( 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 ) .

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(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 . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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(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 ) 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 .

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(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 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 .

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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 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 .

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, 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 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 .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 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

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 ± SEM ; n = 3 . Statistical analysis was performed using two - tailed Student ' s t - test . * * * 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 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 .

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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 ± SEM ; n = 3 .

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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 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 .

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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 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 .

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

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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the TβRI protein level . TβRI and PTPN3 were determined by Western blotting in HaCaT cells 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) and PTPN3 ( L232R ) are similarly expressed in Huh7 stable cell lines . Expression levels of indicated proteins 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 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 .

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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 cells stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice . Fifty days after cell implantation , tumors were dissected and 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 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 .

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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 . 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 .

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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 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 .

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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 the periplasm and GFP ( green ) in the 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 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 .

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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 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 .

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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

O O O O O O O O O O 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) 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 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 .

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

O O O O O O O O O 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) 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 ) 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

(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 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 .

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

(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 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 .

O O O 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 B-CONTROLLED_VAR 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

(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 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 .

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

(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 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 .

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 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 B-MEASURED_VAR 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 ) 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O 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

(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 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 .

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 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 B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

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(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 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 ) .

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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 - 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 cells in vitro . Results represent means ± s . d . ( n = 5 each ) .

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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 cells as in f . Results represent means ± s . d . ( n = 5 each ) . * P < 0 . 05 .

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


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 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 .

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


c , Lentiviral knockdown of hexokinase 2 ( Hk2 ) by short hairpin RNA ( shHk2 ) . d , Inhibition of AMPK by compound C . e , Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir . f , Inhibition of the electron transport chain by antimycin A . * P 0 . 01 , except b ( n = 8 each ) and e , in which * P 0 . 05 .

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

c, Lentiviral knockdown of hexokinase 2 (Hk2) by short hairpin RNA (shHk2). d, Inhibition of AMPK by compound C. e, Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir. f, Inhibition of the electron transport chain by antimycin A. *P  0.01, except b (n = 8 each) and e, in which *P  0.05.


Proteostasis analysis in TIS control versus senescence - incapable lymphoma cells in vitro , as in Fig . 2 , or versus SASP - attenuated TISlymphoma cells . a , Global protein synthesis rate , measured by incorporation of green - fluorescent methionine . Mean fluorescent intensities reflect newly synthesized protein within 2 h ( DAPI used as a counterstain ) . Data represent means ± s . d . ( n = 5 each ) . b , Rate of global protein synthesis ( measured as in a ) in ADR - senescent relative to untreated ( ut ) control ; Bcl2 lymphoma cells ; either empty vector - infected cells or cells stably expressing the NF - kB super - repressor IkBaDN ( SR , ref . 18 ) . Results represent means ± s . d . ( n = 5 each ) . c , Relative intracellular glucose concentrations in cells as in b .

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Proteostasis analysis in TIS control versus senescence-incapable lymphoma cells in vitro, as in Fig. 2, or versus SASP-attenuated TISlymphoma cells. a, Global protein synthesis rate, measured by incorporation of green-fluorescent methionine. Mean fluorescent intensities reflect newly synthesized protein within 2 h (DAPI used as a counterstain). Data represent means ± s.d. (n = 5 each). b, Rate of global protein synthesis (measured as in a) in ADR-senescent relative to untreated (ut) control;Bcl2 lymphoma cells; either empty vector-infected cells or cells stably expressing the NF-kB super-repressor IkBaDN(SR, ref. 18). Results represent means ± s.d. (n = 5 each). c, Relative intracellular glucose concentrations in cells as in b.


d , Relative OCR in cells as in b . e , Relative intracellular ATP concentrations in cells as in

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d, Relative OCR in cells as in b. e, Relative intracellular ATP concentrations in cells as in b


f , Expression of the transcription factors Xbp1 ( by RT - PCR ( PCR with reverse transcription ; top ) ; note that the lower band reflects the UPR - related splice form ) , ATF4 , CHOP , phospho - Jun N - terminal kinase ( JNK - P - Thr183 / Thr185 ) and total JNK ( by immunoblot analysis , with a - tubulin as a loading control ; bottom ) in lymphoma cells as indicated ( n = 3 each , gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) .

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f, Expression of the transcription factors Xbp1 (by RT-PCR (PCR with reverse transcription; top); note that the lower band reflects the UPR-related splice form), ATF4, CHOP, phospho-Jun N-terminal kinase (JNK-P-Thr183/Thr185) and total JNK(by immunoblot analysis, with a-tubulin as a loading control; bottom) in lymphoma cells as indicated (n = 3 each, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).


g , Global protein ubiquitination , expression of p62 / SQSTM1 ( whose transcript levels were also selectively induced in TIS cells by approximately 4 . 8 - fold ( data not shown ) ) and V - ATPase subunit A1 , and conversion of MAP1 - LC3 - I to MAP1 - LC3 - II by immunoblot analysis , demonstrating autophagosome formation in lymphoma cells as in f ( α - tubulin was used as a loading control , gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . * P < 0 . 05 .

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g, Global protein ubiquitination, expression of p62/SQSTM1 (whose transcript levels were also selectively induced in TIS cells by approximately 4.8-fold (data not shown)) and V-ATPase subunit A1, and conversion of MAP1-LC3-I to MAP1-LC3-II by immunoblot analysis, demonstrating autophagosome formation in lymphoma cells as in f (α-tubulin was used as a loading control, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). *P<0.05.


a , Cellular viability of super - repressor - or empty - vector - infected control ; Bcl2 lymphomas treated with ADR or untreated for 5 days , then exposed for 2 days to the inhibitors 2DG or bafilomycin A1 , compared to no inhibitor ( ut ) in vitro . Results represent means ± s . d . ( n = 5 each ) .

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a, Cellular viability of super-repressor- or empty-vector-infected control;Bcl2 lymphomas treated with ADR or untreated for 5 days, then exposed for 2 days to the inhibitors 2DG or bafilomycin A1, compared to no inhibitor (ut) in vitro. Results represent means ± s.d. (n = 5 each).


b , Immunoblot analyses of ATF4 , global protein ubiquitination , p62 / SQSTM1 , and MAP1 - LC3 - I to MAP1 - LC3 - II conversion in control ; Bcl2 lymphomas with no ADR treatment , in TIS cells , and in TIS cells after exposure to 2DG or bafilomycin A1in vitro ; representative blot with α - tubulin as a loading control ( gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . c , Cleavage ( activation ) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control ; Bcl2 lymphomas as in b ; representative blot with α - tubulin as a loading control ( gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) .

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b, Immunoblot analyses of ATF4, global protein ubiquitination, p62/SQSTM1, and MAP1-LC3-I to MAP1-LC3-II conversion in control;Bcl2 lymphomas with no ADR treatment, in TIS cells, and in TIS cells after exposure to 2DG or bafilomycin A1in vitro; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). c, Cleavage (activation) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control;Bcl2 lymphomas as in b; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).


d , Cellular viability of ADR - treated control ; Bcl2 lymphoma cells infected with short - hairpin RNAs against either caspase 12 ( shCasp12 ) or caspase 3 ( shCasp3 ) compared to empty vector - infected lymphoma cells treated as in a . Results represent means ± s . d . ( n = 4 each ) .

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d, Cellular viability of ADR-treated control;Bcl2 lymphoma cells infected with short-hairpin RNAs against either caspase 12 (shCasp12) or caspase 3 (shCasp3) compared to empty vector-infected lymphoma cells treated as in a. Results represent means ± s.d. (n = 4 each).


e , TIS cells in primary human acute myeloid leukaemia ( AML ) samples by SA - b - gal staining of treated ( 0 . 1 µg ml − 1 ADR for 5 days ) versus untreated cells in vitro . Data represent means ± s . d . ( n = 5 each ) .

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e, TIS cells in primary human acute myeloid leukaemia (AML) samples by SA-b-gal staining of treated (0.1 µg ml−1 ADR for 5 days) versus untreated cells in vitro. Data represent means ± s.d. (n = 5 each).


f , Intracellular glucose ( left ) and ATP ( right ) concentrations in senescent AML cells as in e .

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f, Intracellular glucose (left) and ATP (right) concentrations in senescent AML cells as in e.


g , Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a . Results represent means ± s . d . ( n = 5 each ) .

O O O O O O O O O O O O O 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, Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a. Results represent means ± s.d. (n = 5 each).


h , Colour - coded whole - body fluorescence imaging to visualize green fluorescent protein ( GFP ) - tagged tumour burden in time - courses of three representative mice harbouring CTX - senescent control ; Bcl2 lymphomas at day 5 ( see Figure 1a ) , and two days later , after the administration of 2DG ( n = 3 mice ) , bafilomycin A1 ( n = 4 mice ) or mock treatment ( n = 4 mice ) starting from day 5 post CTX ( red , highest intensities ; blue , lowest intensities ) . TdT - mediated dUTP nick end labelling ( assay ) ( TUNEL ) staining of lymph node sections in situ to visualize apoptosis at day 7 . Numbers represent means ± s . d . ( n = 3 ) .

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

h, Colour-coded whole-body fluorescence imaging to visualize green fluorescent protein (GFP)-tagged tumour burden in time-courses of three representative mice harbouring CTX-senescent control;Bcl2 lymphomas at day 5 (see Figure 1a), and two days later, after the administration of 2DG (n = 3 mice), bafilomycin A1 (n = 4 mice) or mock treatment (n = 4 mice) starting from day 5 post CTX (red, highest intensities; blue, lowest intensities). TdT-mediated dUTP nick end labelling (assay) (TUNEL) staining of lymph node sections in situ to visualize apoptosis at day 7. Numbers represent means ± s.d. (n = 3).


i , Overall survival of 5 day - CTX - treated control ; Bcl2 or Suv - 39h1 - ; Bcl2 lymphoma - bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h ( control ; Bcl2 : n = 12 and Suv39h1 - ; Bcl2 : n = 10 ) , presented as matched pair analyses ( P < 0 . 001 for the control ; Bcl2 comparison ; P = 0 . 221 for the Suv39h1 - ; Bcl2 comparison ) . * P < 0 . 05 .

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i, Overall survival of 5 day-CTX-treated control;Bcl2 or Suv-39h1-;Bcl2 lymphoma-bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h (control;Bcl2: n = 12 and Suv39h1-;Bcl2: n = 10), presented as matched pair analyses (P<0.001 for the control; Bcl2 comparison; P = 0.221 for the Suv39h1-;Bcl2 comparison). *P<0.05.


A Control animals ( + / w ; UAS - ralIR / + ) , n = 20 , and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Control animals ( + / w ; UAS - ralS25N / + ) , n = 20 , and those with salivary gland - specific expression of dominant - negative Ral ( fkh - GAL4 / w ; UAS - ralS25N / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . D Quantification of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square test . E Control animals ( ral35d / + ) , n = 25 , and ral hypomorph mutants ( ral35d / Y ) , n = 23 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . F Quantification of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

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

A Control animals (+/w; UAS-ralIR/+), n= 20, and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-ralS25N/+), n= 20, and those with salivary gland-specific expression of dominant-negative Ral (fkh-GAL4/w; UAS-ralS25N/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (ral35d/+), n= 25, and ral hypomorph mutants (ral35d/Y), n= 23, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.