengths were measured from time - lapse microscopy images upon beta - lactam antibiotic treatment . Initial cell length was measured in the first time frame . Elongation ends with cell length shrinkage due to bulging . The longest length of each cell across the time frames was used as the final length . Tracked intermediate cell lengths were shown over time , while initial and final lengths were shown in larger circles . Linear regression of log - scaled lengths over time showed a constant rate of exponential elongation ( solid gray line ) .

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B. Single-cell lengths were measured from time-lapse microscopy images upon beta-lactam antibiotic treatment. Initial cell length was measured in the first time frame. Elongation ends with cell length shrinkage due to bulging. The longest length of each cell across the time frames was used as the final length. Tracked intermediate cell lengths were shown over time, while initial and final lengths were shown in larger circles. Linear regression of log-scaled lengths over time showed a constant rate of exponential elongation (solid gray line).


A . Probability density distributions of final lengths of MG1655 cells exposed to carbenicillin . Individual cell lengths were measured from populations exposed to three doses of carbenicillin ( 20 , 50 , and 100 µg / ml ) at two temperatures ( 27 ° C in blue and 37 ° C in red ) . Line plots show the measured initial and final lengths over time in log scale , and the first 15 cells were picked in each dataset for presentation among n ≥ 100 of the tracked cells in each condition Probability density distributions ( ρL ) of final lengths were collected with the same number of bins within the same final cell length range ( bottom panels ) . The peak of ρL shifted to the left with increasing carbenicillin dose .

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A. Probability density distributions of final lengths of MG1655 cells exposed to carbenicillin. Individual cell lengths were measured from populations exposed to three doses of carbenicillin (20, 50, and 100 µg/ml) at two temperatures (27°C in blue and 37°C in red). Line plots show the measured initial and final lengths over time in log scale, and the first 15 cells were picked in each dataset for presentation among n ≥ 100 of the tracked cells in each condition Probability density distributions (ρL) of final lengths were collected with the same number of bins within the same final cell length range (bottom panels). The peak of ρL shifted to the left with increasing carbenicillin dose.


C . The probability of lysis increased with the filamentation length of bacteria treated with other beta - lactams . MG1655 cells were treated with cefotaxime ( 20 and 100 µg / ml ) and amoxicillin ( 6 . 25 and 25 µg / ml , shown in gray text ) at two temperatures ( 27 ° C in blue and 37 ° C in red ) .

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C. The probability of lysis increased with the filamentation length of bacteria treated with other beta-lactams. MG1655 cells were treated with cefotaxime (20 and 100 µg/ml) and amoxicillin (6.25 and 25 µg/ml, shown in gray text) at two temperatures (27 °C in blue and 37 °C in red).


D . The probability of lysis increased with the filamentation length of E . coli pathogens treated with a beta - lactam . E . coli pathogens expressing extended beta - lactam resistance were treated with amoxicillin ( 6 . 25 or 25 µg / ml , shown in gray text ) and clavulanic acid ( 50 µg / ml ) simultaneously at 37 ° C . Cells were sensitized by clavulanate acid , which inhibits beta - lactamases .

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D. The probability of lysis increased with the filamentation length of E. coli pathogens treated with a beta-lactam. E. coli pathogens expressing extended beta-lactam resistance were treated with amoxicillin (6.25 or 25 µg/ml, shown in gray text) and clavulanic acid (50 µg/ml) simultaneously at 37 °C. Cells were sensitized by clavulanate acid, which inhibits beta-lactamases.


C . Experimental measurements confirm that τE decreases with the increase of antibiotic dose and growth rates . For growth rate modulation , casamino acid at 0 . 02 % ( left panel ) and 0 . 2 % ( right panel ) were added to minimal media . Cells were first cultured without carbenicillin for 3h ( dashed line ) and then exposed to carbenicillin ( antibiotic doses are marked near each plot , µg / ml ) . The average of four technical repeats ( gray lines ) was plotted in a colored line . τE points were marked by finding the first time point at which the averaged OD became equal to or less than the OD at the start of carbenicillin exposure .

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C. Experimental measurements confirm that τE decreases with the increase of antibiotic dose and growth rates. For growth rate modulation, casamino acid at 0.02% (left panel) and 0.2% (right panel) were added to minimal media. Cells were first cultured without carbenicillin for 3h (dashed line) and then exposed to carbenicillin (antibiotic doses are marked near each plot, µg/ml). The average of four technical repeats (gray lines) was plotted in a colored line. τE points were marked by finding the first time point at which the averaged OD became equal to or less than the OD at the start of carbenicillin exposure.


B - C . Stochastic simulations and measurements of survivor length . Simulated survivor lengths were plotted using the predicted LC of lower doses of carbenicillin ( left panels ) at 2h ( B ) and 6h ( C ) . The corresponding single - cell length measurements in vitro were plotted ( right panels ) . In the simulation , only non - antibiotic treated cells were set to divide , when the cell length reaches the division length in the linear model ( Ld = aLi + b , a = 0 . 871 , b = 2 . 7 . In both simulations and measurements , cells in carbenicillin - treated conditions were further elongated than control cells at all time points . At 2h exposure , antibiotic - exposed cells showed similar lengths across antibiotic concentrations since lysis is not highly probable for short filaments . At 6h exposure , survivor lengths decreased with increasing carbenicillin dose . Independent t - tests between non - treated to treated group showed extreme significance , * * * * P < 0 . 0001 . The box represents the middle 50 % , the central line represents the median , and the whiskers represent the lower and upper quartile of the data points ( n > 300 ) .

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B-C. Stochastic simulations and measurements of survivor length. Simulated survivor lengths were plotted using the predicted LC of lower doses of carbenicillin (left panels) at 2h (B) and 6h (C). The corresponding single-cell length measurements in vitro were plotted (right panels). In the simulation, only non-antibiotic treated cells were set to divide, when the cell length reaches the division length in the linear model (Ld= aLi + b, a = 0.871, b = 2.7. In both simulations and measurements, cells in carbenicillin-treated conditions were further elongated than control cells at all time points. At 2h exposure, antibiotic-exposed cells showed similar lengths across antibiotic concentrations since lysis is not highly probable for short filaments. At 6h exposure, survivor lengths decreased with increasing carbenicillin dose. Independent t-tests between non-treated to treated group showed extreme significance, **** P < 0.0001. The box represents the middle 50%, the central line represents the median, and the whiskers represent the lower and upper quartile of the data points (n>300).


Binding of recombinant ( r ) CEACAM1 ( CC1 ) - HIS ( 10 μg / mL ) to ( A ) a panel of Gram - positive bacteria , namely Enterococcus faecalis , Enterococcus faecium , Group A Streptococcus ( GAS ) , Group B Streptococcus ( GBS ) , Group C Streptococcus ( GCS ) , Group G Streptococcus ( GGS ) , Streptococcus pneumoniae , Staphylococcus aureus , ( B ) an expanded collection of GBS isolates , with serotype and carriage of bac gene shown . rCC1 binding to live bacteria was quantified by a secondary anti - His - FITC monoclonal antibody ( mAb ) . Mean and standard deviation ( SD ) values are reported for n = 3 independent experiments . In ( B ) , GBS strains from left to right are A909 , BS39 , 515 , BS22 , H36B , BS29 , 18RS21 , BM110 , COH1 , SBL3066 , NCTC10 / 84 , SB35 , SB10 , SB20 , BS26 .

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Binding of recombinant (r)CEACAM1 (CC1)-HIS (10 μg/mL) to (A) a panel of Gram-positive bacteria, namely Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Group C Streptococcus (GCS), Group G Streptococcus (GGS), Streptococcus pneumoniae, Staphylococcus aureus, (B) an expanded collection of GBS isolates, with serotype and carriage of bac gene shown. rCC1 binding to live bacteria was quantified by a secondary anti-His-FITC monoclonal antibody (mAb). Mean and standard deviation (SD) values are reported for n = 3 independent experiments. In (B), GBS strains from left to right are A909, BS39, 515, BS22, H36B, BS29, 18RS21, BM110, COH1, SBL3066, NCTC10/84, SB35, SB10, SB20, BS26.


Western blot analysis of rCC1 - HIS ( 30 μg / mL ) binding to purified β protein , but not to control GBS surface proteins α and Rib . Panel on the left show staining of proteins after separation by SDS - PAGE .

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Western blot analysis of rCC1-HIS (30 μg/mL) binding to purified β protein, but not to control GBS surface proteins α and Rib. Panel on the left show staining of proteins after separation by SDS-PAGE.


Inhibition of β - IgSF binding to DB . CC1 by anti - CC1 - N mAb ( clone CC1 / 3 / 5 - Sab ) , but not by anti - CC1 - A1B1 mAb ( clone B3 - 17 ) . β - IgSF - biotin was coupled to SPE . Mean and SD values are reported for n = 6 replicates .

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Inhibition of β-IgSF binding to DB.CC1 by anti-CC1-N mAb (clone CC1/3/5-Sab), but not by anti-CC1-A1B1 mAb (clone B3-17). β-IgSF-biotin was coupled to SPE. Mean and SD values are reported for n = 6 replicates.


A ) Representative isothermal titration calorimetry ( ITC ) binding curves of CEACAM - N domains and β - IgSF duplicates . Experiments were performed using β - IgSF and N domains of ( r ) CEACAM1 ( CC1 ) , CEACAM3 ( CC3 ) , CEACAM5 ( CC5 ) , CEACAM6 ( CC6 ) and CEACAM8 ( CC8 ) . B ) Representative flow cytometry plots analyzing the interaction of rCC1 , CC3 , CC5 , CC6 or CC8 ( 10 μg / ml ) with GBS A909 . Fluorescence of bacteria was measured by flow cytometry . Representative data from n = 3 independent experiments .

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A) Representative isothermal titration calorimetry (ITC) binding curves of CEACAM-N domains and β-IgSF duplicates. Experiments were performed using β-IgSF and N domains of (r)CEACAM1 (CC1), CEACAM3 (CC3), CEACAM5 (CC5), CEACAM6 (CC6) and CEACAM8 (CC8). B) Representative flow cytometry plots analyzing the interaction of rCC1, CC3, CC5, CC6 or CC8 (10 μg/ml) with GBS A909. Fluorescence of bacteria was measured by flow cytometry. Representative data from n = 3 independent experiments.


Adhesion of WT , ∆ bac and ∆ bac + pLZ . bac complemented A909 strains to human CC1 - or CC5 - expressing CHO transfectants , or empty vector control cells . Mean and SD from n = 5 independent replicates is displayed .

Adhesion of WT, ∆bac and ∆bac + pLZ.bac complemented A909 strains to human CC1- or CC5-expressing CHO transfectants, or empty vector control cells. Mean and SD from n = 5 independent replicates is displayed.


Binding of rCC1 - N to dynabeads ( DB ) coated with β - IgI3 wild type ( WT ) or mutants . Mutation of β - IgI3 residues 42 , 46 and 53 lead to significant changes in CC1 - N binding capacity .

Binding of rCC1-N to dynabeads (DB) coated with β-IgI3 wild type (WT) or mutants. Mutation of β-IgI3 residues 42, 46 and 53 lead to significant changes in CC1-N binding capacity.


Binding of β - IgI3 coupled to Streptavidin - PE to DB coated with rCC1 - N wildtype or variants . Mutation of CC1 - N residues 29 , 44 , 89 , 91 and 95 lead to significant changes in β - IgI3 binding capacity .

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Binding of β-IgI3 coupled to Streptavidin-PE to DB coated with rCC1-N wildtype or variants. Mutation of CC1-N residues 29, 44, 89, 91 and 95 lead to significant changes in β-IgI3 binding capacity.


Binding of recombinant ( r ) CC1 - HIS to dynabeads ( DB ) coated with β - IgI3 , R28 - IgI3 or control . Mean and SD is shown for n = 6 independent replicates .

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Binding of recombinant (r)CC1-HIS to dynabeads (DB) coated with β-IgI3, R28-IgI3 or control. Mean and SD is shown for n = 6 independent replicates.


Binding of β - IgI3 , R28 - IgI3 or human serum albumin ( HSA ) coupled to PE - conjugated streptavidin ( SPE ) , to DB coated with CC1 ( DB . CC1 ) . Mean and SD is shown for n = 6 independent replicates .

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Binding of β-IgI3, R28-IgI3 or human serum albumin (HSA) coupled to PE-conjugated streptavidin (SPE), to DB coated with CC1 (DB.CC1). Mean and SD is shown for n = 6 independent replicates.


( A ) Representative images of ILEI immunohistochemistry on healthy ( upper panels ) and psoriatic ( lower panels ) skin sections , scale bar 100 µm . Inlets show a magnification of the marked regions , scale bar 50 µm .

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(A) Representative images of ILEI immunohistochemistry on healthy (upper panels) and psoriatic (lower panels) skin sections, scale bar 100 µm. Inlets show a magnification of the marked regions, scale bar 50 µm.


( B ) Distribution of ILEI protein content per keratinocyte ( n = 5000 - 15000 cells per skin sample ) in healthy and psoriatic skin . Box and whiskers plot : Central band shows median , box extends from the 25th to 75th percentiles and whiskers go from the smallest ( min ) to the largest ( max ) value .

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(B) Distribution of ILEI protein content per keratinocyte (n=5000-15000 cells per skin sample) in healthy and psoriatic skin. Box and whiskers plot: Central band shows median, box extends from the 25th to 75th percentiles and whiskers go from the smallest (min) to the largest (max) value.


( C ) Median ILEI content of keratinocytes per person shown as mean ± SEM of healthy ( n = 6 ) and psoriasis ( n = 5 ) conditions . Data information : ( C , Statistical significance was determined by Student ' s t - test and marked with asterisks ( * * p < 0 . 01 ) .

(C) Median ILEI content of keratinocytes per person shown as mean ± SEM of healthy (n=6) and psoriasis (n=5) conditions. Data information: (C, Statistical significance was determined by Student's t-test and marked with asterisks (**p<0.01).


( C ) hematoxylin - eosin staining with scale bar of 50 µm of the back skin of ILEIind and K5 - ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days ( n = 2 - 5 ; 3 independent experiments ) .

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(C) hematoxylin-eosin staining with scale bar of 50 µm of the back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days (n=2-5; 3 independent experiments).


( D ) mean epidermal thickness ± SEM of the back skin of ILEIind and K5 - ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days ( n = 2 - 5 ; 3 independent experiments ) . Statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * * p , 0 . 001 ) .

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(D) mean epidermal thickness ±SEM of the back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days (n=2-5; 3 independent experiments). Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; ***p,0.001).


( E ) Representative images of ILEI immunohistochemistry on sections of back skin of ILEIind and K5 - ILEIind mice kept on normal or doxycycline diet upon 5 days of treatment with acetone or TPA . Scale bar 50 µm . Inlets show a magnification of the marked regions , scale bar 20 µm .

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(E) Representative images of ILEI immunohistochemistry on sections of back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet upon 5 days of treatment with acetone or TPA. Scale bar 50 µm. Inlets show a magnification of the marked regions, scale bar 20 µm.


( A ) Representative images of Ki67 immunohistochemistry on back skin sections of K5 - ILEIind and ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days . Scale bar : 100 µm . ( B ) Percentage of Ki67 positive cells in the epidermis shown as mean ± SEM and quantified on samples described in panel A ( n = 1 - 5 ; 3 independent experiments ) . Data information : Statistical significance was determined by one - way ANOVA ( B at ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p , 0 . 001 ) .

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(A) Representative images of Ki67 immunohistochemistry on back skin sections of K5-ILEIind and ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days. Scale bar: 100 µm. (B) Percentage of Ki67 positive cells in the epidermis shown as mean ±SEM and quantified on samples described in panel A (n=1-5; 3 independent experiments). Data information: Statistical significance was determined by one-way ANOVA (B at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).


( C ) Immunofluorescence for Keratin 5 ( K5 ; red ) and Keratin 10 ( K10 ; green ) expression on TPA - treated skin sections of mice described in panel A . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 50 µm . Inlets show a magnification of the marked regions . Scale bar : 20 µm .

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(C) Immunofluorescence for Keratin 5 (K5; red) and Keratin 10 (K10; green) expression on TPA-treated skin sections of mice described in panel A. Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. Inlets show a magnification of the marked regions. Scale bar: 20 µm.


( D ) Keratin 16 ( K16 ) western blot analysis ( left ) and quantification ( right ) of proteins extracted from back skin of ILEIind and K5 - ILEIind mice kept on doxycycline and treated with acetone or TPA for 5 days ( n = 3 ) . Vinculin was used as loading control . Data information : Statistical significance was determined by one - way ANOVA at ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p , 0 . 001 ) .

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(D) Keratin 16 (K16) western blot analysis (left) and quantification (right) of proteins extracted from back skin of ILEIind and K5-ILEIind mice kept on doxycycline and treated with acetone or TPA for 5 days (n=3). Vinculin was used as loading control. Data information: Statistical significance was determined by one-way ANOVA at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).


( G ) Mean fold change in mRNA expression ± SEM of K5 and K10 in primary keratinocytes described in panel F ( n = 5 - 6 ; 2 independent experiments ) . Data information : Statistical significance was determined by two - way ANOVA ( G ) at ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p , 0 . 001 ) .

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(G) Mean fold change in mRNA expression ±SEM of K5 and K10 in primary keratinocytes described in panel F (n=5-6; 2 independent experiments). Data information: Statistical significance was determined by two-way ANOVA (G) at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).


Mean number ± SEM of ( C ) neutrophils ( MPO + cells ) / mm2 epidermal - dermal area and ( D ) epidermal CD8 + T - cells / cm skin section of mice described in A ( n = 3 - 5 C ) , n = 1 - 5 ( D ) ; 3 independent experiments ) . N . A . , not analyzed . Data information : statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets .

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

Mean number ±SEM of (C) neutrophils (MPO+ cells)/mm2 epidermal-dermal area and (D) epidermal CD8+ T-cells/cm skin section of mice described in A (n=3-5 C), n=1-5 (D); 3 independent experiments). N.A., not analyzed. Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.


Mean fold change ± SEM in mRNA expression of Tnfa , Il1α and Cxcl1 ( E ) in freshly sorted keratinocytes enriched for the inter - follicular epithelium ( IFE ) and hair follicles ( HF ) from acetone and TPA treated back skin in mice supplemented with doxycycline and treated with acetone or TPA for 96 hours ( n = 3 ( E ) , independent experiments ) . Data information : statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets .

Mean fold change ±SEM in mRNA expression of Tnfa, Il1α and Cxcl1 (E) in freshly sorted keratinocytes enriched for the inter-follicular epithelium (IFE) and hair follicles (HF) from acetone and TPA treated back skin in mice supplemented with doxycycline and treated with acetone or TPA for 96 hours (n=3 (E), independent experiments). Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.


( G - I ) Mean fold change ± SEM in mRNA expression of ( G ) Tnfa , ( H ) Il1α and ( I ) Cxcl1 in primary keratinocytes treated with acetone and TPA and with increasing concentrations ( 100ng / ml , 250ng / ml , 500ng / ml , 1000ng / ml ) of murine recombinant ILEI ( mILEI ) or dimerization - disabled ILEI ( mILEICA ) or with empty - vector ( empty ) for 72 hours ( n = 3 , standing for independent keratinocyte cultures from three mice ) . Data information : statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Mean fold change ±SEM in mRNA expression of (G) Tnfa, (H) Il1α and (I) Cxcl1 in primary keratinocytes treated with acetone and TPA and with increasing concentrations (100ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml) of murine recombinant ILEI (mILEI) or dimerization-disabled ILEI (mILEICA) or with empty-vector (empty) for 72 hours (n=3, standing for independent keratinocyte cultures from three mice). Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.


( A - C ) Representative images of ( A ) Phospho - STAT3 ( Tyr727 ) , ( B ) Phospho - Erk1 / 2 and ( C ) Phospho - Akt immunofluorescence on thin sections of acetone and TPA treated back skin of ILEIind and K5 - ILEIind mice kept on doxycycline diet . Scale bar , 50 µm .

(A-C) Representative images of (A) Phospho-STAT3 (Tyr727), (B) Phospho-Erk1/2 and (C) Phospho-Akt immunofluorescence on thin sections of acetone and TPA treated back skin of ILEIind and K5-ILEIind mice kept on doxycycline diet. Scale bar, 50 µm.


( E ) Mean STAT3 ( Ser727 and Tyr705 ) , Akt and Erk1 / 2 phosphorylation levels ± SEM over time in primary keratinocytes upon TPA and recombinant ILEI treatment as described in panel A ( n = 3 , standing for independent keratinocyte cultures , each a pool of isolates from two mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mean STAT3 (Ser727 and Tyr705), Akt and Erk1/2 phosphorylation levels ±SEM over time in primary keratinocytes upon TPA and recombinant ILEI treatment as described in panel A (n=3, standing for independent keratinocyte cultures, each a pool of isolates from two mice).


( F ) Western blot analysis of STAT3 ( Ser727 and Tyr705 ) , Akt and Erk1 / 2 phosphorylation levels in primary Dox - induced K5 - ILEIind keratinocytes treated with acetone or TPA for 4 hours in the presence of the inhibitors STATTIC ( 10µM ) , LY92004 ( 10µM ) and UO126 ( 10µM ) . DMSO was used as vehicle control . Vinculin was used as loading control . Lanes are from non - continuous parts of the same gel .

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

(F) Western blot analysis of STAT3 (Ser727 and Tyr705), Akt and Erk1/2 phosphorylation levels in primary Dox-induced K5-ILEIind keratinocytes treated with acetone or TPA for 4 hours in the presence of the inhibitors STATTIC (10µM), LY92004 (10µM) and UO126 (10µM). DMSO was used as vehicle control. Vinculin was used as loading control. Lanes are from non-continuous parts of the same gel.


( B ) hematoxylin - eosin staining with a scale bar of 50 µm and ( C ) mean epidermal thickness ± SEM of the back skin of ILEIfl / fl and ILEI ∆ Ep mice treated with acetone or TPA for 5 days ( n = 3 - 8 ; 3 independent experiments ) . Data information : In C statistical significance was determined one - way ANOVA with Tukey multiple comparison test ( C marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) hematoxylin-eosin staining with a scale bar of 50 µm and (C) mean epidermal thickness ±SEM of the back skin of ILEIfl/fl and ILEI∆Ep mice treated with acetone or TPA for 5 days (n=3-8; 3 independent experiments). Data information: In C statistical significance was determined one-way ANOVA with Tukey multiple comparison test (C marked with asterisks (*p<0.05; **p<0.01; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.


( F - H ) ( F ) Representative images of MPO + microabscesses and neutrophils , ( G ) mean number ± SEM of MPO - positive microabsesses / cm skin section and ( H ) mean number of neutrophils ( MPO + cells ) / mm2 epidermal - dermal area ± SEM on thin sections of acetone and TPA treated back skin of ILEIfl / fl and ILEI ∆ Ep mice ( n = 6 - 8 ( G ) and n = 2 - 8 ( H ) ; 3 independent experiments ) . Scale bar , 100 µm . Data information : In G and H , statistical significance was determined one - way ANOVA with Tukey multiple comparison test H ) or by Student ' s t - test ( G ) and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) (F) Representative images of MPO+ microabscesses and neutrophils, (G) mean number ±SEM of MPO-positive microabsesses/cm skin section and (H) mean number of neutrophils (MPO+ cells)/mm2 epidermal-dermal area ±SEM on thin sections of acetone and TPA treated back skin of ILEIfl/fl and ILEI∆Ep mice (n=6-8 (G) and n=2-8 (H); 3 independent experiments). Scale bar, 100 µm. Data information: In G and H, statistical significance was determined one-way ANOVA with Tukey multiple comparison test H) or by Student's t-test (G) and marked with asterisks (*p<0.05; **p<0.01; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.


Mean fold change ± SEM in mRNA expression of uPA and uPAR ( D ) in freshly sorted keratinocytes enriched for the interfollicular epithelium from acetone and TPA treated back skin , ( E ) in primary keratinocytes isolated from ILEIind and K5 - ILEIind mice , supplemented with doxycycline and treated with acetone or TPA for 96 hours ( n = 3 ( D ) , n = 6 ( E 1 - 2 independent experiments ) . Data information : In D , E Statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

Mean fold change ±SEM in mRNA expression of uPA and uPAR (D) in freshly sorted keratinocytes enriched for the interfollicular epithelium from acetone and TPA treated back skin, (E) in primary keratinocytes isolated from ILEIind and K5-ILEIind mice, supplemented with doxycycline and treated with acetone or TPA for 96 hours (n=3 (D), n=6 (E 1-2 independent experiments). Data information: In D,E Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


Mean fold change ± SEM in mRNA expression of uPA and uPAR ( F ) in primary wild type keratinocytes treated with acetone and TPA and with murine recombinant wild type ILEI ( mILEI ) for 8 and 96 hours n = 6 F ) ; 1 - 2 independent experiments ) . Data information : Statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

Mean fold change ±SEM in mRNA expression of uPA and uPAR (F) in primary wild type keratinocytes treated with acetone and TPA and with murine recombinant wild type ILEI (mILEI) for 8 and 96 hours n=6 F); 1-2 independent experiments). Data information: Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( L ) ILEI Western blot analysis of conditioned media harvested from primary keratinocytes of ILEIind and K5 - ILEIind mice after 48 - hours doxycycline induction and treatment with TPA and DMSO or indicated concentrations of UK371804 . Loading was normalized to cell count , numbers indicate relative intensities . Lanes are from non - continuous parts of the same gel .

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

(L) ILEI Western blot analysis of conditioned media harvested from primary keratinocytes of ILEIind and K5-ILEIind mice after 48-hours doxycycline induction and treatment with TPA and DMSO or indicated concentrations of UK371804. Loading was normalized to cell count, numbers indicate relative intensities. Lanes are from non-continuous parts of the same gel.


( B ) HEK293T cells were transfected with SFB control , SFB - tagged PR130 and SFB - B56δ , and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot ( WB ) .

O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells were transfected with SFB control, SFB-tagged PR130 and SFB-B56δ, and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot (WB).


( C ) Glutathione Sepharose beads bound with bacterially expressed recombinant GST , GST - B56δ or GST - PPP2R1A proteins were incubated with bacterially purified recombinant MBP - PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody . Interaction of B56δ - PPP2R1A was shown similarly using MBP - B56δ and GST - PPP2R1A proteins . Recombinant protein expression is shown by Coomassie staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Glutathione Sepharose beads bound with bacterially expressed recombinant GST, GST-B56δ or GST-PPP2R1A proteins were incubated with bacterially purified recombinant MBP-PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody. Interaction of B56δ-PPP2R1A was shown similarly using MBP- B56δ and GST-PPP2R1A proteins. Recombinant protein expression is shown by Coomassie staining.


( D ) HEK293T cells were transfected with SFB control , SFB - PPP2CA , SFB - PPP2CB , SFB - PPP2R1A , SFB - PPP2R1B and SFB - B56δ , and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody .

(D) HEK293T cells were transfected with SFB control, SFB- PPP2CA, SFB-PPP2CB, SFB-PPP2R1A, SFB-PPP2R1B and SFB- B56δ, and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody.


( E ) Cell lysates from HEK293T cells expressing SFB vector , SFB - B56α , SFB - B56β , SFB - B56γ , SFB - B56δ and SFB - B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody .

O O O O O O O O O O O O O O O O O O O O O O O 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) Cell lysates from HEK293T cells expressing SFB vector, SFB-B56α, SFB-B56β, SFB-B56γ, SFB-B56δ and SFB-B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody.


( G ) Cells expressing B56δ full length ( FL ) , Δ91 - 102 , L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting .

O O O O O O O O O O O O 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) Cells expressing B56δ full length (FL), Δ91-102, L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting.


( H ) Various mutants of B56δ were transfected along with myc - PPP2R1A and pulled down using streptavidin beads and interaction with myc - PPP2R1A was detected by immunoblotting .

O O O O O O O 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) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting.


( I ) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio - layer interferometry . 6xHis - tagged PPM1G and PPP2R1A were immobilized on Ni - NTA biosensors and incubated with the GST - B56δ wild type ( WT ) or Δ91 - 102 mutant at various concentrations ( 100 - 400 nm ) . Curves represent experimental trace obtained from the BLI experiments . Binding affinities ( Kd ) ( + SD ; n = 3 independent experiments ) for B56δ - PPM1G and B56δ - PPP2R1A are shown .

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

(I) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio-layer interferometry. 6xHis-tagged PPM1G and PPP2R1A were immobilized on Ni-NTA biosensors and incubated with the GST-B56δ wild type (WT) or Δ91-102 mutant at various concentrations (100-400 nm). Curves represent experimental trace obtained from the BLI experiments. Binding affinities (Kd) (+SD; n=3 independent experiments) for B56δ-PPM1G and B56δ-PPP2R1A are shown.


( A ) MCF7 and MDA - MB - 231 cells transfected with MYC - B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization . Representative images from three independent experiments are shown , Scale bars 5 µm . ( B ) Quantification of cytoplasmic to nuclear ( Cyto / Nuc ) ratio of PPM1G intensity per each cell was plotted ( n = 35 for MCF - 7 cells ; n = 25 for MDA - MB - 231 cells ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

(A) MCF7 and MDA-MB-231 cells transfected with MYC-B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization. Representative images from three independent experiments are shown, Scale bars 5 µm. (B) Quantification of cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell was plotted (n=35 for MCF-7 cells; n=25 for MDA-MB-231 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( C ) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies .

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

(C) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies.


( D ) Cells expressing either vector or SFB - B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting . Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively .

O O O O O O O O O O O O O O O O O O O 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) Cells expressing either vector or SFB-B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.


( E ) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting . Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively .

O O O O O O O O O O O O O O O O O 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) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.


( F ) Localization of PPM1G in cells expressing either empty vector ( control ) , full length B56δ ( FL ) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies . Representative images from two independent experiments are shown , Scale bars 10 µm . ( G ) Cytoplasmic to nuclear ( Cyto / Nuc ) ratio of PPM1G intensity per each cell expressing either vector , B56δ full length ( FL ) or mutant versions was quantified and plotted ( n = 10 ) . Error bars represents SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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(F) Localization of PPM1G in cells expressing either empty vector (control), full length B56δ (FL) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies. Representative images from two independent experiments are shown, Scale bars 10 µm. (G) Cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell expressing either vector, B56δ full length (FL) or mutant versions was quantified and plotted (n=10). Error bars represents SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( A ) Endogenous association of PPM1G with α - Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α - Catenin antibody .

O O O O O O 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) Endogenous association of PPM1G with α-Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α-Catenin antibody.


( B ) SFB - PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads . Interaction with α - catenin was determined by western blotting .

O O O O O O O O O O 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) SFB-PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads. Interaction with α-catenin was determined by western blotting.


( C ) In vitro phosphorylated MBP - tagged α - Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant ( D496A ) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent ( A620 nm ) . Data represents mean absorbance from three independent experiments . Error bars indicate SD , * * p < 0 . 01 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

(C) In vitro phosphorylated MBP-tagged α-Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant (D496A) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represents mean absorbance from three independent experiments. Error bars indicate SD, **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( D ) In vitro phosphate release assay was performed using wild type α - Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted , n = 3 , Error bars indicate SD ; * * p < 0 . 01 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

(D) In vitro phosphate release assay was performed using wild type α-Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted, n=3, Error bars indicate SD; **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( E ) MDA - MB - 231 breast cancer cells were transfected with vector , wild type PPM1G or catalytically inactive PPM1G D496A ( D / A ) mutant . Levels of phosphorylated α - catenin were detected by using phospho - specific antibodies .

O O O O O O O O O O O O O O O O O O O O O O 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) MDA-MB-231 breast cancer cells were transfected with vector, wild type PPM1G or catalytically inactive PPM1G D496A (D/A) mutant. Levels of phosphorylated α-catenin were detected by using phospho-specific antibodies.


( F ) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies .

(F) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies.


( G ) Cells transfected with vector , SFB - PR130 and SFB - B56δ and levels of pS641 α - catenin was detected using western blotting .

O O O O 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) Cells transfected with vector, SFB-PR130 and SFB-B56δ and levels of pS641 α-catenin was detected using western blotting.


( H ) 293T Cells transfected with either empty vector , full length B56δ or indicated mutants were lysed and the levels of phosphorylated α - catenin was determined using pS641 α - catenin specific antibody .

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(H) 293T Cells transfected with either empty vector, full length B56δ or indicated mutants were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.


( I ) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α - catenin was determined using pS641 α - catenin specific antibody .

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(I) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.


Cells were transduced with shRNAs as indicated and the effect of ( J ) B56δ expression on α - Catenin phosphorylation was assessed .

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Cells were transduced with shRNAs as indicated and the effect of (J) B56δ expression on α-Catenin phosphorylation was assessed.


, K ) Cells were transduced with shRNAs as indicated and the effect of PPM1G expression on α - Catenin phosphorylation was assessed .

, K) Cells were transduced with shRNAs as indicated and the effect of PPM1G expression on α-Catenin phosphorylation was assessed.


( L ) SFB - PPM1G and SFB - PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells . Lysates were pulled down using streptavidin beads and interaction with α - catenin was detected by western blotting using α - catenin antibody .

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(L) SFB-PPM1G and SFB-PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells. Lysates were pulled down using streptavidin beads and interaction with α-catenin was detected by western blotting using α-catenin antibody.


( M ) Control shRNA and B56δ shRNA cells were transfected with vector , MYC - PPM1G and MYC - PPM1G ΔNLS in indicated combination and pS641 α - catenin levels were determined using western blotting .

(M) Control shRNA and B56δ shRNA cells were transfected with vector, MYC-PPM1G and MYC-PPM1G ΔNLS in indicated combination and pS641 α-catenin levels were determined using western blotting.


( A ) Cells transduced with the indicated shRNAs were fixed and localization of α - catenin and β - catenin was determined by imaging with a confocal microscope after staining with antibodies against α - catenin and β - catenin . Representative images from three independent experiments are shown . Scale bars , 10 μm . ( B ) Quantification of plasma membrane ( PM ) to cytoplasmic ratio of α - catenin intensity per each cell from figure 4A was plotted ( n = 40 cells ) . Error bars indicate SD . * * * p < 0 . 001 , ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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(A) Cells transduced with the indicated shRNAs were fixed and localization of α-catenin and β-catenin was determined by imaging with a confocal microscope after staining with antibodies against α-catenin and β-catenin. Representative images from three independent experiments are shown. Scale bars, 10 μm. (B) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4A was plotted (n=40 cells). Error bars indicate SD. ***p < 0.001, (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( C ) Control , PPM1G and B56δ shRNA transduced cells were transfected with SFB - α - catenin . Cell lysates were pulled down using streptavidin beads and interaction with β - catenin was determined by western blotting .

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(C) Control, PPM1G and B56δ shRNA transduced cells were transfected with SFB-α-catenin. Cell lysates were pulled down using streptavidin beads and interaction with β-catenin was determined by western blotting.


( D ) F - Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope . Scale bars , 20 μm .

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(D) F-Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope. Scale bars, 20 μm.


E ) Localization of α - catenin and β - catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB - PPM1G and SFB - PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies . Representative images are shown , Scale bars , 10 μm . ( F ) Quantification of plasma membrane ( PM ) to cytoplasmic ratio of α - catenin intensity per each cell from figure 4F was plotted ( n = 30 cells ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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E) Localization of α-catenin and β-catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB-PPM1G and SFB-PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies. Representative images are shown, Scale bars, 10 μm. (F) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4F was plotted (n=30 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( A , B ) MDA - MB - 231 cells were transfected with vector control , PPM1G wild type , catalytically inactive PPM1G D496A mutant or α - Catenin and transwell migration assay was performed . Phase contrast microscopic images ( A ) were taken after staining the migrated cells with crystal violet and ( B ) Quantification data for migrated cells per field ( derived from average of 3 different fields ) from three independent experiments is shown . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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(A, B) MDA-MB-231 cells were transfected with vector control, PPM1G wild type, catalytically inactive PPM1G D496A mutant or α-Catenin and transwell migration assay was performed. Phase contrast microscopic images (A) were taken after staining the migrated cells with crystal violet and (B) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( C , D ) MCF - 7 cells were transfected with SFB - vector , SFB - B56δ or indicated mutants and transwell migration assay was performed . Phase contrast microscopic images ( C ) were taken after staining the migrated cells with crystal violet and ( D ) Quantification data for migrated cells per field ( derived from average of 3 different fields ) from three independent experiments is shown . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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(C, D) MCF-7 cells were transfected with SFB-vector, SFB-B56δ or indicated mutants and transwell migration assay was performed. Phase contrast microscopic images (C) were taken after staining the migrated cells with crystal violet and (D) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( E , F ) SFB - B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells . Representative images from transwell migration assay ( E ) are shown and ( F ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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(E, F) SFB-B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells. Representative images from transwell migration assay (E) are shown and (F) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( G , H ) SFB - PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells . Representative images from transwell migration assay ( G ) are shown and ( H ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

(G, H) SFB-PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells. Representative images from transwell migration assay (G) are shown and (H) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( I , J ) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC - PPM1G and MYC - PPM1G ΔNLS . Representative images from transwell migration assay ( I ) performed with these cells are shown and ( J ) quantified data for migrated cells from three independent experiments per field was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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(I, J) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC-PPM1G and MYC-PPM1G ΔNLS. Representative images from transwell migration assay (I) performed with these cells are shown and (J) quantified data for migrated cells from three independent experiments per field was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( K , L ) SFB - tagged α - catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown . Representative images from transwell migration assay ( K ) performed with these cells are shown and ( L ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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(K, L) SFB-tagged α-catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown. Representative images from transwell migration assay (K) performed with these cells are shown and (L) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


Kaplan - Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9 - hSynI - mWwox [ total n = 18 , spontaneously dead n = 6 , mice taken out for electrophysiology / electron microscopy / analysis , are shown in yellow , n = 12 ] compared to mice injected with AAV9 - hSynI - GFP ( n = 6 ) or the non - injected ( n = 8 ( P < 0 . 0001 , Log - rank Mantel - Cox test ) .

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Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hSynI-mWwox [total n=18, spontaneously dead n=6, mice taken out for electrophysiology/electron microscopy/analysis, are shown in yellow, n=12] compared to mice injected with AAV9-hSynI-GFP (n=6) or the non-injected (n=8 (P <0.0001, Log-rank Mantel-Cox test).


Immunofluorescence images showing the expression of WWOX ( red ) in brain tissue ( cortex ) at P17 from WT and Wwox null mice that were injected with AAV9 - hSynI - mWwox ( 2x1010 ) . Neurons are labelled with anti - NeuN antibody ( green ) . The square white box shows the magnified area . White arrows show the transgene expression in NeuN positive cells . Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain ( Cortex , Hippocampus and Cerebellum ) from KO mice injected with AAV - mWwox at P17 . NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV - mWwox injected mice ( n = 3 ) brains .

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Immunofluorescence images showing the expression of WWOX (red) in brain tissue (cortex) at P17 from WT and Wwox null mice that were injected with AAV9-hSynI-mWwox (2x1010). Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area. White arrows show the transgene expression in NeuN positive cells. Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain (Cortex, Hippocampus and Cerebellum) from KO mice injected with AAV-mWwox at P17. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-mWwox injected mice (n=3) brains.


Kaplan - Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9 - hWWOX [ total n = 16 , alive n = 6 , spontaneously dead n = 6 , 4 mice ( shown in yellow ) were taken out for analysis ) compared to the non - injected ( n = 8 ) ] ( P < 0 . 0001 , Log - rank Mantel - Cox test ) .

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Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hWWOX [total n=16, alive n=6, spontaneously dead n=6, 4 mice (shown in yellow) were taken out for analysis) compared to the non-injected (n=8)] (P <0.0001, Log-rank Mantel-Cox test).


D - F . Immunofluorescence images showing the expression of WWOX ( magenta ) in cortex ( D ) , Hippocampus ( E ) and Cerebellum ( F ) at P19 from WT and Wwox null mice that were injected with AAV - hWWOX ( 2x1010 ) or Wwox null alone . Neurons are labelled with anti - NeuN antibody ( green ) . The square white box shows the magnified area . Graph represents the percentage of neurons showing the transgene ( NeuN and WWOX positive ) expression at different parts ( cortex , hippocampus and cerebellum ) of the KO brain at P19 after AAV injection . NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV - hWWOX injected mice ( n = 3 ) brains .

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D-F. Immunofluorescence images showing the expression of WWOX (magenta) in cortex (D), Hippocampus (E) and Cerebellum (F) at P19 from WT and Wwox null mice that were injected with AAV-hWWOX (2x1010) or Wwox null alone. Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area.   Graph represents the percentage of neurons showing the transgene (NeuN and WWOX positive) expression at different parts (cortex, hippocampus and cerebellum) of the KO brain at P19 after AAV injection. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-hWWOX injected mice (n=3) brains.


Representative traces of cell - attached recordings performed in WT ( blue ) , KO ( red ) and KO injected with either with AAV9 - hSynI - mWwox ( KO + A - mW shown in purple ) or AAV9 - hSynI - hWWOX ( KO + A - hW shown in green ) pups at P18 - 21 days . Traces represent spontaneous neocortical activity ( action potentials shown ) . The panels represent 12 s recording with an inset presenting a zoom - in of an 0 . 5 s interval . The graph shows the recordings ( P18 - 21 ) averages over 30 neurons from WT ( n = 3 ) , 30 neurons from KO + A - mWwox ( n = 3 ) , 30 neurons from KO + A - hWWOX ( n = 3 ) , and 45 neurons from KO ( n = 3 ) .

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Representative traces of cell-attached recordings performed in WT (blue), KO (red) and KO injected with either with AAV9-hSynI-mWwox (KO+A-mW shown in purple) or AAV9-hSynI-hWWOX (KO+A-hW shown in green) pups at P18-21 days. Traces represent spontaneous neocortical activity (action potentials shown). The panels represent 12 s recording with an inset presenting a zoom-in of an 0.5 s interval.   The graph shows the recordings (P18-21) averages over 30 neurons from WT (n=3), 30 neurons from KO+A-mWwox (n=3), 30 neurons from KO+A-hWWOX (n=3), and 45 neurons from KO (n=3).


Immunofluorescence staining with anti - GFAP ( shown in green ) and anti - Iba1 ( shown in magenta ) antibodies showing the expression of astrocytes and microglia at P19 from WT , KO and KO + A - hWWOX . The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes . F , G . Graphs represents the quantification of GFAP ( F ) and Iba1 ( G ) positive cells from the images shown in E . The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group ( n = 3 per each genotype ) .

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Immunofluorescence staining with anti-GFAP (shown in green) and anti-Iba1 (shown in magenta) antibodies showing the expression of astrocytes and microglia at P19 from WT, KO and KO+A-hWWOX. The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes. F, G. Graphs represents the quantification of GFAP (F) and Iba1 (G) positive cells from the images shown in E. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group (n=3 per each genotype).


H . Immunocytochemical images showing astrocytes ( anti - GFAP ) and microglia ( anti - Iba1 ) in CA3 region of hippocampus from WT ( n = 3 ) and rescued ( KO + AAV - hWWOX ) ( n = 3 ) mice at 9 months . Magnified area is shown with square box . I , J . Graphs represents the quantification of GFAP ( I ) and Iba1 ( J ) positive cells from the images shown in H . The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections ( paraffin embedded ) from CA3 region per group ( n = 3 per each genotype ) .

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H. Immunocytochemical images showing astrocytes (anti-GFAP) and microglia (anti-Iba1) in CA3 region of hippocampus from WT (n=3) and rescued (KO+AAV-hWWOX) (n=3) mice at 9 months. Magnified area is shown with square box. I, J. Graphs represents the quantification of GFAP (I) and Iba1 (J) positive cells from the images shown in H. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections (paraffin embedded) from CA3 region per group (n=3 per each genotype).


Images of whole brain sagittal sections that were immunolabelled with anti - MBP at P17 from indicated mice ( n = 3 for each group ) . MBP staining ( shown in gray ) in cortex ( green dotted line represents the border of the cortex ) , hippocampus and cerebellum is presented ( magnified from the top panel ) .

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Images of whole brain sagittal sections that were immunolabelled with anti-MBP at P17 from indicated mice (n=3 for each group). MBP staining (shown in gray) in cortex (green dotted line represents the border of the cortex), hippocampus and cerebellum is presented (magnified from the top panel).


Sagittal section of the brain ( P17 ) tissues immunostained for CC1 ( green ) and PDGFRα ( magenta ) . Images showing increased number of matured oligodendrocytes in corpus callosum of the Wwox null after treatment with AAV - mWwox compared to Wwox null injected with AAV - GFP virus . Graph represents the quantification of CC1 and PDGFRα positive cells in corpus callosum ( area 0 . 5 mm2 ) of WT ( n = 3 ) , KO ( n = 3 ) and KO + A - mWwox ( n = 3 ) counted from three similar brain sagittal sections per mouse in each genotype .

Sagittal section of the brain (P17) tissues immunostained for CC1 (green) and PDGFRα (magenta). Images showing increased number of matured oligodendrocytes in corpus callosum of the Wwox null after treatment with AAV-mWwox compared to Wwox null injected with AAV-GFP virus.   Graph represents the quantification of CC1 and PDGFRα positive cells in corpus callosum (area 0.5 mm2) of WT (n=3), KO (n=3) and KO+A-mWwox (n=3) counted from three similar brain sagittal sections per mouse in each genotype.


Electron micrograph ( EM ) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9 - hSynI - mWwox - injected mice compared to Wwox - null mice ( KO ) at P17 . Graph showing average number of myelinated axons in corpus callosum from WT ( n = 3 ) , KO ( n = 3 ) or KO mice injected with AAV9 - hSynI - mWwox ( n = 3 ) per field of view ( FOV ) . g - ratio analysis showing reduced myelin thickness of axons in corpus callosum KO ( n = 3 ) compared to the KO + AAV - Wwox ( n = 3 ) . Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum ( total no of axons n = 350 per each group ) .

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Electron micrograph (EM) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9-hSynI-mWwox-injected mice compared to Wwox-null mice (KO) at P17.   Graph showing average number of myelinated axons in corpus callosum from WT (n=3), KO (n=3) or KO mice injected with AAV9-hSynI-mWwox (n=3) per field of view (FOV).   g-ratio analysis showing reduced myelin thickness of axons in corpus callosum KO (n=3) compared to the KO+AAV-Wwox (n=3). Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum (total no of axons n=350 per each group).


EM images showing increased number of myelinated axons in optic nerve in AAV9 - hSynI - mWwox injected mice compared to KO mice at P17 . Graph showing the average number of myelinated axons ( counted per FOV ) in optic nerve from WT ( n = 3 ) , KO ( n = 3 ) and KO + AAV - mWwox ( n = 3 ) . g - ratio ' s of myelinated axons from optic nerves showing improved myelin thickness ( less g - ratio ) after WWOX restoration ( KO + AAV - mWwox ) compared to KO alone ( n = 3 per genotype , total no of axons n = 200 per each group ) .

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EM images showing increased number of myelinated axons in optic nerve in AAV9-hSynI-mWwox injected mice compared to KO mice at P17.   Graph showing the average number of myelinated axons (counted per FOV) in optic nerve from WT (n=3), KO (n=3) and KO+AAV-mWwox(n=3).   g-ratio's of myelinated axons from optic nerves showing improved myelin thickness (less g-ratio) after WWOX restoration (KO+AAV-mWwox) compared to KO alone (n=3 per genotype, total no of axons n=200 per each group).


Representative images of open field test showing tracking pattern ( open arena ) of WT ( females , n = 7 , males n = 6 ) and AAV - mWwox - injected KO mice ( females n = 7 , males n = 5 ) at 8 - 10 weeks . Dot plot graphs represent the velocity ( B ) , total distance travelled ( C )

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Representative images of open field test showing tracking pattern (open arena) of WT (females, n=7, males n=6) and AAV-mWwox-injected KO mice (females n=7, males n=5) at 8-10 weeks. Dot plot graphs represent the velocity (B), total distance travelled (C)


A . CAF and WI - 38 cells pretreated with EVs from MDA - MB - 231 or MCF10A cells , or PBS for 48 h were unstarved ( U ) or starved ( S ) for 2 h in EBSS prior to addition of a mixture of leucine , arginine , and lysine ( LRK ) or a cocktail of essential AAs ( EAA ) . Cells were analyzed by Western blot analysis in a time course as indicated .

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A. CAF and WI-38 cells pretreated with EVs from MDA-MB-231 or MCF10A cells, or PBS for 48 h were unstarved (U) or starved (S) for 2 h in EBSS prior to addition of a mixture of leucine, arginine, and lysine (LRK) or a cocktail of essential AAs (EAA). Cells were analyzed by Western blot analysis in a time course as indicated.


B . Western blot analysis of puromycin ( Puro ) - labeled fibroblasts ( CAF , normal human mammary fibroblasts , WI - 38 , and NIH3T3 ) after 2 - h starvation and EAA add - back in the presence or absence of cycloheximide ( CHX ) . C . Western blot analysis of Puro - labeled fibroblasts cultured in growth medium .

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B. Western blot analysis of puromycin (Puro)-labeled fibroblasts (CAF, normal human mammary fibroblasts, WI-38, and NIH3T3) after 2-h starvation and EAA add-back in the presence or absence of cycloheximide (CHX). C. Western blot analysis of Puro-labeled fibroblasts cultured in growth medium.


B . TORCAR FRET reporter assay in WI - 38 or NIH3T3 cells after 2 - h starvation and EAA add - back at time 0 ( n = 3 biological replicates for each group ) . Data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( one - way ANOVA ) .

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B. TORCAR FRET reporter assay in WI-38 or NIH3T3 cells after 2-h starvation and EAA add-back at time 0 (n=3 biological replicates for each group). Data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA).


A , B . Decreases of leucine , arginine , and lysine ( A ) as well as production of ammonium ( B ) in the conditioned medium ( CM ) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS ( n = 3 biological replicates for each group ) .

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A,B. Decreases of leucine, arginine, and lysine (A) as well as production of ammonium (B) in the conditioned medium (CM) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS (n=3 biological replicates for each group).


C . Intracellular levels of leucine , arginine , and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells ( n = 3 biological replicates for each group ) .

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C. Intracellular levels of leucine, arginine, and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells (n=3 biological replicates for each group).


A . Western blot analysis of WI - 38 and NIH3T3 cells treated with EVs or PBS for 48 h .

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A. Western blot analysis of WI-38 and NIH3T3 cells treated with EVs or PBS for 48 h.


B . Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h .

B. Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h.


C . Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX .

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C. Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX.


D . Western blot analysis of NIH3T3 cells that had been transfected with a pRK5 - RAGC - HA - GST expression plasmid or control vector and treated with EVs or PBS for 48 h , and then labeled with Puro .

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D. Western blot analysis of NIH3T3 cells that had been transfected with a pRK5-RAGC-HA-GST expression plasmid or control vector and treated with EVs or PBS for 48 h, and then labeled with Puro.


E . Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h .

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E. Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h.


A . Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti - miRNAs or control for 24 h and then treated with EVs or PBS for 48 h .

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A. Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti-miRNAs or control for 24 h and then treated with EVs or PBS for 48 h.


C . Dual luciferase assay of the psiCHECK reporters containing the 3 ' UTR of RRAGC that was wild - type , with all three miR - 105 sites mutated , or with the miR - 204 site mutated . NIH3T3 cells were co - transfected with the reporter and indicated miRNA mimic ( n = 3 biological replicates for each group ) .

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C. Dual luciferase assay of the psiCHECK reporters containing the 3'UTR of RRAGC that was wild-type, with all three miR-105 sites mutated, or with the miR-204 site mutated. NIH3T3 cells were co-transfected with the reporter and indicated miRNA mimic (n=3 biological replicates for each group).


D . Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h ( n = 3 biological replicates for each group ) .

D. Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h (n=3 biological replicates for each group).


D . A heatmap showing the comparison of log2 fold change ( FC ) of the abundance of each identified 13C6 - labeled protein and its corresponding mRNA level determined by RNA - seq .

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D. A heatmap showing the comparison of log2 fold change (FC) of the abundance of each identified 13C6-labeled protein and its corresponding mRNA level determined by RNA-seq.


A , B . A breast cancer tissue array was analyzed for correlations among RagC , miR - 105 , and miR - 204 in tumor stroma by IHC / ISH - determined scores . Representative IHC and ISH images ( A ) and correlations determined by Kendall ' s tau correlation tests ( N = 24 independent tumors ; Kendall ' s tau and two - sided P value as indicated ) ( B ) are shown . Bar = 200 μm .

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A,B. A breast cancer tissue array was analyzed for correlations among RagC, miR-105, and miR-204 in tumor stroma by IHC/ISH-determined scores. Representative IHC and ISH images (A) and correlations determined by Kendall's tau correlation tests (N=24 independent tumors; Kendall's tau and two-sided P value as indicated) (B) are shown. Bar=200 μm.


C , D . Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR - 105 or empty vector were collected from NSG mice for RagC IHC ( n = 5 tumors for each group ) . The relative intensity of RagC stain in tumor stroma was quantified by ImageJ . Bar = 50 μm . In ( D ) , data are presented as mean ± SD . * P < 0 . 05 ( Student ' s t - test ) .

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C,D. Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR-105 or empty vector were collected from NSG mice for RagC IHC (n=5 tumors for each group). The relative intensity of RagC stain in tumor stroma was quantified by ImageJ. Bar=50 μm. In (D), data are presented as mean ± SD. *P<0.05 (Student's t-test).


( A ) BMDMs from WT ( Pml + / + ) and Pml - / - mice were pre - treated with zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 30 min followed by co - treatment with AT - 406 ( 1 μM ) . Cell viability was evaluated by ATP release after 18 h . Data show mean ± SD of four biological replicates . ( B ) WT and Pml - / - BMDM were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) and Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 16 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates .

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(A) BMDMs from WT (Pml+/+) and Pml-/- mice were pre-treated with zVAD (20 μM), Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 30 min followed by co-treatment with AT-406 (1 μM). Cell viability was evaluated by ATP release after 18 h. Data show mean ± SD of four biological replicates. (B) WT and Pml-/- BMDM were treated with TNF (100 ng/ml), zVAD (20 μM) and Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates.


( D ) WT and Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) , and GSK872 ( 10 μM ) , as indicated , for 8 h . Cell viability was determined by ATP assay . Data show mean ± SD of three biological replicates . ( E ) WT and Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , IKK inhibitor BMS - 345541 ( 10 mM ) and Nec - 1 , as indicated , for 6 h and then cell death was assayed . Data show mean ± SD of five biological replicates , except the Nec - 1 - containing experiment ( n = 2 ) .

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(D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates. (E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2).


( A , B ) Pml + / + and Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) ( A ) or TNF + zVAD ( Tz ) ( B ) , as in Fig . 1 , for the indicated times . Contents of RIPK1 , p - RIPK1 [ S166 ] , RIPK3 , p - RIPK3 , MLKL , and p - MLKL in cell lysates was analysed by immunoblots . Bottom panels , quantitation of p - RIPK1 [ S166 ] from three biological replicates using normalized intensity of p - RIPK1 [ S166 ] in WT cells at 4 h as 1 . Data show mean ± SD .

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(A, B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) (A) or TNF + zVAD (Tz) (B), as in Fig. 1, for the indicated times. Contents of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates was analysed by immunoblots. Bottom panels, quantitation of p-RIPK1[S166] from three biological replicates using normalized intensity of p-RIPK1[S166] in WT cells at 4 h as 1. Data show mean ± SD.


( D ) BMDMs were treated with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) for 18 h , and TNF generated was then analyzed by ELISA . Data show mean ± SD of four biological replicates . ( E ) BMDMs were treated with zVAD + AT - 406 ( 1 μM ) , with or without anti - TNF for 18 h , and cell viability quantitated . Data show mean ± SD of three biological replicates .

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(D) BMDMs were treated with zVAD (20 μM) +AT-406 (1 μM) for 18 h, and TNF generated was then analyzed by ELISA. Data show mean ± SD of four biological replicates. (E) BMDMs were treated with zVAD + AT-406 (1 μM), with or without anti-TNF for 18 h, and cell viability quantitated. Data show mean ± SD of three biological replicates.


( A ) Pml + / + and Pml - / - BMDMs were treated with zVAD for 30 min , followed by AT - 406 ( zA ) treatment at the indicated time - points , before preparing total cell lysates ( TCL ) . TCL were immunoprecipitated with anti - FADD and the amounts of p - RIPK1 [ S166 ] , RIPK1 and caspase - 8 in the precipitates and TCL were analysed . ( B ) Pml + / + and Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) as in ( A ) , and TCL were then harvested . TCL were immunoprecipitated with anti - caspase - 8 , and the contents of p - RIPK1 [ S166 ] and RIPK1 in the precipitates and TCL was analysed .

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(A) Pml+/+ and Pml-/- BMDMs were treated with zVAD for 30 min, followed by AT-406 (zA) treatment at the indicated time-points, before preparing total cell lysates (TCL). TCL were immunoprecipitated with anti-FADD and the amounts of p-RIPK1[S166], RIPK1 and caspase-8 in the precipitates and TCL were analysed. (B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) as in (A), and TCL were then harvested. TCL were immunoprecipitated with anti-caspase-8, and the contents of p-RIPK1[S166] and RIPK1 in the precipitates and TCL was analysed.


( A , B ) WT ( Pml + / + ) and Pml - / - mice ( n = 12 in each group ) were injected with mouse TNF ( 1 . 5 μg / g body weight ) , and rectal body temperature ( A ) and survival ( B ) were determined at the indicated time - points . # , the temperature of only six mice was measured at the indicated time - points . Mean ± S . E . M is shown . * P < 0 . 05 as determined by two - way ANOVA ( A ) , or by Log - rank ( Mantel - Cox ) test ( B ) .

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(A, B) WT (Pml+/+) and Pml-/- mice (n = 12 in each group) were injected with mouse TNF (1.5 μg/g body weight), and rectal body temperature (A) and survival (B) were determined at the indicated time-points. #, the temperature of only six mice was measured at the indicated time-points. Mean ± S.E.M is shown. *P < 0.05 as determined by two-way ANOVA (A), or by Log-rank (Mantel-Cox) test (B).


( A ) Pml + / + and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated timeframes . The contents of p - IKKα / β , IKKα / β , IκBα , p - MK2 and MK2 was analysed by immunoblotting . ( B ) Pml + / + and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated times , and levels of p - RIPK1 [ S321 ] , RIPK1 , p - JNK , JNK , p - ERK , ERK , p - p38 , and p38 at the indicated time points were then determined .

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(A) Pml+/+ and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated timeframes. The contents of p-IKKα/β, IKKα/β, IκBα, p-MK2 and MK2 was analysed by immunoblotting. (B) Pml+/+and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times, and levels of p-RIPK1[S321], RIPK1, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 at the indicated time points were then determined.