evaluation of S100A9 + neutrophil CD8 + T infiltration in histologic sections of human CRC tumors . of S100A9 + ( upper panel ) CD8 + T in different areas on CRC sections containing border with adjacent benign mucosa as determined by a specialized pathologist : " center " was tissue within > 200μm from border , " border " was tissue within < 100μm from border " Adjacent benign mucosa " was mucosa tissue outside < 100μm from border . Per section , identifiable border adjacent benign areas , as well as three arbitrary center areas were scored . Sections were derived from 10 .

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

evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. Quantification of S100A9+ cells (upper panel) and CD8+ T cells in different areas on CRC tumor sections containing tumor border with adjacent benign mucosa as determined by a specialized pathologist: "Tumor center" was tissue within tumor >200μm from tumor border, "Tumor border" was tissue within tumor <100μm from tumor border and "Adjacent benign mucosa" was mucosa tissue outside tumor <100μm from tumor border. Per section, all identifiable tumor border and adjacent benign areas, as well as three arbitrary tumor center areas were scored. Sections were derived from 10 individual patients.


Gene expression scores were generated as described in material methods . Boxes are lower upper quartiles with median as solid lines ; horizontal lines define minimum maximum ; dots define outliers . , Neutrophil scores in samples categorized according to CMS subtypes .

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-CELL_TYPE O O O O O O O O O

Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. , Neutrophil scores in tumor samples categorized according to CMS subtypes.


Gene expression scores were generated as described in material methods . Boxes are lower upper quartiles with median as solid lines ; horizontal lines define minimum maximum ; dots define outliers . Comparison of T gene expression scores in CMS4 tumors within two publicly available CRC gene expression datasets ( median , lower upper quartiles ; horizontal lines define minimum maximum ; dots define outliers ) . Samples are categorized according to medians ( high = > median ; low = neutrophil TGFβ gene expression scores . Upper panel : TCGA dataset . Lower panel GSE39582 dataset .

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

Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. Comparison of T cell gene expression scores in CMS4 tumors within two publicly available CRC gene expression datasets (median, lower and upper quartiles; horizontal lines define minimum and maximum; dots define outliers). Samples are categorized according to the medians (high = >median; low = neutrophil and TGFβ gene expression scores. Upper panel: TCGA dataset. Lower panel GSE39582 dataset.


Proliferation of activated blood T in vitro in co - culture with increasing ratios of CRC - derived autologous blood neutrophils . CD3 + T CD66b + neutrophils were isolated cultured individually for each . Bar graphs display relative numbers of proliferated T after three days of co - culture . Each dot per condition represents a neutrophil sample from an ( n = 13 ) . Bars represent mean + SD .

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

Proliferation of activated blood T cells in vitro in co-culture with increasing ratios of CRC patient-derived autologous blood neutrophils. CD3+ T cells and CD66b+ neutrophils were isolated and cultured individually for each patient. Bar graphs display relative numbers of proliferated T cells after three days of co-culture. Each dot per condition represents a neutrophil sample from an individual patient (n=13). Bars represent mean + SD.


Proliferation of activated blood T in vitro in co - culture with autologous blood neutrophils . were derived from both CRC ( n = 2 ) healthy volunteers ( n = 5 ) . T neutrophils were cultured at a 1 : 1 treated with vehicle ( T + Ne ) or 10µm Galunisertib ( T + Ne + TGFBRi ) . Each represents a neutrophil sample from an healthy volunteer .

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

Proliferation of activated blood T cells in vitro in co-culture with autologous blood neutrophils. Cells were derived from both CRC patients (n=2) and healthy volunteers (n=5). T cells and neutrophils were cultured at a 1:1 ratio and treated with vehicle (T + Ne) or 10µm Galunisertib (T + Ne + TGFBRi). Each line represents a neutrophil sample from an individual healthy volunteer.


Jurkat T were treated with either DMSO ( vehicle ) or with 100μM of Dieldrin for indicated times . Abundance of mRNA for indicated genes or HERVs was assessed by RT - qPCR .

B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of Dieldrin for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.


Jurkat T were treated with either DMSO ( vehicle ) or with 100μM of PMA for indicated times . Abundance of mRNA for indicated genes or HERVs was assessed by RT - qPCR .

Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of PMA for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.


Jurkat T were treated with either DMSO ( vehicle ) or with 100μM of either Dieldrin or PMA for indicated times . Abundance of mRNA for indicated genes by RT - qPCR .

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

Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of either Dieldrin or PMA for the indicated times. Abundance of mRNA for the indicated genes by RT-qPCR.


Jurkat were exposed to either DMSO , PMA in DMSO , or dieldrin in DMSO for 30 min in triplicates . - seq was then performed on cDNA libraries prepared with poly ( A ) selection : principal component on 485 genes most affected by DMSO , PMA , or Dieldrin .

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

Jurkat cells were exposed to either DMSO, PMA in DMSO, or dieldrin in DMSO for 30 min in triplicates. RNA-seq was then performed on cDNA libraries prepared with poly(A) selection: principal component analysis on the 485 genes the most affected by DMSO, PMA, or Dieldrin.


Jurkat were exposed to either DMSO , or dieldrin in DMSO for 30 min . - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of Jurkat to dieldrin . Top tracks show coverage from sequencing of poly ( A ) - enriched libraries for comparison . coverage tracks are at same scale sequencing result files are of comparable . Blue red reads ( rds ) are in opposite orientation . CAGE peaks use same color code . Reads mapping inside a series of 4686 HERVs located away ( 30Kb ) from - coding genes were quantified both in poly ( A ) - enriched ribo - depleted data . Read from each replicate were then summed plotted .

B-CELL_LINE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O 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 O O O O O O O O O O B-ORGANISM O O O O O O O O O B-CELL_LINE O B-SMALL_MOLECULE 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 B-EXP_ASSAY 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 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

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of the Jurkat cells to dieldrin. Top tracks show coverage from sequencing of poly(A)-enriched libraries for comparison. All coverage tracks are at the same scale and all the sequencing result files are of comparable size. Blue and red reads (rds) are in opposite orientation. CAGE peaks use the same color code. Reads mapping inside a series of 4686 HERVs located away (30Kb) from protein-coding genes were quantified both in the poly(A)-enriched and the ribo-depleted data. Read counts from each replicate were then summed and plotted.


Jurkat were exposed to either DMSO , or dieldrin in DMSO for 30 min . - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Distribution of regions annotated " LTR " in RepeatMasker relative to sites of divergent transcription in Jurkat treated with either DMSO or dieldrin in DMSO .

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

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Distribution of regions annotated "LTR" in RepeatMasker relative to sites of divergent transcription in Jurkat cells treated with either DMSO or dieldrin in DMSO.


Jurkat were exposed to either DMSO , or dieldrin in DMSO for 30 min . - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Jurkat T were treated with either DMSO ( vehicle ) or 100μM dieldrin for indicated times . Abundance of IL2 mRNA was assessed by RT - qPCR . Data shown are means + / - from four independent experiments . Significance ( p - value ) was estimated using two - sided student t appropriate for small sample numbers . * * * : p - value < 0 . 001 .

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

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Jurkat T cells were treated with either DMSO (vehicle) or 100μM dieldrin for the indicated times. Abundance of IL2 mRNA was assessed by RT-qPCR. Data shown are means +/- SEM from four independent experiments. Significance (p-value) was estimated using the two-sided student t test appropriate for small sample numbers. ***: p-value<0.001.


Jurkat were exposed to either DMSO , or dieldrin in DMSO for 30 min . - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Screen captures from IGV showing increased divergent transcription inside a HERV upon exposure of Jurkat to dieldrin in intergenic region between MUC21 MUC22 . Color code as in ( A ) .

B-CELL_LINE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O 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 O O O O O O O O O O O B-ORGANISM O O O B-CELL_LINE O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O O O O O

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing increased divergent transcription inside a HERV sequence upon exposure of the Jurkat cells to dieldrin in the intergenic region between MUC21 and MUC22. Color code as in (A).


A human phospho - kinase antibody array was probed with extracts from Jurkat treated with either DMSO , dieldrin , or PMA for 30 min as indicated . array processed as a western revealed by ECL . Graphic shows selected antibodies . Values shown are means + / - from two measures .

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

A human phospho-kinase antibody array was probed with extracts from Jurkat cells treated with either DMSO, dieldrin, or PMA for 30 min as indicated. The array processed as a western and revealed by ECL. Graphic shows selected antibodies. Values shown are means +/- SEM from two measures.


Jurkat were treated with either DMSO ( vehicle ) , dieldrin , or PMA for indicated times . extracts were resolved by PAGE western blotting was carried out with indicated antibodies . Nearest marker is indicated to right ( kDa ) .

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

Jurkat cells were treated with either DMSO (vehicle), dieldrin, or PMA for the indicated times. Cell extracts were resolved by PAGE and western blotting was carried out with the indicated antibodies. Nearest size marker is indicated to the right (kDa).


Jurkat were cultured in absence or in presence of indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min . Abundance of mRNA for indicated genes or HERVs was assessed by RT - qPCR .

B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.


Jurkat treated as in ( C ) were subject to fractionation as indicated in Material Method section to distinguish a fraction with mostly RNAPII S2p ( F1 ) , one with mostly histone H3 ( F3 ) , one with both RNAPII S2p histone H3 ( P ) . Fractions were resolved by PAGE , western blots were carried out with indicated antibodies .

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

Jurkat cells treated as in (C) were subject to fractionation as indicated in the Material and Method section to distinguish a fraction with mostly RNAPII S2p (F1), one with mostly histone H3 (F3), and one with both RNAPII S2p and histone H3 (P). Fractions were resolved by PAGE, and western blots were carried out with the indicated antibodies.


( A ) Anti - acetyllysine Western blot analyses total stains of E . coli extracts . extracts before after induction of His6 - MBP - GNAT expression were separated on 12 % acrylamide gels immunoblotted by using an anti - acetyllysine antibody or stained with Coomassie Dye . GNAT1 , 2 , 5 , 6 , 7 10 as well as His6 - MBP control were expressed in E . coli BL21 ( DE3 ) pLysS . GNAT3 4 were expressed in Rosetta ( DE3 ) . In total stains , recombinant GNAT constructs were highlighted by blue arrowheads . expression before ( ni ) after ( i ) induction with IPTG is indicated . luminescence signal , indicating acetylated , was usually recorded after 40 - 120 seconds . Since MBP - GNAT4 expression resulted in a saturated signal , luminescence was additionally recorded for 10 seconds ( indicated by an asterisk ) .

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

(A) Anti-acetyllysine Western blot analyses and total protein stains of E. coli cell extracts. Cellular protein extracts before and after induction of His6-MBP-GNAT expression were separated on 12 % acrylamide gels and immunoblotted by using an anti-acetyllysine antibody or stained with Coomassie Dye. GNAT1, 2, 5, 6, 7 and 10 as well as His6-MBP control were expressed in E. coli BL21(DE3)pLysS. GNAT3 and 4 were expressed in Rosetta(DE3). In the total protein stains, recombinant GNAT protein constructs were highlighted by blue arrowheads. Protein expression before (ni) and after (i) induction with IPTG is indicated. The luminescence signal, indicating acetylated proteins, was usually recorded after 40-120 seconds. Since MBP-GNAT4 expression resulted in a saturated signal, the luminescence was additionally recorded for 10 seconds (indicated by an asterisk).


( A ) Number of characterized substrates with significant increases of NTA yields . NTA yields of retrieved N - termini upon a given GNAT expression was divided into four categories each column is result of sum of categories : 50 - 100 % , green ; 20 - 50 % , green ; 10 - 20 % , orange ; 5 - 10 % , red . number of occurrence with a yield higher than 30 % is also displayed to visualize most significant acetylation status of each GNAT . respective overexpression level of each GNAT as assessed from gel electrophoresis ( + + + + is for very high expression , + + + for high , + + for medium , + for intermediate ) .

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

(A) Number of characterized substrates with significant increases of NTA yields. The NTA yields of all retrieved N-termini upon a given GNAT expression was divided into four categories and each column is the result of the sum of all categories: 50-100%, dark green; 20-50%, light green; 10-20%, orange; 5-10%, red. The number of protein occurrence with a yield higher than 30% is also displayed to visualize the most significant acetylation status of each GNAT. The respective overexpression level of each GNAT as assessed from gel electrophoresis (++++ is for very high expression, +++ for high, ++ for medium, + for intermediate).


( C ) Comparison of NTA yield of retrieved N - termini of starting at position 1 or 2 . majority of these , corresponding mostly to components , undergoing or not to N - terminal methionine excision , were not affected by inactivation of GNAT2 . For statistical analyses nsi - 1 nsi - 2 were pooled compared to wild type . Two independent technical replicates of four biological replicates for each of WT , nsi - 1 nsi - 2 samples were analysed . Error bars are ± SD . ( D ) Comparison of NTA yield of retrieved N - termini of starting at positions > 2 . Clear alteration of NTA yield was observed in nsi mutant lines in pool of - encoded plastid . nsi - 1 nsi - 2 samples were treated as in D . Error bars are ± SD ( E ) Comparison of NTA yields of retrieved N - termini in plastid . Similar variation as in panel D was observed when NTA of only plastid was analyzed .

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 B-SMALL_MOLECULE 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 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 O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O

(C) Comparison of NTA yield of retrieved N-termini of proteins starting at position 1 or 2. The majority of these proteins, corresponding mostly to cytosolic components, undergoing or not to N-terminal methionine excision, were not affected by inactivation of GNAT2. For statistical analyses nsi-1 and nsi-2 were pooled and compared to the wild type. Two independent technical replicates of four biological replicates for each of the WT, nsi-1 and nsi-2 samples were analysed. Error bars are ± SD. (D) Comparison of NTA yield of retrieved N-termini of proteins starting at positions > 2. Clear alteration of NTA yield was observed in nsi mutant lines in the pool of nuclear-encoded plastid proteins. nsi-1 and nsi-2 samples were treated as in D. Error bars are ± SD (E) Comparison of NTA yields of retrieved N-termini in plastid proteins. Similar variation as in panel D was observed when NTA of only plastid proteins was analyzed.


( F ) Volcano plot representing NTA analyses of nsi knockout lines ( treated together ) wild type . For this , p - value was calculated using Excel ' s two - t - function , for two - sample with equal variance . most impacted are shown in green . N is related to number of quantified N - termini .

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

(F) Volcano plot representing NTA analyses of nsi knockout lines (treated together) and wild type. For this analysis, the p-value was calculated using Excel's two-tailed t-test function, for two-sample with equal variance. The most impacted proteins are shown in green. N is related to the number of quantified N-termini.


A , PTPN3 promotes TGF - β - induced CAGA - luc reporter gene activity . HaCaT were transfected with expression plasmids for CAGA - luc reporter gene , Renilla - luc reporter gene PTPN3 as indicated treated with TGF - β ( 2 ng / mL , 8 h ) . Relative luciferase activity was measured Data are shown as mean ± ; n = 3 . Statistical was performed using two - Student ' s t - . * * * P < 0 . 001 .

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

A, PTPN3 promotes TGF-β-induced CAGA-luc reporter gene activity. HaCaT cells were transfected with expression plasmids for CAGA-luc reporter gene, Renilla-luc reporter gene and PTPN3 as indicated and treated with TGF-β (2 ng/mL, 8 h). Relative luciferase activity was measured Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.


, Knockdown of PTPN3 decreases TGF - β - induced CAGA - luc reporter gene activity . HaCaT were transfected with siRNA against PTPN3 or control siRNA . 24 h post - transfection , expression plasmids for CAGA - luc reporter Renilla - luc reporter were transfected as indicated . were harvested for relative luciferase after another 24 h with TGF - β treatment ( 2 ng / mL , 8 h ) . Data are shown as mean ± ; n = 3 . Statistical was performed using two - Student ' s t - . * * * P < 0 . 001 .

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

, Knockdown of PTPN3 decreases TGF-β-induced CAGA-luc reporter gene activity. HaCaT cells were transfected with siRNA against PTPN3 or control siRNA. 24 h post-transfection, expression plasmids for CAGA-luc reporter and Renilla-luc reporter were transfected as indicated. Cells were harvested for relative luciferase assay after another 24 h with TGF-β treatment (2 ng/mL, 8 h). Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.


C , Knockdown of PTPN3 blocks TGF - β - induced p21 transcription . HaCaT were transfected with two independent siRNAs against PTPN3 treated with TGF - β ( 2 ng / mL ) for 8 h before harvested . Total mRNA was analyzed by qRT - PCR using primers specific to p21 . Data are shown as mean ± ; n = 3 . Statistical was performed using two - Student ' s t - . * * P < 0 . 01 , * * * P < 0 . 001 .

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

C, Knockdown of PTPN3 blocks TGF-β-induced p21 transcription. HaCaT cells were transfected with two independent siRNAs against PTPN3 and treated with TGF-β (2 ng/mL) for 8 h before harvested. Total mRNA was analyzed by qRT-PCR using primers specific to p21. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. **P<0.01, ***P < 0.001.


D , Knockdown of PTPN3 blocks TGF - β - induced PAI - 1 gene transcription . Experiment was done with qRT - PCR primers specific to PAI - 1 . Data are shown as mean ± ; n = 3 . Statistical was performed using two - Student ' s t - . * * * P < 0 . 001 .

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D, Knockdown of PTPN3 blocks TGF-β-induced PAI-1 gene transcription. Experiment was done with qRT-PCR primers specific to PAI-1. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.


E , Knockdown of PTPN3 abolishes TGF - β - induced p21 transcription . HaCaT were transfected with siRNA then treated with TGF - β ( 2 ng / mL ) for 0 , 4 , 8 or 24 h as indicated . qRT - PCR was carried out Data are shown as mean ± ; n = 3 .

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E, Knockdown of PTPN3 abolishes TGF-β-induced p21 transcription. HaCaT cells were transfected with siRNA and then treated with TGF-β (2 ng/mL) for 0, 4, 8 or 24 h as indicated. qRT-PCR was carried out Data are shown as mean ± SEM; n = 3.


F , Knockdown of PTPN3 abolishes TGF - β - induced transcription of PAI - 1 . Experiment was done Data are shown as mean ± ; n = 3 .

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

F, Knockdown of PTPN3 abolishes TGF-β-induced transcription of PAI-1. Experiment was done Data are shown as mean ± SEM; n = 3.


G , Depletion of PTPN3 abolishes TGF - β - induced target expression . HaCaT with stable knockdown of PTPN3 were treated with TGF - β ( 2 ng / mL ) for 6 h before harvested . were harvested for Western blotting with anti - p21 , anti - PAI - 1 , anti - PTPN3 anti - β - Actin antibodies .

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G, Depletion of PTPN3 abolishes TGF-β-induced target protein expression. HaCaT cells with stable knockdown of PTPN3 were treated with TGF-β (2 ng/mL) for 6 h before harvested. Cells were harvested for Western blotting with anti-p21, anti-PAI-1, anti-PTPN3 and anti-β-Actin antibodies.


H , PTPN3 enhances TGF - β - induced target expression . HaCaT stably expressing HA - PTPN3 were stimulated with 2 ng / mL of TGF - β for 6 h then harvested for lysates . were examined by Western blotting with appropriate antibodies as indicated .

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

H, PTPN3 enhances TGF-β-induced target protein expression. HaCaT cells stably expressing HA-PTPN3 were stimulated with 2 ng/mL of TGF-β for 6 h and then harvested for cell lysates. Protein levels were examined by Western blotting with appropriate antibodies as indicated.


I , Depletion of PTPN3 attenuates TGF - β global gene responses . Venn diagram shows number of differential expression genes ( DEGs ) with TGF - β treatment in control or PTPN3 - depleted HaCaT by - Seq . overlap represents TGF - β target genes that are commonly regulated in both lines . HaCaT transfected with siControl or siPTPN3 were harvested for - Seq after treated with or without TGF - β ( 2 ng / mL , 8 h ) .

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

I, Depletion of PTPN3 attenuates TGF-β global gene responses. Venn diagram shows the number of differential expression genes (DEGs) with TGF-β treatment in control or PTPN3-depleted HaCaT cells by RNA-Seq analysis. The overlap represents TGF-β target genes that are commonly regulated in both cell lines. HaCaT cells transfected with siControl or siPTPN3 were harvested for RNA-Seq after treated with or without TGF-β (2 ng/mL, 8 h).


J , Loss of PTPN3 markedly decreases TGF - β - dependent upregulation or downregulation of target genes . Heat map of DEGs in control HaCaT ( siControl ) or HaCaT depleted of PTPN3 ( siPTPN3 ) . Log2FC data represent fold change of TGF - β ( 2 ng / mL ) treatment over vehicle for 8 h .

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J, Loss of PTPN3 markedly decreases TGF-β-dependent upregulation or downregulation of target genes. Heat map of DEGs in control HaCaT cells (siControl) or HaCaT cells depleted of PTPN3 (siPTPN3). Log2FC data represent fold change of TGF-β (2 ng/mL) treatment over vehicle for 8 h.


K , Heat map of Log2FC of select TGF - β target genes .

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K, Heat map of Log2FC of select TGF-β target genes.


B , Both PTPN3 PTPN3 ( L232R ) , but not phosphatase - dead mutants PTPN3 ( D811A ) PTPN3 ( C842S ) , dephosphorylate P - EGFR ( Y1173 ) . Wildtype PTPN3 or its mutants were co - transfected with EGFR into HEK293T as indicated . 24 h later , were harvested for Western blotting . of P - EGFR ( Y1173 ) , EGFR , PTPN3 its mutants were examined by appropriate antibodies .

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B, Both PTPN3 and PTPN3 (L232R), but not the phosphatase-dead mutants PTPN3 (D811A) and PTPN3 (C842S), dephosphorylate P-EGFR (Y1173). Wildtype PTPN3 or its mutants were co-transfected with EGFR into HEK293T cells as indicated. 24 h later, cells were harvested for Western blotting. Levels of P-EGFR (Y1173), EGFR, PTPN3 and its mutants were examined by appropriate antibodies.


C , PTPN3 PTPN3 ( D811A ) enhance TGF - β - induced target expression . HaCaT stably expressing HA - PTPN3 or HA - PTPN3 ( D811A ) were stimulated with 2 ng / mL of TGF - β for 6 h then harvested for lysates . were examined by Western blotting with appropriate antibodies as indicated .

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C, PTPN3 and PTPN3 (D811A) enhance TGF-β-induced target protein expression. HaCaT cells stably expressing HA-PTPN3 or HA-PTPN3 (D811A) were stimulated with 2 ng/mL of TGF-β for 6 h and then harvested for cell lysates. Protein levels were examined by Western blotting with appropriate antibodies as indicated.


D , PTPN3 its catalytically inactive mutants PTPN3 ( D811A ) PTPN3 ( C842S ) equally enhance TGF - β - induced reporter gene activity . HaCaT were transfected with expression plasmids for each PTPN3 variant CAGA - luc reporter Renilla - luc reporter . were harvested for relative luciferase after TGF - β ( 2 ng / mL , 8 h ) treatment . Data are shown as mean ± ; n = 3 . Statistical was performed using two - Student ' s t - . * * * P < 0 . 001 .

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

D, PTPN3 and its catalytically inactive mutants PTPN3 (D811A) and PTPN3 (C842S) equally enhance TGF-β-induced reporter gene activity. HaCaT cells were transfected with expression plasmids for each PTPN3 variant and CAGA-luc reporter and Renilla-luc reporter. Cells were harvested for relative luciferase assay after TGF-β (2 ng/mL, 8 h) treatment. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.


E , PTPN3 FERM domain enhances TGF - β - induced reporter gene activity as potently as wildtype PTPN3 . HaCaT were transfected with expression plasmids for PTPN3 truncations CAGA - luc reporter , internal control Renilla - luc reporter . were harvested for relative luciferase with TGF - β treatment ( 2 ng / mL , 8 h ) . Data are shown as mean ± ; n = 3 . Statistical was performed using two - Student ' s t - . * * P < 0 . 01 , * * * P < 0 . 001 . NS , non - significant .

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

E, PTPN3 FERM domain enhances TGF-β-induced reporter gene activity as potently as wildtype PTPN3. HaCaT cells were transfected with expression plasmids for PTPN3 truncations and CAGA-luc reporter, and internal control Renilla-luc reporter. Cells were harvested for relative luciferase assay with TGF-β treatment (2 ng/mL, 8 h). Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. **P<0.01, ***P<0.001. NS, non-significant.


A , PTPN3 PTPN3 ( D811A ) stabilize TβRI . TβRI PTPN3 were detected by Western blotting with appropriate antibodies as indicated in HaCaT stably expressing PTPN3 or PTPN3 ( D811A ) .

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A, PTPN3 and PTPN3 (D811A) stabilize TβRI. TβRI and PTPN3 were detected by Western blotting with appropriate antibodies as indicated in HaCaT cells stably expressing PTPN3 or PTPN3 (D811A).


B , shRNA PTPN3 - mediated reduction of endogenous TβRI level is restored by MG132 . HaCaT expressing shRNA PTPN3 - 1 , shRNA PTPN3 - 2 or control shRNA were treated with MG132 ( 20 μM ) for 4 h . lysates were analyzed by Western blotting with anti - TβRI , anti - PTPN3 anti - GAPDH antibodies .

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B, shRNA PTPN3-mediated reduction of endogenous TβRI level is restored by MG132. HaCaT cells expressing shRNA PTPN3-1, shRNA PTPN3-2 or control shRNA were treated with MG132 (20 μM) for 4 h. Cell lysates were analyzed by Western blotting with anti-TβRI, anti-PTPN3 and anti-GAPDH antibodies.


C , PTPN3 rescues degradation of TβRI induced by Smurf2 . HEK293T were transfected with indicated plasmids . were harvested after 24 h post - transfection . Western blotting was done with indicated antibodies .

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C, PTPN3 rescues the degradation of TβRI induced by Smurf2. HEK293T cells were transfected with the indicated plasmids. Cells were harvested after 24 h post-transfection. Western blotting was done with the indicated antibodies.


D , PTPN3 PTPN3 ( D811A ) attenuate ubiquitination of TβRI . HEK293T were transfected with indicated plasmids . 24 h after transfection , IP immunoblotting were performed with anti - FLAG antibody . of TβRI ubiquitination other were examined by Western blotting with indicated antibodies .

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D, PTPN3 and PTPN3 (D811A) attenuate ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids. 24 h after transfection, IP and immunoblotting analysis were performed with the anti-FLAG antibody. Levels of TβRI ubiquitination and other proteins were examined by Western blotting with the indicated antibodies.


E , PTPN3 blocks Smurf2 - mediated ubiquitination of TβRI . HEK293T were transfected with indicated plasmids , treated with MG132 ( 20 μM , 4 h ) before harvest . TβRI IP was performed with anti - HA antibody , TβRI ubiquitination was determined by Western blotting with anti - Myc antibody . were examined by Western blotting with indicated antibodies .

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E, PTPN3 blocks Smurf2-mediated ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids, and treated with MG132 (20 μM, 4 h) before harvest. TβRI IP was performed with the anti-HA antibody, and TβRI ubiquitination was determined by Western blotting with the anti-Myc antibody. Protein levels were examined by Western blotting with the indicated antibodies.


F , Depletion of Smurf2 reverses shPTPN3 - induced degradation of TβRI . HEK293T were transfected with expression plasmids for shPTPN3 , shSmurf2 HA - TβRI . 48 h post - transfection , were harvested for immunoblotting . TβRI level was determined by Western blotting with anti - HA antibody . of other were done by Western blotting with indicated antibodies .

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F, Depletion of Smurf2 reverses the shPTPN3-induced degradation of TβRI. HEK293T cells were transfected with expression plasmids for shPTPN3, shSmurf2 and HA-TβRI. 48 h post-transfection, cells were harvested for immunoblotting analysis. TβRI level was determined by Western blotting with the anti-HA antibody. Levels of other proteins were done by Western blotting with the indicated antibodies.


G , Dominant negative Smurf2 mutant C716A prevents shPTPN3 - induced TβRI degradation . HaCaT stably expressing shPTPN3 were transfected with FLAG - Smurf2 ( C716A ) . were harvested for Western blotting with anti - TβRI , anti - FLAG , anti - PTPN3 anti - GAPDH antibodies .

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G, Dominant negative Smurf2 mutant C716A prevents shPTPN3-induced TβRI degradation. HaCaT cells stably expressing shPTPN3 were transfected with FLAG-Smurf2 (C716A). Cells were harvested for Western blotting with anti-TβRI, anti-FLAG, anti-PTPN3 and anti-GAPDH antibodies.


A , PTPN3 interacts with TβRI at endogenous . HaCaT were harvested after TGF - β treatment ( 2 ng / mL , 1 h ) then subjected to IP by PTPN3 antibody or control IgG . PTPN3 TβRI were detected by Western blotting .

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A, PTPN3 interacts with TβRI at endogenous levels. HaCaT cells were harvested after TGF-β treatment (2 ng/mL, 1 h) and then subjected to IP by PTPN3 antibody or control IgG. PTPN3 and TβRI were detected by Western blotting.


B , Both PTPN3 PTPN3 ( D811A ) interact with TβRI . FLAG - TβRI was co - transfected with HA - PTPN3 or HA - PTPN3 ( D811A ) into HEK293T . lysates were harvested subjected to IP with FLAG antibody . PTPN3 , PTPN3 ( D811A ) TβRI were detected by Western blotting with appropriate antibodies .

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B, Both PTPN3 and PTPN3 (D811A) interact with TβRI. FLAG-TβRI was co-transfected with HA-PTPN3 or HA-PTPN3 (D811A) into HEK293T cells. Cell lysates were harvested and subjected to IP with FLAG antibody. PTPN3, PTPN3 (D811A) and TβRI were detected by Western blotting with appropriate antibodies.


C , FERM domain of PTPN3 is necessary for its binding to TβRI . FLAG - TβRI HA - PTPN3 ( wildtype or truncations ) were co - transfected into HEK293T . were harvested after 24 h subjected to IP by HA antibody . TβRI , PTPN3 PTPN3 truncations were detected by Western blotting with appropriate antibodies .

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C, The FERM domain of PTPN3 is necessary for its binding to TβRI. FLAG-TβRI and HA-PTPN3 (wildtype or truncations) were co-transfected into HEK293T cells. Cells were harvested after 24 h and subjected to IP by HA antibody. TβRI, PTPN3 and PTPN3 truncations were detected by Western blotting with appropriate antibodies.


E , Knockdown of endogenous PTPN3 promotes Smad7 - TβRI association . HEK293T were transfected with indicated plasmids treated with MG132 ( 20 µM ) for 4 h before harvested . SFB - Smad7 were retrieved with Streptavidin pulldown . of TβRI other were examined by Western blotting with indicated antibodies .

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E, Knockdown of endogenous PTPN3 promotes the Smad7-TβRI association. HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 µM) for 4 h before harvested. SFB-Smad7 proteins were retrieved with the Streptavidin pulldown. Levels of TβRI and other proteins were examined by Western blotting with the indicated antibodies.


F , PTPN3 PTPN3 ( D811A ) abolish interaction between TβRI Smurf2 in HEK293T . HEK293T were co - transfected with plasmids as indicated . 20 h later , were treated with MG132 ( 20 μM ) for 4 h , followed by IP Western blotting .

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F, PTPN3 and PTPN3 (D811A) abolish the interaction between TβRI and Smurf2 in HEK293T cells. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h, followed by IP and Western blotting.


A , PTPN3 wildtype , phosphatase - dead D811A ICC - derived mutant L232R exhibit similar patterns of subcellular localization co - localization with TβRI . HaCaT were transfected with HA - TβRI Flag - PTPN3 ( wildtype or its mutants ) . 24 h later , were harvested for immunofluorescence with indicated antibodies . Scale bar = 10 μm .

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

A, PTPN3 wildtype, phosphatase-dead D811A and ICC-derived mutant L232R exhibit similar patterns of subcellular localization and co-localization with TβRI. HaCaT cells were transfected with HA-TβRI and Flag-PTPN3 (wildtype or its mutants). 24 h later, cells were harvested for immunofluorescence with the indicated antibodies. Scale bar = 10 μm.


B , PTPN3 ( L232R ) does not increase TβRI level . TβRI PTPN3 were determined by Western blotting in HaCaT stably expressing PTPN3 , PTPN3 ( D811A ) or PTPN3 ( L232R ) .

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B, PTPN3 (L232R) does not increase the TβRI protein level. TβRI and PTPN3 were determined by Western blotting in HaCaT cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R).


C , PTPN3 ( L232R ) fails to rescue Smurf2 - mediated degradation of TβRI . HEK293T were transfected with FLAG - TβRI , Myc - Smurf2 increasing anount of PTPN3 ( wildtype mutants ) . were harvested 24 h post - transfection . were examined by Western blotting with indicated antibodies .

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C, PTPN3 (L232R) fails to rescue the Smurf2-mediated degradation of TβRI. HEK293T cells were transfected with FLAG- TβRI, Myc-Smurf2 and the increasing anount of PTPN3 (wildtype and mutants). Cells were harvested 24 h post-transfection. Protein levels were examined by Western blotting with the indicated antibodies.


D , PTPN3 ( L232R ) does not block interaction between TβRI Smurf2 . HEK293T were co - transfected with plasmids as indicated . 20 h later , were treated with MG132 ( 20 μM ) for 4 h analyzed by IP - Western blotting .

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D, PTPN3 (L232R) does not block the interaction between TβRI and Smurf2. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h and analyzed by IP-Western blotting.


E , Ectopic expression of PTPN3 wildtype PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , completely abolishes Smad7 - TβRI interaction . HEK293T were co - transfected with plasmids as indicated . 20 h later , were treated with MG132 ( 20 µM ) for 4 h , followed by IP Western blotting .

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E, Ectopic expression of PTPN3 wildtype and PTPN3 (D811A), but not PTPN3 (L232R), completely abolishes the Smad7-TβRI interaction. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 µM) for 4 h, followed by IP and Western blotting.


F , PTPN3 PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , attenuate ubiquitination of TβRI . HEK293T were transfected with indicated plasmids . 24 h after transfection , IP - Western blotting was performed with anti - Flag antibody IP followed by Western blotting with indicated antibodies .

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F, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuate ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids. 24 h after transfection, IP-Western blotting analysis was performed with anti-Flag antibody IP and followed by Western blotting with the indicated antibodies.


A , PTPN3 PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , enhance Smad2 Smad3 phosphorylation . HaCaT were stimulated with TGF - β ( 2 ng / mL ) for 1 h . of P - Smad2 , P - Smad3 , Smad2 , Smad3 , PTPN3 GAPDH were detected by Western blotting .

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A, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance Smad2 and Smad3 phosphorylation. HaCaT cells were stimulated with TGF-β (2 ng/mL) for 1 h. Levels of P-Smad2, P-Smad3, Smad2, Smad3, PTPN3 and GAPDH were detected by Western blotting.


B , PTPN3 ( L232R ) inhibits TGF - β - induced reporter gene activity . HepG2 were transfected with expression plasmids for PTPN3 , PTPN3 mutants CAGA - luc reporter , Renilla - luc reporter . were harvested for luciferase after 8 h of TGF - β ( 2 ng / mL ) treatment . Data are shown as mean ± ; n = 3 . Statistical was performed using two - Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 .

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B, PTPN3 (L232R) inhibits TGF-β-induced reporter gene activity. HepG2 cells were transfected with expression plasmids for PTPN3, PTPN3 mutants and CAGA-luc reporter, and Renilla-luc reporter. Cells were harvested for luciferase assay after 8 h of TGF-β (2 ng/mL) treatment. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. *P<0.05, **P<0.01.


C , PTPN3 PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , enhance TGF - β - induced growth inhibitory effect . HepG2 stably expressing PTPN3 , PTPN3 ( D811A ) or PTPN3 ( L232R ) were treated with or without TGF - β ( 1 ng / mL ) for indicated days . were analyzed for proliferation by MTS . Data are shown as mean ± ; n = 3 .

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C, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by MTS assay. Data are shown as mean ± SEM; n = 3.


D , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect . HepG2 stably expressing shPTPN3 were treated with or without TGF - β ( 1 ng / mL ) for indicated days . were analyzed for proliferation by using MTS . Data are shown as mean ± ; n = 3 .

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D, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing shPTPN3 were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by using MTS assay. Data are shown as mean ± SEM; n = 3.


E , PTPN3 ( L232R ) attenuates TGF - β - induced growth inhibition on colony formation in HepG2 . were treated with or without TGF - β ( 1 ng / mL ) treatment for 10 days . Clonogenic of results in clonogenic assays was analyzed by Image J . Data are shown as mean ± ; n = 3 .

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E, PTPN3 (L232R) attenuates TGF-β-induced growth inhibition on colony formation in HepG2 cells. Cells were treated with or without TGF-β (1 ng/mL) treatment for 10 days. Clonogenic assay Quantification of the results in clonogenic assays was analyzed by Image J. Data are shown as mean ± SEM; n = 3.


F , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect in HepG2 . with stable knockdown of PTPN3 were treated with or without TGF - β ( 1 ng / mL ) for 10 days . Clonogenic of results in clonogenic assays were analyzed by Image J . Knockdown efficiency of PTPN3 in HepG2 were analyzed by Western blotting with anti - PTPN3 antibody . Data are shown as mean ± ; n = 3 . G , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect in Huh7 . Experiments were similarly done as in ( F ) . Data are shown as mean ± ; n = 3 .

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F, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in HepG2 cells. Cells with stable knockdown of PTPN3 were treated with or without TGF-β (1 ng/mL) for 10 days. Clonogenic assay Quantification of the results in clonogenic assays were analyzed by Image J. Knockdown efficiency of PTPN3 in HepG2 cells were analyzed by Western blotting with the anti-PTPN3 antibody. Data are shown as mean ± SEM; n = 3. G, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in Huh7 cells. Experiments and analysis were similarly done as in (F). Data are shown as mean ± SEM; n = 3.


A , PTPN3 , PTPN3 ( D811A ) PTPN3 ( L232R ) are similarly expressed in Huh7 stable lines . Expression of indicated were detected with appropriate antibodies in Western blotting .

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A, PTPN3, PTPN3 (D811A) and PTPN3 (L232R) are similarly expressed in Huh7 stable cell lines. Expression levels of indicated proteins were detected with appropriate antibodies in Western blotting.


B , PTPN3 PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , attenuates tumorigenesis . Luciferase - harboring Huh7 ( 2 × 106 / mouse ) expressing PTPN3 , PTPN3 ( D811A ) or PTPN3 ( L232R ) or empty vector ( served as negative control ) were subcutaneously injected into 5 - week - old nude mice . Twenty days after injection , mice were analyzed by bioluminescence using Xenogen IVIS imaging system . Tumors expressing luciferase is indicated by radiance bar ( p / sec / cm2 / sr ) . C , Quantitation of bioluminescence with data from ( B ) . Data are shown as mean ± ; n = 5 .

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B, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuates tumorigenesis. Luciferase-harboring Huh7 tumor cells (2 × 106 cells/mouse) expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) or empty vector (served as negative control) were subcutaneously injected into 5-week-old nude mice. Twenty days after injection, mice were analyzed by bioluminescence using Xenogen IVIS imaging system. Tumors expressing luciferase is indicated by radiance bar (p/sec/cm2/sr). C, Quantitation of bioluminescence with data from (B). Data are shown as mean ± SEM; n = 5.


D , Depletion of PTPN3 accelerates tumorigenesis . HepG2 stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice . Fifty days after implantation , tumors were dissected photographed .

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D, Depletion of PTPN3 accelerates tumorigenesis. HepG2 cells stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice. Fifty days after cell implantation, tumors were dissected and photographed.


E , Depletion of PTPN3 increased sizes . volume was measured at indicated days post - implantation . Data are shown as mean ± ; n = 6 for each group . F , Depletion of PTPN3 increased . from tumors was recorded from both control shPTPN3 groups . Data are shown as mean ± ; n = 6 for each group .

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E, Depletion of PTPN3 increased tumor sizes. Tumor volume was measured at indicated days post-implantation. Data are shown as mean ± SEM; n = 6 for each group. F, Depletion of PTPN3 increased tumor weight. Weight from all tumors was recorded from both control and shPTPN3 groups. Data are shown as mean ± SEM; n = 6 for each group.


, PTPN3 depletion impairs TGF - β signaling in tumors . of PTPN3 , GAPDH TGF - β signaling associated , such as TβRI , P - Smad2 , P - Smad3 , Smad2 / 3 , Smad4 , PAI - 1 , c - Myc p15 in tumors were analyzed by Western blotting .

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, PTPN3 depletion impairs TGF-β signaling in tumors. Levels of PTPN3, GAPDH and TGF-β signaling associated proteins, such as TβRI, P-Smad2, P-Smad3, Smad2/3, Smad4, PAI-1, c-Myc and p15 in tumors were analyzed by Western blotting analysis.


H , Clinical significance of PTPN3 expression in LIHC on TCGA database . In boxplots , Y - axis represents expression level of PTPN3 in normal samples . boxes show median ( horizontal in box ) ± 1 quartile ; upper lower box limits represent upper lower quartiles , respectively . whiskers ( vertical lines ) extend from hinge to smallest or larget value within 1 . 5 interquartile range from box boundaries .

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H, Clinical significance of PTPN3 expression in LIHC based on TCGA database. In the boxplots, Y-axis represents the expression level of PTPN3 in normal and tumor samples. The boxes show the median (horizontal line in the box) ± 1 quartile; the upper and lower box limits represent the upper and lower quartiles, respectively. The whiskers (vertical lines) extend from the hinge to the smallest or larget value within 1.5 interquartile range from the box boundaries.


( B ) Structured illumination microscopy image of perimCherry / cytoGFP E . coli confirming localization of mCherry ( red ) in periplasm GFP ( green ) in cytosol . Scale bar = 3 µm .

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(B) Structured illumination microscopy image of perimCherry/cytoGFP E. coli confirming localization of mCherry (red) in the periplasm and GFP (green) in the cytosol. Scale bar = 3 µm.


( C ) Outer damage ( mCherry ) inner damage ( % Sytox positive ) of perimCherry / cytoGFP E . coli exposed to ( different concentrations of ) human serum . Inner damage correlates with killing ( samples where are killed are indicated with gray shadings a cross , see CFU data in Fig EV1b ) . Data information : ( C - E ) Data represent mean ± SD of 3 independent experiments .

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(C) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of perimCherry/cytoGFP E. coli bacteria exposed to (different concentrations of) human serum. Inner membrane damage correlates with killing (samples where bacteria are killed are indicated with gray shadings and a cross, see CFU data in Fig EV1b). Data information: (C-E) Data represent mean ± SD of 3 independent experiments.


( D ) Serum - induced inner damage ( % Sytox positive ) of different Gram - negative strains depends on MAC components C5 C8 , but not on lysozyme ( 10 % serum ) . Dotted represents detection limit of . Data information : ( D , E ) cfu / ml ( Fig . 1E , 2C ) sytox measurements ( Fig . 1D , 2E ) of " Buffer " , " Serum " , " ΔC5 " , " ΔC8 " " Δlysozym " " C5b6MAC " were generated from same experiment . ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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(D) Serum-induced inner membrane damage (% Sytox positive) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


, E Serum - induced killing ( CFU / ml ) of different Gram - negative strains depends on MAC components C5 C8 , but not on lysozyme ( 10 % serum ) . Dotted represents detection limit of . Data information : ( D , E ) cfu / ml ( Fig . 1E , 2C ) sytox measurements ( Fig . 1D , 2E ) of " Buffer " , " Serum " , " ΔC5 " , " ΔC8 " " Δlysozym " " C5b6MAC " were generated from same experiment . ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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, E Serum-induced killing (CFU/ml) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( B ) Lysis of human erythrocytes after exposure to a range of preassembled C5b6 in presence of 100 nM C7 . After washing , erythrocytes were exposed to 20 nM C8 100 nM C9 for 30 minutes after which OD405 nm of supernatant was measured . ( B - E ) Data represent mean ± SD of 3 independent experiments .

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(B) Lysis of human erythrocytes after exposure to a concentration range of preassembled C5b6 in the presence of 100 nM C7. After washing, erythrocytes were exposed to 20 nM C8 and 100 nM C9 for 30 minutes after which the OD405 nm of the supernatant was measured. (B-E) Data represent mean ± SD of 3 independent experiments.


C ) viability of three Gram - negative strains after exposure to buffer , 10 % human serum or C5b6MAC . Buffer serum conditions are same as Fig . 1E .

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C) Bacterial viability of three Gram-negative strains after exposure to buffer, 10% human serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1E.


( D ) Permeabilization of outer , but not inner of perimCherry / cytoGFP E . coli exposed to C5b6MAC ( different concentrations of C5b6 with fixed concentrations of C7 - C9 ) . ( B - E ) Data represent mean ± SD of 3 independent experiments .

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(D) Permeabilization of the outer, but not inner membrane of perimCherry/cytoGFP E. coli cells exposed to C5b6MAC (different concentrations of C5b6 with fixed concentrations of C7-C9). (B-E) Data represent mean ± SD of 3 independent experiments.


E ) Inner damage of three Gram - negative strains exposed to buffer , 10 % serum or C5b6MAC . Buffer serum conditions are same as Fig . 1D .

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E) Inner membrane damage of three Gram-negative strains exposed to buffer, 10% serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1D.


( B ) viability of convertase - labeled strains exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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(B) Bacterial viability of convertase-labeled bacterial strains exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( C ) viability of convertase - labeled E . coli MG1655 exposed to a range of C5 in presence of 100 nM C6 , 100 nM C7 , 20 nM C8 100 nM C9 . ' Ctrl ' indicates that are pretreated with heat - inactivated ΔC5 serum . Dotted represents detection limit of . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments .

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(C) Bacterial viability of convertase-labeled E. coli MG1655 exposed to a concentration range of C5 in the presence of 100 nM C6, 100 nM C7, 20 nM C8 and 100 nM C9. 'Ctrl' indicates bacteria that are pretreated with heat-inactivated ΔC5 serum. Dotted line represents the detection limit of the assay. Data information: (B-E) Data represent mean ± SD of 3 independent experiments.


( D ) viability of convertase labeled E . coli MG1655 exposed to C5 - C9 or conditions lacking one MAC component . As an extra control , convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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

(D) Bacterial viability of convertase labeled E. coli MG1655 exposed to C5-C9 or conditions lacking one MAC component. As an extra control, convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin. Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( E ) viability of E . coli MG1655 exposed to FB depleted serum in presence of 20 µg / ml OmCI ( to deposit C4b C3b without Bb ) . After washing , were exposed to C5 - C9 in presence or absence of C1 C2 ( to generate classical pathway C5 convertases , C4b2aC3b ) . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O B-TISSUE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial viability of E. coli MG1655 exposed to FB depleted serum in the presence of 20 µg/ml OmCI (to deposit C4b and C3b without Bb). After washing, bacteria were exposed to C5-C9 in the presence or absence of C1 and C2 (to generate classical pathway C5 convertases, C4b2aC3b). Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( A ) Outer damage ( mCherry ) inner damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli incubated with a range of C5 fixed concentrations of C6 - C9 . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments .

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

(A) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli cells incubated with a concentration range of C5 and fixed concentrations of C6-C9. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.


( B ) Inner damage of perimCherry / cytoGFP E . coli exposed to a range of ΔC5 serum , after washing , to C5 - C9 . As controls , were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to ΔC5 serum to block C3b deposition . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments .

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

(B) Inner membrane damage of perimCherry/cytoGFP E. coli exposed to a concentration range of ΔC5 serum and, after washing, to C5-C9. As controls, bacteria were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to the ΔC5 serum to block C3b deposition. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.


( C ) Inner damage of three different convertase - labeled exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments . ( C ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 or * * p ≤ 0 . 01 .

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

(C) Inner membrane damage of three different convertase-labeled bacteria exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (A-C) Data represent mean ± SD of 3 independent experiments. (C) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05 or ** p≤0.01.


( D ) Confocal microscopy of convertase - labeled perimCherry / cytoGFP E . coli exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Unlabeled exposed to 1 % serum served as control . Green = GFP , Red = To - pro - 3 dye . Scale bars = 3 µm .

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

(D) Confocal microscopy images of convertase-labeled perimCherry/cytoGFP E. coli exposed to buffer (Conv) or C5-C9 (Conv-MAC). Unlabeled bacteria exposed to 1% serum served as control. Green = GFP, Red = To-pro-3 DNA dye. Scale bars = 3 µm.


( B ) viability of convertase - labeled E . coli MG1655 exposed to Buffer ( Conv ) , preassembled C5b6 ( Conv + C5b6MAC ) or a mixture of C5 C6 ( Conv - MAC ) , in presence of C7 , C8 C9 . Dotted represents detection limit of . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical was done using a paired two - t - displayed only when significant as * * p ≤ 0 . 01 .

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

(B) Bacterial viability of convertase-labeled E. coli MG1655 exposed to Buffer (Conv), preassembled C5b6 (Conv + C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of C7, C8 and C9. Dotted line represents the detection limit of the assay. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


( C ) Inner damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a range of preassembled C5b6 or a mixture of C5 C6 , in presence of 100 nM C7 . After washing , were exposed to 20 nM C8 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical was done using a paired two - t - displayed only when significant as * * p ≤ 0 . 01 .

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

(C) Inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


( D ) outer damage ( mCherry ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a range of preassembled C5b6 or a mixture of C5 C6 , in presence of 100 nM C7 . After washing , were exposed to 20 nM C8 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical was done using a paired two - t - displayed only when significant as * * p ≤ 0 . 01 .

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

(D) outer membrane damage (mCherry) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


A , B Step - wise assembly of MAC on convertase - labeled . Convertase - labeled were incubated with C5 / C6 or C5 / C6 / C7 for 15 minutes , subsequently washed ( @ ) or treated with 10 μg / ml Eculizumab ( Ecu ) . Then , remaining MAC components ( C7 - 9 for C5 / C6 or C8 - 9 for C5 / C6 / C7 respectively ) were added to incubation mixture . In control conditions ( Conv - MAC ) , remaining MAC components were added to incubation mixture without washing or adding an inhibitor . ( A ) Outer ( mCherry ) ( B ) inner damage ( % Sytox positive ) were determined . Data information : ( A - B ) Data represent mean ± SD of 3 independent experiments . Statistical was done using a paired two - t - in which conditions were compared to Conv - MAC displayed only when significant as * p ≤ 0 . 05 .

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

A, B Step-wise assembly of MAC on convertase-labeled bacteria. Convertase-labeled bacteria were incubated with C5/C6 or C5/C6/C7 for 15 minutes, and subsequently washed (@) or treated with 10 μg/ml Eculizumab (Ecu). Then, the remaining MAC components (C7-9 for C5/C6 or C8-9 for C5/C6/C7 respectively) were added to the incubation mixture. In the control conditions (Conv-MAC), the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor. (A) Outer membrane (mCherry) and (B) inner membrane damage (% Sytox positive) were determined. Data information: (A-B) Data represent mean ± SD of 3 independent experiments. Statistical analysis was done using a ratio paired two-tailed t-test in which the test conditions were compared to Conv-MAC and displayed only when significant as * p≤0.05.


( A ) Outer inner damage of convertase - labeled exposed to different combinations of MAC components . ' @ ' indicates a washing step . Data information : Data represent mean ± SD of 3 independent experiments . ( A ) Statistical was done using a one - way ANOVA displayed only when significant as * * p ≤ 0 . 01 or * * * * p ≤ 0 . 0001 .

O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Outer and inner membrane damage of convertase-labeled bacteria exposed to different combinations of MAC components. '@' indicates a washing step. Data information: Data represent mean ± SD of 3 independent experiments. (A) Statistical analysis was done using a one-way ANOVA and displayed only when significant as ** p≤0.01 or **** p≤0.0001.


( B ) Outer inner damage of convertase - labeled exposed to C5 - C8 after washing , to a range of C9 . Data information : Data represent mean ± SD of 3 independent experiments .

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

(B) Outer and inner membrane damage of convertase-labeled bacteria exposed to C5-C8 and after washing, to a concentration range of C9. Data information: Data represent mean ± SD of 3 independent experiments.


( A ) Convertase - labeled were exposed to a range of either preassembled C5b6 ( C5b6MAC ) or a mixture of C5 C6 ( Conv - MAC ) , in presence of 100 nM C7 . After washing , 20 nM C8 100 nM C9 - Cy3 was added . Controls at 0 nM C5b6 or C5 - C6 confirm that detected C9 - Cy3 deposition is specifically related to MAC formation ( solid lines ) . Proper insertion of pores was assessed by a previously described shaving method with trypsin ( Moskovich & Fishelson , 2007 ) . were first incubated with MAC components for 30 minutes subsequently treated with 20 µg / ml trypsin for 15 min at 37 ° C ( dotted lines ) . Data information : ( A ) Data represent mean ± SD of 3 independent experiments .

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

(A) Convertase-labeled bacteria were exposed to a concentration range of either preassembled C5b6 (C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of 100 nM C7. After washing, 20 nM C8 and 100 nM C9-Cy3 was added. Controls at 0 nM C5b6 or C5-C6 confirm that the detected C9-Cy3 deposition is specifically related to MAC formation (solid lines). Proper insertion of pores was assessed by a previously described shaving method with trypsin (Moskovich & Fishelson, 2007). Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg/ml trypsin for 15 min at 37°C (dotted lines). Data information: (A) Data represent mean ± SD of 3 independent experiments.


( B ) Convertase labeled perimCherry / cytoGFP ( Green ) exposed to C5b6MAC or Conv - MAC . Conditions were similar to those in ( A ) , however C9 - Cy5 was used to detect MAC pores ( Red ) . 100 nM of C5 C6 or C5b6 were used in combination with 100 nM C7 , 20 nM C8 100 nM C9 - Cy5 . Conv + C5b6MAC conv - MAC were taken in separate experiments in which settings were adjusted to of C9 - Cy5 to properly visualize pore distribution . Scale bars = 3 µm .

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

(B) Convertase labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv-MAC. Conditions were similar to those in (A), however C9-Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7, 20 nM C8 and 100 nM C9-Cy5. Conv + C5b6MAC and conv-MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9-Cy5 to properly visualize pore distribution. Scale bars = 3 µm.


( C Atomic force microscopy of E . coli BL21 MG1655 immobilized using Poly - L - Lysine protocol . ( C ) Entire high - resolution comparisons of untreated convertase - labeled E . coli BL21 exposed to C5 - C9 ( Conv - MAC ) for 10 minutes . Scale bars : 800 nm ( left ) 30 nm ( right ) . Height Scales : 1 . 01 µm ( left ) , 8 nm ( top right ) , 22 nm ( bottom right ) . Width of magnification boxes ( left ) : 42 nm , height scales : 8 nm ( top left ) 13 nm ( bottom left ) . Arrows highlight E . coli porin structures , an asterisk highlights C5b - 7 stalk . Height profiles ( bottom ) are shown for dashed lines in .

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

(C Atomic force microscopy analysis of E. coli BL21 and MG1655 immobilized using the Poly-L-Lysine protocol. (C) Entire bacteria and high-resolution comparisons of untreated and convertase-labeled E. coli BL21 exposed to C5-C9 (Conv-MAC) for 10 minutes. Scale bars: 800 nm (left) and 30 nm (right). Height Scales: 1.01 µm (left), 8 nm (top right), 22 nm (bottom right). Width of magnification boxes (left): 42 nm, height scales: 8 nm (top left) and 13 nm (bottom left). Arrows highlight E. coli porin structures, an asterisk highlights the C5b-7 stalk. Height profiles (bottom) are shown for the white dashed lines in the images.


( D ) Atomic force microscopy ( height phase ) of convertase - labeled E . coli MG1655 exposed to a mixture of C5 C6 ( Conv - MAC ) or preassembled C5b6 ( Conv + C5b6MAC ) , in presence of C7 , C8 , C9 , FB FD . were generated in same experiment . Scale bars : 50 nm . Height scales : 15 nm .

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

(D) Atomic force microscopy (height and phase images) of convertase-labeled E. coli MG1655 exposed to a mixture of C5 and C6 (Conv-MAC) or preassembled C5b6 (Conv + C5b6MAC), in the presence of C7, C8, C9, FB and FD. Images were generated in the same experiment. Scale bars: 50 nm. Height scales: 15 nm.


BrdU incorporation reflecting synthesis SA - b - gal activity as markers of CTX - induced senescence in Em - myc transgeniccontrol ; Bcl2 versus Suv39h12 ; Bcl2lymphomas in vivo . Numbers represent mean percentages of positive cells6s . d . ( n55 each ) .

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

BrdU incorporation reflecting DNA synthesis and SA-b-gal activity as markers of CTX-induced senescence in Em-myc transgeniccontrol;Bcl2 versus Suv39h12;Bcl2lymphomas in vivo. Numbers represent mean percentages of positive cells6s.d. (n55 each).


b , Representative whole - FLT - FDG - PET scans of an control ; Bcl2 lymphoma - bearing mouse at tumour manifestation ( untreated ) at day 6 after a single dose of CTX . Red circles , areas of specific tracer retention ; green circles , matching areas with lost retentions . Specific tracer uptake was assessed by calculation of tumour - to - background ratios . Data represent mean ratios ± s . d . ( n = 5 mice for FLT - PET scans n = 6 mice for FDG - PET scans ) . Insets show representative Ki67 colour - calibrated ( red , highest intensities ; blue , lowest intensities ) bioluminescence of glucose in tissue sections of respective groups .

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

b, Representative whole-body FLT- and FDG-PET scans of an individual control;Bcl2 lymphoma-bearing mouse at tumour manifestation (untreated) and at day 6 after a single dose of CTX. Red circles, areas of specific tracer retention; green circles, matching areas with lost retentions. Specific tracer uptake was assessed by calculation of tumour-to-background ratios. Data represent mean ratios ± s.d. (n = 5 mice for FLT-PET scans and n = 6 mice for FDG-PET scans). Insets show representative Ki67 staining and colour-calibrated (red, highest intensities; blue, lowest intensities) bioluminescence images of glucose levels in tissue sections of the respective groups.


c , Intracellular glucose concentrations in untreated ADR - treated control ; Bcl2 ( that is , senescent ) versus Suv39h1 - ; Bcl2 ( that is , non - senescent ) lymphomas in vitro ( as in d - g ) . Results represent means ± s . d . ( n = 5 each ) .

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

c, Intracellular glucose concentrations in untreated and ADR-treated control;Bcl2 (that is, senescent) versus Suv39h1-;Bcl2 (that is, non-senescent) lymphomas in vitro (as in d-g). Results represent means ± s.d. (n = 5 each).


d , Extracellular of specified metabolite - to - glucose ratios in indicated lymphoma groups relative to untreated control ; Bcl2 lymphoma after 18 h of cultivation . Data represent mean ratios ± s . d . ( n = 4 each , measurements were carried out in duplicate ) . Note that glucose - 6 - phosphate is not detectable ( n . d . ) in medium . Insets show ( ADR - treated relative to untreated ) intracellular of respective metabolites after labelling with 13C6 - glucose for one representative lymphoma from each group . Data represent mean ratios ( see Supplementary Fig . 5 for an assessment of heterogeneity of TIS - associated hypermetabolism a comparison of metabolic parameters of lymphomas ) .

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

d, Extracellular levels of the specified metabolite-to-glucose ratios in the indicated lymphoma cells groups relative to untreated control;Bcl2 lymphoma cells after 18 h of cultivation. Data represent mean ratios ± s.d. (n = 4 each, measurements were carried out in duplicate). Note that glucose-6-phosphate is not detectable (n.d.) in the medium. Insets show (ADR-treated relative to untreated) intracellular levels of the respective metabolites after labelling with 13C6-glucose for one representative lymphoma from each group. Data represent mean ratios (see Supplementary Fig. 5 for an assessment of the heterogeneity of the TIS-associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas).


e , Immunoblot analyses of phosphorylated AMP - activated kinase ( pAMPK ) , total AMPK , pyruvate kinase isoforms M1 M2 in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma in vitro ( n = 3 each ; α - tubulin as a loading control . Gels were cropped to ease presentation ; full - blots are provided in Supplementary Fig . 16 ) .

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

e, Immunoblot analyses of phosphorylated AMP-activated protein kinase (pAMPK), total AMPK, pyruvate kinase isoforms M1 and M2 in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells in vitro (n = 3 each; α-tubulin as a loading control. Gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).


f , Mean OCRs in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 − ; Bcl2 lymphoma in vitro . Results represent means ± s . d . ( n = 5 each ) .

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

f, Mean OCRs in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells in vitro. Results represent means ± s.d. (n = 5 each).


g , Intracellular ATP concentrations in as in f . Results represent means ± s . d . ( n = 5 each ) . * P < 0 . 05 .

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

g, Intracellular ATP concentrations in cells as in f. Results represent means ± s.d. (n = 5 each). *P<0.05.


a - e , viability by trypan blue dye exclusion in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma at day 5 compared to untreated condition in various biochemical , pharmacological or genetic interventional settings in vitro . results in this figure represent means ± s . d . ( n = 5 each ) . a , Inhibition of glucose metabolism by phloretin , cytochalasin B , sodium oxamate .

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

a-e, Cell viability by trypan blue dye exclusion in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical, pharmacological or genetic interventional settings in vitro. All results in this figure represent means ± s.d. (n = 5 each). a, Inhibition of glucose metabolism by phloretin, cytochalasin B, and sodium oxamate.


b , Exposure to 2DG or switch to glucose - free medium with or without glucose replenishment .

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

b, Exposure to 2DG or switch to glucose-free medium with or without glucose replenishment.