f4a ) Induction of autophagy in response to 5 - FU in the GFP : : LGG - 1 reporter strain fed on RNAi targeting the indicated DNA repair genes ( scale bars , 10 μm ) . ( b ) The fraction ( as % of untreated control ) of hermaphrodites having embryos with excessive GFP expression following 5 - FU treatment . Autophagy induction in independent experiments ( circles ) and the mean ( line ) are presented . ( c ) F1 survival after control ( black diamonds ) or bec - 1 RNAi ( red squares ) in N2 and msh - 6 ( pk2504 ) mutants . The survival curve shows the mean ± s . d . for each data point from three independent experiments .

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f4a) Induction of autophagy in response to 5-FU in the GFP::LGG-1 reporter strain fed on RNAi targeting the indicated DNA repair genes (scale bars, 10 μm).(b) The fraction (as % of untreated control) of hermaphrodites having embryos with excessive GFP expression following 5-FU treatment. Autophagy induction in independent experiments (circles) and the mean (line) are presented.(c) F1 survival after control (black diamonds) or bec-1 RNAi (red squares) in N2 and msh-6(pk2504) mutants. The survival curve shows the mean±s.d. for each data point from three independent experiments.


f5 ( a ) Expression of ATF - 2 : : GFP in 5 - FU - treated hermaphrodites fed control or RNAi targeting the indicated genes ( scale bars , 20 μm ) . ( b ) The number of animals harbouring embryos with excessive ATF - 2 : : GFP expression was scored in at least 150 animals and given as the fraction ( % ) of the total . ( c ) N2 or msh - 6 ( pk2504 ) embryos were continuously monitored under differential interference contrast microscopy ( scale bars , 5 μm ) from the three - cell stage until completion of the four - cell stage . Magnified images of representative nuclei are shown . ( d ) The fraction of embryos with visible , enlarged nucleoli ( arrows ) after 5 - FU treatment was scored . Bar graphs represent mean ± s . d . from three independent experiments .

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f5(a) Expression of ATF-2::GFP in 5-FU-treated hermaphrodites fed control or RNAi targeting the indicated genes (scale bars, 20 μm). (b) The number of animals harbouring embryos with excessive ATF-2::GFP expression was scored in at least 150 animals and given as the fraction (%) of the total.(c) N2 or msh-6(pk2504) embryos were continuously monitored under differential interference contrast microscopy (scale bars, 5 μm) from the three-cell stage until completion of the four-cell stage. Magnified images of representative nuclei are shown. (d) The fraction of embryos with visible, enlarged nucleoli (arrows) after 5-FU treatment was scored. Bar graphs represent mean±s.d. from three independent experiments.


f6 ( a ) Western blots showing autophagy induction by monitoring LC3 levels in whole - cell extracts from U2OS cells treated or not with 5 - FU alone or in combination with Mx for the indicated time . Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase - 3 ( 17 and 19 kDa ) and poly ( ADP - ribose ) polymerase 1 ( PARP1 ; ΔPARP , 85 kDa ) . α − Actin was used as loading control . ( b ) Quantification of LC3 - II levels in cells treated as in a . Intensities of the lower LC3 band ( LC3 - II ) was normalized to those of actin . . ( c ) Autophagy induction demonstrated by accumulation of LC3 - positive puncta in U2OS cells treated or not with 5 - FU alone or in combination with Mx for 48 h . The cells were fixed and prepared for immunofluorescence of LC3 ( green ) . DAPI staining is shown in blue . Scale bar , 20 μm . ( d ) Quantification of LC3 - positive puncta in cells treated as in c . In each separate experiment , 200 - 1 , 600 cells were scored per condition . e ) Western blots showing absence of LC3 - II accumulation in U2OS cells transfected with siRNA against MSH - 2 . The same blot probed with anti - MSH - 2 antibodies confirm efficient knockdown . ( f ) Quantification of LC3 - II levels in cells transfected with control or MSH - 2 siRNA with or without FU treatment for 24 h . ( g ) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH - 2 . ( h ) Quantification of LC3 - positive puncta in cells treated as in g . ( i ) Quantification of normalized RPS3 protein level ( black bars ) or RPS3 mRNA level ( white bars ) in U2OS cells after 48 h 5 - FU treatment . RPS3 mRNA levels were determined by quantitative real - time reverse transcriptase - PCR .

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f6(a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time. Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase-3 (17 and 19 kDa) and poly (ADP-ribose) polymerase 1 (PARP1; ΔPARP, 85 kDa). α−Actin was used as loading control. (b) Quantification of LC3-II levels in cells treated as in a. Intensities of the lower LC3 band (LC3-II) was normalized to those of actin.. (c) Autophagy induction demonstrated by accumulation of LC3-positive puncta in U2OS cells treated or not with 5-FU alone or in combination with Mx for 48 h. The cells were fixed and prepared for immunofluorescence of LC3 (green). DAPI staining is shown in blue. Scale bar, 20 μm. (d) Quantification of LC3-positive puncta in cells treated as in c. In each separate experiment, 200-1,600 cells were scored per condition.e) Western blots showing absence of LC3-II accumulation in U2OS cells transfected with siRNA against MSH-2. The same blot probed with anti-MSH-2 antibodies confirm efficient knockdown. (f) Quantification of LC3-II levels in cells transfected with control or MSH-2 siRNA with or without FU treatment for 24 h.(g) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH-2. (h) Quantification of LC3-positive puncta in cells treated as in g.(i) Quantification of normalized RPS3 protein level (black bars) or RPS3 mRNA level (white bars) in U2OS cells after 48 h 5-FU treatment. RPS3 mRNA levels were determined by quantitative real-time reverse transcriptase-PCR.


Figure 1A , 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 . 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 . 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 . 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 . 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 . K , Heat map of Log2FC of select TGF - β target genes .

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Figure 1A, 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.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.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.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.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.K, Heat map of Log2FC of select TGF-β target genes.


Figure 2B , 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 . 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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Figure 2B, 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.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.


Figure 3A , 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 . 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 . 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 . 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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Figure 3A, 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.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.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.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.


Figure 4A , 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 . 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 . 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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Figure 4A, 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.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.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.


Figure 5A , 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 ) . 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 . 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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Figure 5A, 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).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.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.


Figure 6A , 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 . 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 . 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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Figure 6A, 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.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.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.


Figure 7A , 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 . 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 . , 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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Figure 7A, 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. 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. , 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.


Figure 1 ( b ) Flow cytometry - based stratification of cells in different cell cycle phases and in G0 ( quiescence , 2n DNA - and low RNA content ) , shown for SKOV3 cells in serum starvation conditions at 8 and 96 hours , respectively . Cells were detached , fixed with ethanol and stained with Hoechst 33342 and Pyronin Y prior to flow cytometry analysis . ( c ) Distribution of cell cycle phases in six cell lines exposed to quiescence - inducing conditions ( see panel a ) for 8 and 96 hours , respectively . Each stacked bar plot reports the fraction of cells in G0 , G1 , S and G2 / M phases , estimated using the assay depicted in panel b . ( d ) Cell confluence of adherent cell cultures in quiescence - inducing conditions ( i . e . serum starvation and glutamine limitation ) was monitored using time - lapse microscopy in a plate reader ( TECAN Spark 10M ) in 1 . 5 - hour intervals . Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach . Data points and shaded areas indicate mean ± standard deviation ( SD ) over three biological replicates . Approximately 9500 , 4500 , 19500 , 6700 , 19500 and 9500 cells / well were seeded for glutamine limitation and serum starvation of A549 , CCD1070Sk , HCT116 , HFL1 , MCF7 and SKOV3 cells , respectively .

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Figure 1(b) Flow cytometry-based stratification of cells in different cell cycle phases and in G0 (quiescence, 2n DNA- and low RNA content), shown for SKOV3 cells in serum starvation conditions at 8 and 96 hours, respectively. Cells were detached, fixed with ethanol and stained with Hoechst 33342 and Pyronin Y prior to flow cytometry analysis.(c) Distribution of cell cycle phases in six cell lines exposed to quiescence-inducing conditions (see panel a) for 8 and 96 hours, respectively. Each stacked bar plot reports the fraction of cells in G0, G1, S and G2/M phases, estimated using the assay depicted in panel b.(d) Cell confluence of adherent cell cultures in quiescence-inducing conditions (i.e. serum starvation and glutamine limitation) was monitored using time-lapse microscopy in a plate reader (TECAN Spark 10M) in 1.5-hour intervals. Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach. Data points and shaded areas indicate mean ± standard deviation (SD) over three biological replicates. Approximately 9500, 4500, 19500, 6700, 19500 and 9500 cells/well were seeded for glutamine limitation and serum starvation of A549, CCD1070Sk, HCT116, HFL1, MCF7 and SKOV3 cells, respectively.


Figure 3 ( b ) Growth phenotypic changes induced by TMZ treatment in A549 and HCT116 cells grown in full growth medium ( RPMI - 1640 with 5 % dialyzed FBS ) . Cell confluence as a proxy of cell numbers was monitored using automated time - lapse microscopy in a plate reader ( TECAN Spark 10M ) . Data points and shaded areas indicate mean ± SD over three biological replicates . ( c ) Growth phenotypic changes induced by TMZ treatment in quiescence - inducing conditions ( serum starvation ) in A549 and HCT116 cancer cells . Growth was monitored via cell confluence as described in panel b . At 96 hours , cells were stimulated to exit quiescence by replacing serum - free media with fresh growth medium without TMZ ( regrowth ) . Data points and shaded areas indicate mean ± SD over three biological replicates . ( d ) Viability of quiescence - induced cells treated with TMZ , or with TMZ plus 10 µM etomoxir was assessed using a fluorescence imaging assay involving two fluorescent dyes : Hoechst 33342 ( all nuclei ) and propidium iodide ( dead cells ) . Data points and error bars indicate mean ± SD over three biological replicates . ( e ) Dynamic changes in the intracellular levels of citrate in A549 and HCT116 cells induced to enter quiescence by serum starvation with or without TMZ . Fold - changes were estimated relative to steady - state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates . ( f ) Volcano plot showing TMZ - induced metabolic changes ( 1 mM , 48 hours ) in A549 cells in serum starvation Shown are differences in log2 fold - change between TMZ treatment and serum starvation alone ( x - axis ) , and their statistical significance ( y - axis , adjusted p - value , t - test and Benjamini - Hochberg correction ) . ( h ) Comparison of relative changes ( log2 fold - change ) in the abundance of 1791 putatively annotated metabolites upon TMZ treatment ( 1 mM , y - axis ) against untreated quiescence - induced A549 cells ( x - axis ) . Fold - changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations ( Spearman , p < 0 . 01 ) with cell death at 96 hours are highlighted in blue and yellow , respectively . ( i ) Fractions of early apoptotic cells in A549 or HCT116 cancer cell cultures in quiescence - inducing conditions ( serum starvation , left panels ) or full growth medium ( 5 % dFBS , right panels ) , treated with TMZ ( 0 . 4 or 1 mM ) , 5 - fluorouracil ( 5 - FU , 0 . 5 mM , positive control ) or medium ( untreated ) . Early apoptotic cells were detected using ApoTracker Green under co - staining with Hoechst 33342 ( nuclei ) and propidium iodide ( dead cells ) . Cell staining was continuously monitored in 3 - hours intervals for 96 hours using automated fluorescence microscopy in a TECAN Spark Cyto . Data points and shaded areas indicate mean ± SD over three biological replicates . Approximately 3400 and 7500 cells / well were seeded of A549 and HCT116 cells , respectively .

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Figure 3(b) Growth phenotypic changes induced by TMZ treatment in A549 and HCT116 cells grown in full growth medium (RPMI-1640 with 5% dialyzed FBS). Cell confluence as a proxy of cell numbers was monitored using automated time-lapse microscopy in a plate reader (TECAN Spark 10M). Data points and shaded areas indicate mean ± SD over three biological replicates. (c) Growth phenotypic changes induced by TMZ treatment in quiescence-inducing conditions (serum starvation) in A549 and HCT116 cancer cells. Growth was monitored via cell confluence as described in panel b. At 96 hours, cells were stimulated to exit quiescence by replacing serum-free media with fresh growth medium without TMZ (regrowth). Data points and shaded areas indicate mean ± SD over three biological replicates.(d) Viability of quiescence-induced cells treated with TMZ, or with TMZ plus 10 µM etomoxir was assessed using a fluorescence imaging assay involving two fluorescent dyes: Hoechst 33342 (all nuclei) and propidium iodide (dead cells). Data points and error bars indicate mean ± SD over three biological replicates.(e) Dynamic changes in the intracellular levels of citrate in A549 and HCT116 cells induced to enter quiescence by serum starvation with or without TMZ. Fold-changes were estimated relative to steady-state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates.(f) Volcano plot showing TMZ-induced metabolic changes (1 mM, 48 hours) in A549 cells in serum starvation Shown are differences in log2 fold-change between TMZ treatment and serum starvation alone (x-axis), and their statistical significance (y-axis, adjusted p-value, t-test and Benjamini-Hochberg correction).(h) Comparison of relative changes (log2 fold-change) in the abundance of 1791 putatively annotated metabolites upon TMZ treatment (1 mM, y-axis) against untreated quiescence-induced A549 cells (x-axis). Fold-changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations (Spearman, p < 0.01) with cell death at 96 hours are highlighted in blue and yellow, respectively.(i) Fractions of early apoptotic cells in A549 or HCT116 cancer cell cultures in quiescence-inducing conditions (serum starvation, left panels) or full growth medium (5% dFBS, right panels), treated with TMZ (0.4 or 1 mM), 5-fluorouracil (5-FU, 0.5 mM, positive control) or medium (untreated). Early apoptotic cells were detected using ApoTracker Green under co-staining with Hoechst 33342 (nuclei) and propidium iodide (dead cells). Cell staining was continuously monitored in 3-hours intervals for 96 hours using automated fluorescence microscopy in a TECAN Spark Cyto. Data points and shaded areas indicate mean ± SD over three biological replicates. Approximately 3400 and 7500 cells/well were seeded of A549 and HCT116 cells, respectively.


Figure 1B . Single - cell lengths were measured from time - lapse microscopy images upon beta - lactam antibiotic treatment . Initial cell length was measured in the first time frame . Elongation ends with cell length shrinkage due to bulging . The longest length of each cell across the time frames was used as the final length . Tracked intermediate cell lengths were shown over time , while initial and final lengths were shown in larger circles . Linear regression of log - scaled lengths over time showed a constant rate of exponential elongation ( solid gray line ) .

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Figure 1B. Single-cell lengths were measured from time-lapse microscopy images upon beta-lactam antibiotic treatment. Initial cell length was measured in the first time frame. Elongation ends with cell length shrinkage due to bulging. The longest length of each cell across the time frames was used as the final length. Tracked intermediate cell lengths were shown over time, while initial and final lengths were shown in larger circles. Linear regression of log-scaled lengths over time showed a constant rate of exponential elongation (solid gray line).


Figure 2A . Probability density distributions of final lengths of MG1655 cells exposed to carbenicillin . Individual cell lengths were measured from populations exposed to three doses of carbenicillin ( 20 , 50 , and 100 µg / ml ) at two temperatures ( 27 ° C in blue and 37 ° C in red ) . Line plots show the measured initial and final lengths over time in log scale , and the first 15 cells were picked in each dataset for presentation among n ≥ 100 of the tracked cells in each condition Probability density distributions ( ρL ) of final lengths were collected with the same number of bins within the same final cell length range ( bottom panels ) . The peak of ρL shifted to the left with increasing carbenicillin dose . C . The probability of lysis increased with the filamentation length of bacteria treated with other beta - lactams . MG1655 cells were treated with cefotaxime ( 20 and 100 µg / ml ) and amoxicillin ( 6 . 25 and 25 µg / ml , shown in gray text ) at two temperatures ( 27 ° C in blue and 37 ° C in red ) . D . The probability of lysis increased with the filamentation length of E . coli pathogens treated with a beta - lactam . E . coli pathogens expressing extended beta - lactam resistance were treated with amoxicillin ( 6 . 25 or 25 µg / ml , shown in gray text ) and clavulanic acid ( 50 µg / ml ) simultaneously at 37 ° C . Cells were sensitized by clavulanate acid , which inhibits beta - lactamases .

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Figure 2A. Probability density distributions of final lengths of MG1655 cells exposed to carbenicillin. Individual cell lengths were measured from populations exposed to three doses of carbenicillin (20, 50, and 100 µg/ml) at two temperatures (27°C in blue and 37°C in red). Line plots show the measured initial and final lengths over time in log scale, and the first 15 cells were picked in each dataset for presentation among n ≥ 100 of the tracked cells in each condition Probability density distributions (ρL) of final lengths were collected with the same number of bins within the same final cell length range (bottom panels). The peak of ρL shifted to the left with increasing carbenicillin dose.C. The probability of lysis increased with the filamentation length of bacteria treated with other beta-lactams. MG1655 cells were treated with cefotaxime (20 and 100 µg/ml) and amoxicillin (6.25 and 25 µg/ml, shown in gray text) at two temperatures (27 °C in blue and 37 °C in red).D. The probability of lysis increased with the filamentation length of E. coli pathogens treated with a beta-lactam. E. coli pathogens expressing extended beta-lactam resistance were treated with amoxicillin (6.25 or 25 µg/ml, shown in gray text) and clavulanic acid (50 µg/ml) simultaneously at 37 °C. Cells were sensitized by clavulanate acid, which inhibits beta-lactamases.


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

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


Figure 5B - C . Stochastic simulations and measurements of survivor length . Simulated survivor lengths were plotted using the predicted LC of lower doses of carbenicillin ( left panels ) at 2h ( B ) and 6h ( C ) . The corresponding single - cell length measurements in vitro were plotted ( right panels ) . In the simulation , only non - antibiotic treated cells were set to divide , when the cell length reaches the division length in the linear model ( Ld = aLi + b , a = 0 . 871 , b = 2 . 7 . In both simulations and measurements , cells in carbenicillin - treated conditions were further elongated than control cells at all time points . At 2h exposure , antibiotic - exposed cells showed similar lengths across antibiotic concentrations since lysis is not highly probable for short filaments . At 6h exposure , survivor lengths decreased with increasing carbenicillin dose . Independent t - tests between non - treated to treated group showed extreme significance , * * * * P < 0 . 0001 . The box represents the middle 50 % , the central line represents the median , and the whiskers represent the lower and upper quartile of the data points ( n > 300 ) .

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Figure 5B-C. Stochastic simulations and measurements of survivor length. Simulated survivor lengths were plotted using the predicted LC of lower doses of carbenicillin (left panels) at 2h (B) and 6h (C). The corresponding single-cell length measurements in vitro were plotted (right panels). In the simulation, only non-antibiotic treated cells were set to divide, when the cell length reaches the division length in the linear model (Ld= aLi + b, a = 0.871, b = 2.7. In both simulations and measurements, cells in carbenicillin-treated conditions were further elongated than control cells at all time points. At 2h exposure, antibiotic-exposed cells showed similar lengths across antibiotic concentrations since lysis is not highly probable for short filaments. At 6h exposure, survivor lengths decreased with increasing carbenicillin dose. Independent t-tests between non-treated to treated group showed extreme significance, **** P < 0.0001. The box represents the middle 50%, the central line represents the median, and the whiskers represent the lower and upper quartile of the data points (n>300).


Figure 1Binding of recombinant ( r ) CEACAM1 ( CC1 ) - HIS ( 10 μg / mL ) to ( A ) a panel of Gram - positive bacteria , namely Enterococcus faecalis , Enterococcus faecium , Group A Streptococcus ( GAS ) , Group B Streptococcus ( GBS ) , Group C Streptococcus ( GCS ) , Group G Streptococcus ( GGS ) , Streptococcus pneumoniae , Staphylococcus aureus , ( B ) an expanded collection of GBS isolates , with serotype and carriage of bac gene shown . rCC1 binding to live bacteria was quantified by a secondary anti - His - FITC monoclonal antibody ( mAb ) . Mean and standard deviation ( SD ) values are reported for n = 3 independent experiments . In ( B ) , GBS strains from left to right are A909 , BS39 , 515 , BS22 , H36B , BS29 , 18RS21 , BM110 , COH1 , SBL3066 , NCTC10 / 84 , SB35 , SB10 , SB20 , BS26 . Western blot analysis of rCC1 - HIS ( 30 μg / mL ) binding to purified β protein , but not to control GBS surface proteins α and Rib . Panel on the left show staining of proteins after separation by SDS - PAGE .

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Figure 1Binding of recombinant (r)CEACAM1 (CC1)-HIS (10 μg/mL) to (A) a panel of Gram-positive bacteria, namely Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Group C Streptococcus (GCS), Group G Streptococcus (GGS), Streptococcus pneumoniae, Staphylococcus aureus, (B) an expanded collection of GBS isolates, with serotype and carriage of bac gene shown. rCC1 binding to live bacteria was quantified by a secondary anti-His-FITC monoclonal antibody (mAb). Mean and standard deviation (SD) values are reported for n = 3 independent experiments. In (B), GBS strains from left to right are A909, BS39, 515, BS22, H36B, BS29, 18RS21, BM110, COH1, SBL3066, NCTC10/84, SB35, SB10, SB20, BS26.Western blot analysis of rCC1-HIS (30 μg/mL) binding to purified β protein, but not to control GBS surface proteins α and Rib. Panel on the left show staining of proteins after separation by SDS-PAGE.


Figure 2Inhibition of β - IgSF binding to DB . CC1 by anti - CC1 - N mAb ( clone CC1 / 3 / 5 - Sab ) , but not by anti - CC1 - A1B1 mAb ( clone B3 - 17 ) . β - IgSF - biotin was coupled to SPE . Mean and SD values are reported for n = 6 replicates .

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Figure 2Inhibition of β-IgSF binding to DB.CC1 by anti-CC1-N mAb (clone CC1/3/5-Sab), but not by anti-CC1-A1B1 mAb (clone B3-17). β-IgSF-biotin was coupled to SPE. Mean and SD values are reported for n = 6 replicates.


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

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


Figure 4Adhesion of WT , ∆ bac and ∆ bac + pLZ . bac complemented A909 strains to human CC1 - or CC5 - expressing CHO transfectants , or empty vector control cells . Mean and SD from n = 5 independent replicates is displayed .

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Figure 4Adhesion of WT, ∆bac and ∆bac + pLZ.bac complemented A909 strains to human CC1- or CC5-expressing CHO transfectants, or empty vector control cells. Mean and SD from n = 5 independent replicates is displayed.


Figure 6Binding of rCC1 - N to dynabeads ( DB ) coated with β - IgI3 wild type ( WT ) or mutants . Mutation of β - IgI3 residues 42 , 46 and 53 lead to significant changes in CC1 - N binding capacity . Binding of β - IgI3 coupled to Streptavidin - PE to DB coated with rCC1 - N wildtype or variants . Mutation of CC1 - N residues 29 , 44 , 89 , 91 and 95 lead to significant changes in β - IgI3 binding capacity .

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Figure 6Binding of rCC1-N to dynabeads (DB) coated with β-IgI3 wild type (WT) or mutants. Mutation of β-IgI3 residues 42, 46 and 53 lead to significant changes in CC1-N binding capacity.Binding of β-IgI3 coupled to Streptavidin-PE to DB coated with rCC1-N wildtype or variants. Mutation of CC1-N residues 29, 44, 89, 91 and 95 lead to significant changes in β-IgI3 binding capacity.


Figure 7Binding of recombinant ( r ) CC1 - HIS to dynabeads ( DB ) coated with β - IgI3 , R28 - IgI3 or control . Mean and SD is shown for n = 6 independent replicates . Binding of β - IgI3 , R28 - IgI3 or human serum albumin ( HSA ) coupled to PE - conjugated streptavidin ( SPE ) , to DB coated with CC1 ( DB . CC1 ) . Mean and SD is shown for n = 6 independent replicates .

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Figure 7Binding of recombinant (r)CC1-HIS to dynabeads (DB) coated with β-IgI3, R28-IgI3 or control. Mean and SD is shown for n = 6 independent replicates.Binding of β-IgI3, R28-IgI3 or human serum albumin (HSA) coupled to PE-conjugated streptavidin (SPE), to DB coated with CC1 (DB.CC1). Mean and SD is shown for n = 6 independent replicates.


Figure 1 ( A ) Representative images of ILEI immunohistochemistry on healthy ( upper panels ) and psoriatic ( lower panels ) skin sections , scale bar 100 µm . Inlets show a magnification of the marked regions , scale bar 50 µm . ( B ) Distribution of ILEI protein content per keratinocyte ( n = 5000 - 15000 cells per skin sample ) in healthy and psoriatic skin . Box and whiskers plot : Central band shows median , box extends from the 25th to 75th percentiles and whiskers go from the smallest ( min ) to the largest ( max ) value . ( C ) Median ILEI content of keratinocytes per person shown as mean ± SEM of healthy ( n = 6 ) and psoriasis ( n = 5 ) conditions . Data information : ( C , Statistical significance was determined by Student ' s t - test and marked with asterisks ( * * p < 0 . 01 ) .

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Figure 1(A) Representative images of ILEI immunohistochemistry on healthy (upper panels) and psoriatic (lower panels) skin sections, scale bar 100 µm. Inlets show a magnification of the marked regions, scale bar 50 µm.(B) Distribution of ILEI protein content per keratinocyte (n=5000-15000 cells per skin sample) in healthy and psoriatic skin. Box and whiskers plot: Central band shows median, box extends from the 25th to 75th percentiles and whiskers go from the smallest (min) to the largest (max) value.(C) Median ILEI content of keratinocytes per person shown as mean ± SEM of healthy (n=6) and psoriasis (n=5) conditions. Data information: (C, Statistical significance was determined by Student's t-test and marked with asterisks (**p<0.01).


Figure 2 ( C ) hematoxylin - eosin staining with scale bar of 50 µm of the back skin of ILEIind and K5 - ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days ( n = 2 - 5 ; 3 independent experiments ) . ( D ) mean epidermal thickness ± SEM of the back skin of ILEIind and K5 - ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days ( n = 2 - 5 ; 3 independent experiments ) . Statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * * p , 0 . 001 ) . ( E ) Representative images of ILEI immunohistochemistry on sections of back skin of ILEIind and K5 - ILEIind mice kept on normal or doxycycline diet upon 5 days of treatment with acetone or TPA . Scale bar 50 µm . Inlets show a magnification of the marked regions , scale bar 20 µm .

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Figure 2(C) hematoxylin-eosin staining with scale bar of 50 µm of the back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days (n=2-5; 3 independent experiments).(D) mean epidermal thickness ±SEM of the back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days (n=2-5; 3 independent experiments). Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; ***p,0.001).(E) Representative images of ILEI immunohistochemistry on sections of back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet upon 5 days of treatment with acetone or TPA. Scale bar 50 µm. Inlets show a magnification of the marked regions, scale bar 20 µm.


Figure 3 ( A ) Representative images of Ki67 immunohistochemistry on back skin sections of K5 - ILEIind and ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days . Scale bar : 100 µm . ( B ) Percentage of Ki67 positive cells in the epidermis shown as mean ± SEM and quantified on samples described in panel A ( n = 1 - 5 ; 3 independent experiments ) . Data information : Statistical significance was determined by one - way ANOVA ( B at ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p , 0 . 001 ) . ( C ) Immunofluorescence for Keratin 5 ( K5 ; red ) and Keratin 10 ( K10 ; green ) expression on TPA - treated skin sections of mice described in panel A . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 50 µm . Inlets show a magnification of the marked regions . Scale bar : 20 µm . ( D ) Keratin 16 ( K16 ) western blot analysis ( left ) and quantification ( right ) of proteins extracted from back skin of ILEIind and K5 - ILEIind mice kept on doxycycline and treated with acetone or TPA for 5 days ( n = 3 ) . Vinculin was used as loading control . Data information : Statistical significance was determined by one - way ANOVA at ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p , 0 . 001 ) . ( G ) Mean fold change in mRNA expression ± SEM of K5 and K10 in primary keratinocytes described in panel F ( n = 5 - 6 ; 2 independent experiments ) . Data information : Statistical significance was determined by two - way ANOVA ( G ) at ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p , 0 . 001 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative images of Ki67 immunohistochemistry on back skin sections of K5-ILEIind and ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days. Scale bar: 100 µm. (B) Percentage of Ki67 positive cells in the epidermis shown as mean ±SEM and quantified on samples described in panel A (n=1-5; 3 independent experiments). Data information: Statistical significance was determined by one-way ANOVA (B at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).(C) Immunofluorescence for Keratin 5 (K5; red) and Keratin 10 (K10; green) expression on TPA-treated skin sections of mice described in panel A. Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. Inlets show a magnification of the marked regions. Scale bar: 20 µm.(D) Keratin 16 (K16) western blot analysis (left) and quantification (right) of proteins extracted from back skin of ILEIind and K5-ILEIind mice kept on doxycycline and treated with acetone or TPA for 5 days (n=3). Vinculin was used as loading control. Data information: Statistical significance was determined by one-way ANOVA at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).(G) Mean fold change in mRNA expression ±SEM of K5 and K10 in primary keratinocytes described in panel F (n=5-6; 2 independent experiments). Data information: Statistical significance was determined by two-way ANOVA (G) at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).


Figure 4Mean number ± SEM of ( C ) neutrophils ( MPO + cells ) / mm2 epidermal - dermal area and ( D ) epidermal CD8 + T - cells / cm skin section of mice described in A ( n = 3 - 5 C ) , n = 1 - 5 ( D ) ; 3 independent experiments ) . N . A . , not analyzed . Data information : statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets . Mean fold change ± SEM in mRNA expression of Tnfa , Il1α and Cxcl1 ( E ) in freshly sorted keratinocytes enriched for the inter - follicular epithelium ( IFE ) and hair follicles ( HF ) from acetone and TPA treated back skin in mice supplemented with doxycycline and treated with acetone or TPA for 96 hours ( n = 3 ( E ) , independent experiments ) . Data information : statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets . ( G - I ) Mean fold change ± SEM in mRNA expression of ( G ) Tnfa , ( H ) Il1α and ( I ) Cxcl1 in primary keratinocytes treated with acetone and TPA and with increasing concentrations ( 100ng / ml , 250ng / ml , 500ng / ml , 1000ng / ml ) of murine recombinant ILEI ( mILEI ) or dimerization - disabled ILEI ( mILEICA ) or with empty - vector ( empty ) for 72 hours ( n = 3 , standing for independent keratinocyte cultures from three mice ) . Data information : statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Mean number ±SEM of (C) neutrophils (MPO+ cells)/mm2 epidermal-dermal area and (D) epidermal CD8+ T-cells/cm skin section of mice described in A (n=3-5 C), n=1-5 (D); 3 independent experiments). N.A., not analyzed. Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.Mean fold change ±SEM in mRNA expression of Tnfa, Il1α and Cxcl1 (E) in freshly sorted keratinocytes enriched for the inter-follicular epithelium (IFE) and hair follicles (HF) from acetone and TPA treated back skin in mice supplemented with doxycycline and treated with acetone or TPA for 96 hours (n=3 (E), independent experiments). Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.(G-I) Mean fold change ±SEM in mRNA expression of (G) Tnfa, (H) Il1α and (I) Cxcl1 in primary keratinocytes treated with acetone and TPA and with increasing concentrations (100ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml) of murine recombinant ILEI (mILEI) or dimerization-disabled ILEI (mILEICA) or with empty-vector (empty) for 72 hours (n=3, standing for independent keratinocyte cultures from three mice). Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.


Figure 5 ( A - C ) Representative images of ( A ) Phospho - STAT3 ( Tyr727 ) , ( B ) Phospho - Erk1 / 2 and ( C ) Phospho - Akt immunofluorescence on thin sections of acetone and TPA treated back skin of ILEIind and K5 - ILEIind mice kept on doxycycline diet . Scale bar , 50 µm . ( E ) Mean STAT3 ( Ser727 and Tyr705 ) , Akt and Erk1 / 2 phosphorylation levels ± SEM over time in primary keratinocytes upon TPA and recombinant ILEI treatment as described in panel A ( n = 3 , standing for independent keratinocyte cultures , each a pool of isolates from two mice ) . ( F ) Western blot analysis of STAT3 ( Ser727 and Tyr705 ) , Akt and Erk1 / 2 phosphorylation levels in primary Dox - induced K5 - ILEIind keratinocytes treated with acetone or TPA for 4 hours in the presence of the inhibitors STATTIC ( 10µM ) , LY92004 ( 10µM ) and UO126 ( 10µM ) . DMSO was used as vehicle control . Vinculin was used as loading control . Lanes are from non - continuous parts of the same gel .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-C) Representative images of (A) Phospho-STAT3 (Tyr727), (B) Phospho-Erk1/2 and (C) Phospho-Akt immunofluorescence on thin sections of acetone and TPA treated back skin of ILEIind and K5-ILEIind mice kept on doxycycline diet. Scale bar, 50 µm.(E) Mean STAT3 (Ser727 and Tyr705), Akt and Erk1/2 phosphorylation levels ±SEM over time in primary keratinocytes upon TPA and recombinant ILEI treatment as described in panel A (n=3, standing for independent keratinocyte cultures, each a pool of isolates from two mice).(F) Western blot analysis of STAT3 (Ser727 and Tyr705), Akt and Erk1/2 phosphorylation levels in primary Dox-induced K5-ILEIind keratinocytes treated with acetone or TPA for 4 hours in the presence of the inhibitors STATTIC (10µM), LY92004 (10µM) and UO126 (10µM). DMSO was used as vehicle control. Vinculin was used as loading control. Lanes are from non-continuous parts of the same gel.


Figure 6 ( B ) hematoxylin - eosin staining with a scale bar of 50 µm and ( C ) mean epidermal thickness ± SEM of the back skin of ILEIfl / fl and ILEI ∆ Ep mice treated with acetone or TPA for 5 days ( n = 3 - 8 ; 3 independent experiments ) . Data information : In C statistical significance was determined one - way ANOVA with Tukey multiple comparison test ( C marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets . ( F - H ) ( F ) Representative images of MPO + microabscesses and neutrophils , ( G ) mean number ± SEM of MPO - positive microabsesses / cm skin section and ( H ) mean number of neutrophils ( MPO + cells ) / mm2 epidermal - dermal area ± SEM on thin sections of acetone and TPA treated back skin of ILEIfl / fl and ILEI ∆ Ep mice ( n = 6 - 8 ( G ) and n = 2 - 8 ( H ) ; 3 independent experiments ) . Scale bar , 100 µm . Data information : In G and H , statistical significance was determined one - way ANOVA with Tukey multiple comparison test H ) or by Student ' s t - test ( G ) and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) . If significance levels were different for the pairwise comparisons with combined marking , asterisks , valid only for a subset of the pairs were put into brackets .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) hematoxylin-eosin staining with a scale bar of 50 µm and (C) mean epidermal thickness ±SEM of the back skin of ILEIfl/fl and ILEI∆Ep mice treated with acetone or TPA for 5 days (n=3-8; 3 independent experiments). Data information: In C statistical significance was determined one-way ANOVA with Tukey multiple comparison test (C marked with asterisks (*p<0.05; **p<0.01; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.(F-H) (F) Representative images of MPO+ microabscesses and neutrophils, (G) mean number ±SEM of MPO-positive microabsesses/cm skin section and (H) mean number of neutrophils (MPO+ cells)/mm2 epidermal-dermal area ±SEM on thin sections of acetone and TPA treated back skin of ILEIfl/fl and ILEI∆Ep mice (n=6-8 (G) and n=2-8 (H); 3 independent experiments). Scale bar, 100 µm. Data information: In G and H, statistical significance was determined one-way ANOVA with Tukey multiple comparison test H) or by Student's t-test (G) and marked with asterisks (*p<0.05; **p<0.01; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.


Figure 7Mean fold change ± SEM in mRNA expression of uPA and uPAR ( D ) in freshly sorted keratinocytes enriched for the interfollicular epithelium from acetone and TPA treated back skin , ( E ) in primary keratinocytes isolated from ILEIind and K5 - ILEIind mice , supplemented with doxycycline and treated with acetone or TPA for 96 hours ( n = 3 ( D ) , n = 6 ( E 1 - 2 independent experiments ) . Data information : In D , E Statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . Mean fold change ± SEM in mRNA expression of uPA and uPAR ( F ) in primary wild type keratinocytes treated with acetone and TPA and with murine recombinant wild type ILEI ( mILEI ) for 8 and 96 hours n = 6 F ) ; 1 - 2 independent experiments ) . Data information : Statistical significance was determined by one - way ANOVA with Tukey multiple comparison test and marked with asterisks ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . ( L ) ILEI Western blot analysis of conditioned media harvested from primary keratinocytes of ILEIind and K5 - ILEIind mice after 48 - hours doxycycline induction and treatment with TPA and DMSO or indicated concentrations of UK371804 . Loading was normalized to cell count , numbers indicate relative intensities . Lanes are from non - continuous parts of the same gel .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Mean fold change ±SEM in mRNA expression of uPA and uPAR (D) in freshly sorted keratinocytes enriched for the interfollicular epithelium from acetone and TPA treated back skin, (E) in primary keratinocytes isolated from ILEIind and K5-ILEIind mice, supplemented with doxycycline and treated with acetone or TPA for 96 hours (n=3 (D), n=6 (E 1-2 independent experiments). Data information: In D,E Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).Mean fold change ±SEM in mRNA expression of uPA and uPAR (F) in primary wild type keratinocytes treated with acetone and TPA and with murine recombinant wild type ILEI (mILEI) for 8 and 96 hours n=6 F); 1-2 independent experiments). Data information: Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).(L) ILEI Western blot analysis of conditioned media harvested from primary keratinocytes of ILEIind and K5-ILEIind mice after 48-hours doxycycline induction and treatment with TPA and DMSO or indicated concentrations of UK371804. Loading was normalized to cell count, numbers indicate relative intensities. Lanes are from non-continuous parts of the same gel.


Figure 1 ( B ) HEK293T cells were transfected with SFB control , SFB - tagged PR130 and SFB - B56δ , and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot ( WB ) . ( C ) Glutathione Sepharose beads bound with bacterially expressed recombinant GST , GST - B56δ or GST - PPP2R1A proteins were incubated with bacterially purified recombinant MBP - PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody . Interaction of B56δ - PPP2R1A was shown similarly using MBP - B56δ and GST - PPP2R1A proteins . Recombinant protein expression is shown by Coomassie staining . ( D ) HEK293T cells were transfected with SFB control , SFB - PPP2CA , SFB - PPP2CB , SFB - PPP2R1A , SFB - PPP2R1B and SFB - B56δ , and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody . ( E ) Cell lysates from HEK293T cells expressing SFB vector , SFB - B56α , SFB - B56β , SFB - B56γ , SFB - B56δ and SFB - B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody . ( G ) Cells expressing B56δ full length ( FL ) , Δ91 - 102 , L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting . ( H ) Various mutants of B56δ were transfected along with myc - PPP2R1A and pulled down using streptavidin beads and interaction with myc - PPP2R1A was detected by immunoblotting . ( I ) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio - layer interferometry . 6xHis - tagged PPM1G and PPP2R1A were immobilized on Ni - NTA biosensors and incubated with the GST - B56δ wild type ( WT ) or Δ91 - 102 mutant at various concentrations ( 100 - 400 nm ) . Curves represent experimental trace obtained from the BLI experiments . Binding affinities ( Kd ) ( + SD ; n = 3 independent experiments ) for B56δ - PPM1G and B56δ - PPP2R1A are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) HEK293T cells were transfected with SFB control, SFB-tagged PR130 and SFB-B56δ, and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot (WB).(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST, GST-B56δ or GST-PPP2R1A proteins were incubated with bacterially purified recombinant MBP-PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody. Interaction of B56δ-PPP2R1A was shown similarly using MBP- B56δ and GST-PPP2R1A proteins. Recombinant protein expression is shown by Coomassie staining.(D) HEK293T cells were transfected with SFB control, SFB- PPP2CA, SFB-PPP2CB, SFB-PPP2R1A, SFB-PPP2R1B and SFB- B56δ, and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody.(E) Cell lysates from HEK293T cells expressing SFB vector, SFB-B56α, SFB-B56β, SFB-B56γ, SFB-B56δ and SFB-B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody.(G) Cells expressing B56δ full length (FL), Δ91-102, L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting.(H) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting.(I) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio-layer interferometry. 6xHis-tagged PPM1G and PPP2R1A were immobilized on Ni-NTA biosensors and incubated with the GST-B56δ wild type (WT) or Δ91-102 mutant at various concentrations (100-400 nm). Curves represent experimental trace obtained from the BLI experiments. Binding affinities (Kd) (+SD; n=3 independent experiments) for B56δ-PPM1G and B56δ-PPP2R1A are shown.