Transwell invasion assays showed that shcircFNTA can increase J82 cells invasion ( I ) more profoundly than oelinearFNTA as compared with the oevector group . Scale bar , 100 μm .

Transwell invasion assays showed that shcircFNTA can increase J82 cells invasion (I) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.


J ) MTT assays showed that oecircFNTA enhances J82 cells chemo - resistance to cisplatin ( 0 , 1 , 2 , 5 , 10 μg / ml were tested ) . This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group ( upper panel ) . MTT assays showed that shcircFNTA increases UMUC3 cells chemo - sensitivity to cisplatin ( lower panel ) .

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J) MTT assays showed that oecircFNTA enhances J82 cells chemo-resistance to cisplatin (0, 1, 2, 5, 10 μg/ml were tested). This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group (upper panel). MTT assays showed that shcircFNTA increases UMUC3 cells chemo-sensitivity to cisplatin (lower panel).


K ) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para - tumor tissues ( N = 41 patients ) and representative images are shown . Scale bar , 250 μm .

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K) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para-tumor tissues (N=41 patients) and representative images are shown. Scale bar, 250 μm.


L ) The qRT - PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para - tumor tissues ( N = 23 patients ) .

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L) The qRT-PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para-tumor tissues (N=23 patients).


( A ) The protein expression of AR and FNTA ( circFNTA host gene ) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells .

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(A) The protein expression of AR and FNTA (circFNTA host gene) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells.


( B ) qRT - PCR results showed that mRNA levels of FNTA are not changed dramatically in UMUC3 cells with shAR and J82 cells with oeAR .

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(B) qRT-PCR results showed that mRNA levels of FNTA are not changed dramatically in UMUC3 cells with shAR and J82 cells with oeAR.


( C ) The mRNA levels of 4 RNA editing genes ( ADAR , ADAR2 , DHX9 , and QKI ) were tested with manipulated AR ( UMUC3 cells in left panel and J82 cells in right panel ) . Results showed ADAR2 mRNA is negatively correlated with AR expression .

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(C) The mRNA levels of 4 RNA editing genes (ADAR, ADAR2, DHX9, and QKI) were tested with manipulated AR (UMUC3 cells in left panel and J82 cells in right panel). Results showed ADAR2 mRNA is negatively correlated with AR expression.


( D ) Western blot analysis also confirmed that ADAR2 can be negatively regulated by AR at the protein level .

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(D) Western blot analysis also confirmed that ADAR2 can be negatively regulated by AR at the protein level.


( E ) Knock down efficiency of AR and ADAR2 ( shADAR2 # 1 and shADAR # 2 ) in UMUC3 cells , as determined by qRT - PCR .

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(E) Knock down efficiency of AR and ADAR2 (shADAR2#1 and shADAR#2) in UMUC3 cells, as determined by qRT-PCR.


( F ) shADAR2 # 1 and shADAR # 2 could both reverse shAR - dependent circFNTA reduction in UMUC3 cells .

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(F) shADAR2#1 and shADAR#2 could both reverse shAR-dependent circFNTA reduction in UMUC3 cells.


( G ) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR - dependent UMUC3 cells invasion reduction . Scale bar , 100 μm .

(G) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR-dependent UMUC3 cells invasion reduction. Scale bar, 100 μm.


( H ) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR - dependent effects on cisplatin chemo - resistance ( 2 μg / ml ) in UMUC3 cells .

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(H) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR-dependent effects on cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.


( J ) ChIP assays confirmed that AR can directly bind to ADAR2 AREI / II ( - 572nt to - 555nt ) .

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(J) ChIP assays confirmed that AR can directly bind to ADAR2 AREI/II (-572nt to -555nt).


( K ) ChIP pull - down DNA products were amplified by qRT - PCR reaction . The IgG antibody was used as a negative control .

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(K) ChIP pull-down DNA products were amplified by qRT-PCR reaction. The IgG antibody was used as a negative control.


( M ) Co - transfection of ARE Wild type ( WT ) or Mutant ( Mut ) ADAR2 promoter pGL3 - Luciferase constructs into UMUC3 cells with / without shAR ( left panel ) , and J82 cells with / without oeAR ( right panel ) . The luciferase assay was used to detect promoter activity .

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(M) Co-transfection of ARE Wild type (WT) or Mutant (Mut) ADAR2 promoter pGL3-Luciferase constructs into UMUC3 cells with/without shAR (left panel), and J82 cells with/without oeAR (right panel). The luciferase assay was used to detect promoter activity.


( A ) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells ( upper panel ) and oecircFNTA can increase FNTA expression , and that this was more profound than using oelinearFNTA in J82 cells ( lower panel ) .

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(A) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells (upper panel) and oecircFNTA can increase FNTA expression, and that this was more profound than using oelinearFNTA in J82 cells (lower panel).


( B ) A RNA pull - down assay was used to test the binding of circFNTA to 11 miRNAs ( predicted by online database ) in UMUC3 cells . The results showed that miR - 328 - 5p , miR - 370 - 3p and miR - 920 can be pulled - down by circFNTA .

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(B) A RNA pull-down assay was used to test the binding of circFNTA to 11 miRNAs (predicted by online database) in UMUC3 cells. The results showed that miR-328-5p, miR-370-3p and miR-920 can be pulled-down by circFNTA.


( C ) Relative miRNA expressions were quantified by qRT - PCR to test miRNA overexpression efficacy .

(C) Relative miRNA expressions were quantified by qRT-PCR to test miRNA overexpression efficacy.


Transwell invasion assay ( D ) confirmed that only oemiR - 370 - 3p can reverse oecircFNTA induced invasion in J82 cells ,

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Transwell invasion assay (D) confirmed that only oemiR-370-3p can reverse oecircFNTA induced invasion in J82 cells,


MTT assays ( E ) confirmed that oemiR - 370 - 3p can reverse oecircFNTA induced cisplatin chemo - resistance ( 2 μg / ml ) in J82 cells . Scale bar , 100 μm .

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MTT assays (E) confirmed that oemiR-370-3p can reverse oecircFNTA induced cisplatin chemo-resistance (2 μg/ml) in J82 cells. Scale bar, 100 μm.


Transducing the miR - 370 - 3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot ( F )

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Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot (F)


Transducing the miR - 370 - 3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Transwell assay ( G ) .

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Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Transwell assay (G).


( H ) MTT assays also confirmed that transducing with a miR - 370 - 3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo - resistance ( 2 μg / ml ) reduction by shcircFNTA .

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(H) MTT assays also confirmed that transducing with a miR-370-3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo-resistance (2 μg/ml) reduction by shcircFNTA.


( J ) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT - PCR .

(J) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT-PCR.


( K ) Transfection with oecircFNTA WT could increase J82 cells invasion , while oecircFNTA Mut cannot . Scale bar , 100 μm .

(K) Transfection with oecircFNTA WT could increase J82 cells invasion, while oecircFNTA Mut cannot. Scale bar, 100 μm.


( L ) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo - resistance ( 2 μg / ml ) , while oecircFNTA Mut did not have this effect .

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(L) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo-resistance (2 μg/ml), while oecircFNTA Mut did not have this effect.


( N ) Luciferase reporter activity after transfection of WT and Mut FNTA 3 ' UTR reporter constructs into UMUC3 cells ( left panel ) , comparing transduced miR - 370 - 3p inhibitor vs Ctrl and J82 cells ( right panel ) , and comparing oemiR - 370 - 3p vs control PLV .

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(N) Luciferase reporter activity after transfection of WT and Mut FNTA 3'UTR reporter constructs into UMUC3 cells (left panel), comparing transduced miR-370-3p inhibitor vs Ctrl and J82 cells (right panel), and comparing oemiR-370-3p vs control PLV.


( A ) Western blots show the knock - down efficiency of shFNTA in J82 and UMUC3 cells .

(A) Western blots show the knock-down efficiency of shFNTA in J82 and UMUC3 cells.


( B ) Transwell assays indicated that shFNTA can partly reverse oecircFNTA - increased invasion in J82 and UMUC3 cells . Quantifications are shown on the right . Scale bar , 100 μm .

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(B) Transwell assays indicated that shFNTA can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Quantifications are shown on the right. Scale bar, 100 μm.


( C ) MTT assays also confirmed that shFNTA can reverse oecircFNTA - increased chemo - resistance to cisplatin ( 2 μg / ml ) in J82 and UMUC3 cells .

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(C) MTT assays also confirmed that shFNTA can reverse oecircFNTA-increased chemo-resistance to cisplatin (2 μg/ml) in J82 and UMUC3 cells.


Transwell assays ( D , with quantitations at the right ) indicated that treatment with Tipifarnib ( farnesyltransferase inhibitor ) can partly reverse oecircFNTA - increased invasion in J82 and UMUC3 cells . Scale bar , 100 μm .

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Transwell assays (D, with quantitations at the right) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.


MTT assays ( E ) indicated that treatment with Tipifarnib ( farnesyltransferase inhibitor ) can partly reverse oecircFNTA - increased invasion in J82 and UMUC3 cells . Scale bar , 100 μm .

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MTT assays (E) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.


( F ) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 cells did not change KRAS protein level significantly .

(F) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 cells did not change KRAS protein level significantly.


( G ) Raf - 1 RBD GST - pull - down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 cells ( upper panel ) , and oecircFNTA can increase KRAS activity in J82 cells ( lower panel ) .

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(G) Raf-1 RBD GST-pull-down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 cells (upper panel), and oecircFNTA can increase KRAS activity in J82 cells (lower panel).


( H ) Western blots showed that shcircFNTA can decrease ERK1 / 2 and MEK1 / 2 phosphorylation in UMUC3 cells ( left panel ) , and oecircFNTA can increase their phosphorylation in J82 cells ( right panel ) .

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(H) Western blots showed that shcircFNTA can decrease ERK1/2 and MEK1/2 phosphorylation in UMUC3 cells (left panel), and oecircFNTA can increase their phosphorylation in J82 cells (right panel).


( A ) UMUC3 - PLKO and UMUC3 - shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model , and cisplatin ( 1 mg / kg ) was intraperitoneally administered . Images of tumors are shown after mice were sacrificed ( N = 6 ) . ( B ) Compared with the vector ( PLKO ) group , the tumor growth rate was significantly inhibited in shcircFNTA nude mice . ( C ) Tumor weights also decreased in the shcircFNTA group .

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(A) UMUC3-PLKO and UMUC3-shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model, and cisplatin (1 mg/kg) was intraperitoneally administered. Images of tumors are shown after mice were sacrificed (N= 6). (B) Compared with the vector (PLKO) group, the tumor growth rate was significantly inhibited in shcircFNTA nude mice. (C) Tumor weights also decreased in the shcircFNTA group.


( D - E ) The qRT - PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced ( D ) , and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group ( E ) .

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(D-E) The qRT-PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced (D), and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group (E).


( F ) Representative immunohistochemistry ( IHC ) images detecting FNTA , p - ERK1 / 2 and p - MEK1 / 2 in PLKO tumurs as compared to shcircFNTA xenografted tumors . Scale bars : 100 μm ( upper panels ) and 25 μm ( lower panels ) . ( G ) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA , p - ERK1 / 2 and p - MEK1 / 2 .

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(F) Representative immunohistochemistry (IHC) images detecting FNTA, p-ERK1/2 and p-MEK1/2 in PLKO tumurs as compared to shcircFNTA xenografted tumors. Scale bars: 100 μm (upper panels) and 25 μm (lower panels). (G) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA, p-ERK1/2 and p-MEK1/2.


( H ) The tumor tail vein injection model was established to observe lung metastasis ( N = 6 ) . Representative images of lung metastasis foci in gross and microscopic HE examination . Scale bars : 400 μm ( upper panels ) and 100 μm ( lower panels ) . ( I ) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group . ( J ) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group .

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(H) The tumor tail vein injection model was established to observe lung metastasis (N=6). Representative images of lung metastasis foci in gross and microscopic HE examination. Scale bars: 400 μm (upper panels) and 100 μm (lower panels). (I) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group. (J) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group.


A GS ∆ 44 myoblasts were transfected with an empty vector ( CTRL ) or with a CELF2a overexpressing construct ( OE CELF2A ) ( Martone et al , 2016 ) . Cells were collected after 10 days in differentiation medium ( DM ) and the RNA was analysed by RT - PCR for CELF2a expression and DMD exon 45 skipping . GAPDH was used as loading control . The arrow indicates the DMD exon 45 skipped band . Representative results are shown ( n = 3 ) .

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A GS∆44 myoblasts were transfected with an empty vector (CTRL) or with a CELF2a overexpressing construct (OE CELF2A) (Martone et al, 2016). Cells were collected after 10 days in differentiation medium (DM) and the RNA was analysed by RT-PCR for CELF2a expression and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).


B Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI staining ( in blue ) of iPSCs obtained from WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) and ∆ 44 edited clones ( UP ∆ 44 # 3 . 1 and UP ∆ 44 # 3 . 5 ) differentiated for 9 days into myocytes by MYOD / BAF60c overexpression . Scale bar 100 μm .

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B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT (WT#1), UP∆44 (UP∆44#3) and ∆44 edited clones (UP∆44#3.1 and UP∆44#3.5) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm.


C Histogram represents fusion index quantification . At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2 ) . Data are presented as mean ± SD of the biological replicates .

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C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.


D RNA extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) and edited UP ∆ 44 iPSC ( UP ∆ 44 # 3 . 1 ∆ CELF2a and UP ∆ 44 # 3 . 5 ∆ CELF2a ) clones was analysed by RT - PCR for CELF2a , CELF2b , CELF2c and DMD exon 45 skipping . GAPDH was used as loading control . The arrow indicates the DMD exon 45 skipped band . Representative results are shown ( n = 3 ) .

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D RNA extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).


E Western blot on proteins ( 40 μg ) extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) and edited UP ∆ 44 iPSC ( UP ∆ 44 # 3 . 1 ∆ CELF2a and UP ∆ 44 # 3 . 5 ∆ CELF2a ) clones probed with antibodies against dystrophin ( DMD ) . Actinin ( ACTN ) was used as a loading control . For the WT 5 % sample 2 μg of proteins were diluted in 38 μg of proteins belonging to UP ∆ 44 # 3 to reach a total amount of 40 μg . Representative results are shown ( n = 3 ) .

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E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample 2 μg of proteins were diluted in 38 μg of proteins belonging to UP∆44#3 to reach a total amount of 40 μg. Representative results are shown (n=3).


A iPSCs obtained from a control ( WT # 1 ; ( Lenzi et al , 2015 ) , GS ∆ 44 ( GS ∆ 44 # 2 , GS ∆ 44 # 8 ) and GSM ( GSM # 1 ) were differentiated into myocytes by MYOD / BAF60c overexpression . Cells were collected before the induction ( iPSC ) and after 9 days in DM ( Myo ) and RNA was analysed by RT - PCR for CELF2a , CELF2b , CELF2c and DMD expression . GAPDH was used as loading control . Representative results are shown ( n = 3 ) .

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A iPSCs obtained from a control (WT#1; (Lenzi et al, 2015), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) were differentiated into myocytes by MYOD/BAF60c overexpression. Cells were collected before the induction (iPSC) and after 9 days in DM (Myo) and RNA was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD expression. GAPDH was used as loading control. Representative results are shown (n=3).


B Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI staining ( in blue ) of iPSCs obtained from WT # 1 , GS ∆ 44 ( GS ∆ 44 # 8 ) and GS ∆ 44 edited clones ( GS ∆ 44 # 8 . 2 ) differentiated for 9 days into myocytes by MYOD / BAF60c overexpression . Scale bar 100 μm . C Histogram represents fusion index quantification . At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2 ) . Data are presented as mean ± SD of the biological replicates .

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B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.


D RNA extracted from myocytes obtained by differentiation of WT # 1 , GS ∆ 44 ( GS ∆ 44 # 8 ) and GS ∆ 44 edited clones ( GS ∆ 44 # 8 . 2 ∆ CELF2a ) was analysed by RT - PCR for CELF2a , CELF2b , CELF2c and DMD exon 45 skipping . GAPDH was used as loading control . The arrow indicates the DMD exon 45 skipped band . Representative results are shown ( n = 3 ) .

D RNA extracted from myocytes obtained by differentiation of WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2 ∆CELF2a) was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).


E Western blot on proteins ( 40 μg ) extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , GS ∆ 44 ( GS ∆ 44 # 8 ) and edited GS ∆ 44 ( GS ∆ 44 # 8 . 2 ∆ CELF2a ) iPSC clones probed with antibodies against dystrophin ( DMD ) . Actinin ( ACTN ) was used as a loading control . For the WT 5 % sample , 2 μg of proteins were diluted in 38 μg of proteins belonging from GS ∆ 44 # 8 to reach the total amount of 40 μg . Representative results are shown ( n = 3 ) .

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E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), GS∆44 (GS∆44#8) and edited GS∆44 (GS∆44#8.2 ∆CELF2a) iPSC clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins were diluted in 38 μg of proteins belonging from GS∆44#8 to reach the total amount of 40 μg. Representative results are shown (n=3).


A DNA accessibility ( ATAC - sequencing ) and chromatin marks ( ChIP - seq ) signals in the Celf2 region ( + / - 50kb ) using the UCSC genome browser . ATAC - seq peaks called in the two conditions are reported below each track . The differential peaks specific for one of the two conditions are reported in the tracks ATACseq _ specific peaks _ WT and ATACseq _ specific peaks _ GS ∆ 44 . ChIP - seq signals for H3K4me3 , H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts ( WT myoblasts in light orange ; GSΔ44 myoblasts in light blue ) . ChIP - seq for MyoD in control cells was obtained from ( MacQuarrie et al , 2013 ) ( GSM1218849 ) . Orange boxes highlight the regions containing the first exon of each isoform ( a , b and c ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 DNA accessibility (ATAC-sequencing) and chromatin marks (ChIP-seq) signals in the Celf2 region (+/- 50kb) using the UCSC genome browser. ATAC-seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GS∆44. ChIP-seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts (WT myoblasts in light orange; GSΔ44 myoblasts in light blue). ChIP-seq for MyoD in control cells was obtained from (MacQuarrie et al, 2013) (GSM1218849). Orange boxes highlight the regions containing the first exon of each isoform (a, b and c).


D Chromatins extracted from Control ( WT ) and GS ∆ 44 myoblasts were immunoprecipitated with anti - MyoD and control IgG antibodies . Regions corresponding to the MyoD binding sites present in Peak A ( see Figure EV3D ) and in the MAFA and LSTZ2 genes were analysed by qPCR . Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation ( IP ) are plotted for WT and GS ∆ 44 samples . The mean ± SEM of triplicates from one representative among three experiment is shown . Statistical significance of differences between means was assessed by a two - tailed t - test and P < 0 . 05 was considered significant . * P = 0 . 0339 , * * P = 0 . 0067 .

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D Chromatins extracted from Control (WT) and GS∆44 myoblasts were immunoprecipitated with anti-MyoD and control IgG antibodies. Regions corresponding to the MyoD binding sites present in Peak A (see Figure EV3D) and in the MAFA and LSTZ2 genes were analysed by qPCR. Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation (IP) are plotted for WT and GS∆44 samples. The mean ± SEM of triplicates from one representative among three experiment is shown. Statistical significance of differences between means was assessed by a two-tailed t-test and P<0.05 was considered significant. *P=0.0339, **P=0.0067.


B RT - PCR analyses of indicated RNAs in control ( WT ) , ∆ 44 and in GS ∆ 44 myocytes collected after 9 days upon the induction of differentiation . GAPDH was used as control . Representative results are shown ( 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 O O O O O O O O O O O O O O O O O

B RT-PCR analyses of indicated RNAs in control (WT), ∆44 and in GS∆44 myocytes collected after 9 days upon the induction of differentiation. GAPDH was used as control. Representative results are shown (n=3).


C RT - PCR analyses for CELF2a and DUXAP8 expression in WT and GS ∆ 44 myoblasts ( GM ) and myocytes ( DM ) . GAPDH was used as control . Representative results are shown ( 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 O O O O O O O O O O O

C RT-PCR analyses for CELF2a and DUXAP8 expression in WT and GS∆44 myoblasts (GM) and myocytes (DM). GAPDH was used as control. Representative results are shown (n=3).


D RT - PCR analysis of DUXAP8 expression in RNA extracted from GS ∆ 44 , GS ∆ 44 ' s mother ( GSM ) and GS ∆ 44 ' s father ( F ) fibroblasts . GAPDH was used as control . Lane ( - ) indicates the negative control . Representative results are shown ( 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 O O O O O O O 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 RT-PCR analysis of DUXAP8 expression in RNA extracted from GS∆44, GS∆44's mother (GSM) and GS∆44's father (F) fibroblasts. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


E RT - PCR analysis for DUXAP8 expression in RNA extracted from WT and GS ∆ 44 myocytes and from WT ( WT # 1 ) , GS ∆ 44 ( GS ∆ 44 # 2 , GS ∆ 44 # 8 ) and GSM ( GSM # 1 ) iPSCs induced to differentiation for 9 days . GAPDH was used as control . Lane ( - ) indicates the negative control . Representative results are shown ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RT-PCR analysis for DUXAP8 expression in RNA extracted from WT and GS∆44 myocytes and from WT (WT#1), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) iPSCs induced to differentiation for 9 days. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


F RT - PCR for the indicated RNAs on extracts from GS ∆ 44 myoblasts treated with either control siRNAs ( siSCR ) or siRNAs against DUXAP8 ( siDUXAP8 ) and collected two days after transfection . GAPDH was used as control . Lane ( - ) indicates the negative control . Representative results are shown ( n = 3 ) .

F RT-PCR for the indicated RNAs on extracts from GS∆44 myoblasts treated with either control siRNAs (siSCR) or siRNAs against DUXAP8 (siDUXAP8) and collected two days after transfection. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 ( OE DUXAP8 ) or an empty vector ( Empty ) , and collected after 96h . Relative mRNA levels were calculated with the delta delta Ct method . qPCR reactions were normalized against an internal control ( GAPDH ) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1 . The mean ± SEM of triplicates from one representative experiment is shown ( n = 3 ) .

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G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 (OE DUXAP8) or an empty vector (Empty), and collected after 96h. Relative mRNA levels were calculated with the delta delta Ct method. qPCR reactions were normalized against an internal control (GAPDH) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown (n=3).


( A ) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h , detected by fluorescent microplate reader and confocal microscopy , respectively . Scale bar 20 µm .

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(A) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h, detected by fluorescent microplate reader and confocal microscopy, respectively. Scale bar 20 µm.


( B ) Representative confocal microscopy images of pPMNs that were treated without ( control ) or with 50 nM PMA for 3 h , and stained for lamin B and DNA as described in panel B . The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope . Scale bar 10 µm .

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(B) Representative confocal microscopy images of pPMNs that were treated without (control) or with 50 nM PMA for 3 h, and stained for lamin B and DNA as described in panel B. The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope. Scale bar 10 µm.


( D ) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice . Vinculin served as loading control .

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(D) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice. Vinculin served as loading control.


( E ) Summary analysis of PAF - induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h , then fixed by 2 % PFA and stained by Sytox Green , following by detection with fluorescent microplate reader .

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(E) Summary analysis of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, then fixed by 2% PFA and stained by Sytox Green, following by detection with fluorescent microplate reader.


( F ) The endpoint analysis of NET - DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without ( control ) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the 3 h time point . The NET - DNA release index was reported in comparison to an assigned value of 100 % for the total DNA released by neutrophils lysed by 0 . 5 % ( v / v ) Triton - X - 100 .

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(F) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.


( G , H ) Representative images ( G ) and summary analysis ( H ) of PAF - induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h , and stained with both cell permeable SYTO Red and cell impermeable Sytox Green , without fixation . Images were taken by Olympus confocal microscopy , followed by automated quantification of NETs on 5 - 6 non - overlapping area per well using ImageJ for calculation of % cells with NET formation . ( G ) Scale bars , 40 μm .

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 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

(G,H) Representative images (G) and summary analysis (H) of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red and cell impermeable Sytox Green, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (G) Scale bars, 40 μm.


( A , B ) Representative and summary of immunoblots of full - length lanes of lamin B in human dPMNs that were treated with PMA for 0 , 0 . 5 , 1 , 2 , 3 h during NET formation , or apoptotic dPMNs that were induced by PMA for longer term ( 12 h ) treatment ( A ) . β - actin served as loading

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

(A,B) Representative and summary of immunoblots of full-length lanes of lamin B in human dPMNs that were treated with PMA for 0, 0.5, 1, 2, 3 h during NET formation, or apoptotic dPMNs that were induced by PMA for longer term (12 h) treatment (A). β-actin served as loading control


( C , D ) Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0 , 0 . 5 , 1 , 2 , 3 h during NET formation . β - actin served as loading

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(C,D) Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0, 0.5, 1, 2, 3 h during NET formation. β-actin served as loading control


( E ) Representative confocal microscopy image of a group of pPMNs with NET formation ( enclosed by a light blue trapezoid ) and their extracellular NETs ( indicated by a purple rectangle square ) released by these cells , as well as the immunoblot analysis of un - cleaved lamin B and cleaved histone H3 with the whole cell lysates , as well as immunoblot analysis of un - cleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment . The anti - human lamin B was used in all immunoblots confocal image ( E ) , and the latter was further detected by FITC - labeled 2nd Ab , scale bar , 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(E) Representative confocal microscopy image of a group of pPMNs with NET formation (enclosed by a light blue trapezoid) and their extracellular NETs (indicated by a purple rectangle square) released by these cells, as well as the immunoblot analysis of un-cleaved lamin B and cleaved histone H3 with the whole cell lysates, as well as immunoblot analysis of un-cleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment. The anti-human lamin B was used in all immunoblots confocal image (E), and the latter was further detected by FITC-labeled 2nd Ab, scale bar, 20 μm.


( F ) Representative immunoblots with full - length lanes for lamin B analysis , and pro - caspase - 3 and its activated form caspase - 3 in human dPMNs with NET formation that were treated with PMA for 0 , 0 . 5 , 1 , 2 , 3 h . Anti - human caspase - 3 was used ( F , G ) , and β - actin served as loading

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(F) Representative immunoblots with full-length lanes for lamin B analysis, and pro-caspase-3 and its activated form caspase-3 in human dPMNs with NET formation that were treated with PMA for 0, 0.5, 1, 2, 3 h. Anti-human caspase-3 was used (F,G), and β-actin served as loading control


( G ) Representative immunoblots display full - length lamin B ( 69 kDa ) and their cleaved fragments ( 45 and 25 kDa ) , and pro - caspase - 3 and its activated form caspase - 3 in the apoptotic human dPMNs that were treated with PMA for 12 , 24h . Anti - human caspase - 3 was used ( F , G ) , and β - actin served as loading

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(G) Representative immunoblots display full-length lamin B (69 kDa) and their cleaved fragments (45 and 25 kDa), and pro-caspase-3 and its activated form caspase-3 in the apoptotic human dPMNs that were treated with PMA for 12, 24h. Anti-human caspase-3 was used (F,G), and β-actin served as loading control


( A ) Representative images for the time course of PKCα nuclear translocation , subsequent nuclear envelope rupture , and DNA release in human dPMNs exposed to 50 nM PMA for 5 , 15 , 30 , 60 , and 180 min , then stained concomitantly for DNA ( DAPI ) , nuclear lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , and PKCα ( primary anti - human PKCα , and PE - labeled secondary antibody ) , following by confocal fluorescent microscopy analysis . White - empty arrows indicate cytoplasmic distribution of PKCα at 5 , 15 , and 30 min , yellow arrows indicate site of discontinuity / rupture of nuclear envelope at 60 min , light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min ( A ) . Scale bars , 20 μm . The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining .

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

(A) Representative images for the time course of PKCα nuclear translocation, subsequent nuclear envelope rupture, and DNA release in human dPMNs exposed to 50 nM PMA for 5, 15, 30, 60, and 180 min, then stained concomitantly for DNA (DAPI), nuclear lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and PKCα (primary anti-human PKCα, and PE-labeled secondary antibody), following by confocal fluorescent microscopy analysis. White-empty arrows indicate cytoplasmic distribution of PKCα at 5, 15, and 30 min, yellow arrows indicate site of discontinuity/rupture of nuclear envelope at 60 min, light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min (A). Scale bars, 20 μm. The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining.


( B ) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti - lamin B , and FITC - labeled secondary antibody .

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(B) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti-lamin B, and FITC-labeled secondary antibody.


( C Representative immunoblots of total PKCα and p - PKCα in primary human pPMNs that were treated by PMA ( C ) for 0 , 0 . 5 , 1 , 2 , 3 h .

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(C Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PMA (C) for 0, 0.5, 1, 2, 3 h.


D ) Representative immunoblots of total PKCα and p - PKCα in primary human pPMNs that were treated by PAF ( D ) for 0 , 0 . 5 , 1 , 2 , 3 h .

D) Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PAF (D) for 0, 0.5, 1, 2, 3 h.


( A ) Representative immunoblots of p - PKCα and total PKCα of isolated nuclei and the corresponding status of lamin B in human dPMNs that were treated with PMA for 0 or 2 h .

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(A) Representative immunoblots of p-PKCα and total PKCα of isolated nuclei and the corresponding status of lamin B in human dPMNs that were treated with PMA for 0 or 2 h.


( B ) Representative sectional images of nuclei of two neutrophils treated by PMA , stained for lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , p - PKCα ( primary anti - human p - PKCαS657 , and PE - labeled secondary antibody ) , and nuclear DNA ( DAPI ) . The accumulation of p - PKCα ( red arrow pointed ) at the site of ruptured / interrupted ( green arrow ) nuclear lamin B , and swollen / de - condensed DNA ( blue arrow ) were indicated ( b1 ) . A neutrophil ( white empty arrow ) with NET formation that contained the mixture of DNA with p - PKCα and lamin B ( white arrow ) was also demonstrated ( b2 ) . Scale bars , 10 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 O 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 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 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

(B) Representative sectional images of nuclei of two neutrophils treated by PMA, stained for lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), p-PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody), and nuclear DNA (DAPI). The accumulation of p-PKCα (red arrow pointed) at the site of ruptured/interrupted (green arrow) nuclear lamin B, and swollen/de-condensed DNA (blue arrow) were indicated (b1). A neutrophil (white empty arrow) with NET formation that contained the mixture of DNA with p-PKCα and lamin B (white arrow) was also demonstrated (b2). Scale bars, 10 µm.


( C ) Immunoblot images of lamin B , then stripped and probed for p - PKCα , of the homogenates of NETs isolated from conditioned medium of primary human pPMNs with NET formation that were induced by PMA .

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(C) Immunoblot images of lamin B, then stripped and probed for p-PKCα, of the homogenates of NETs isolated from conditioned medium of primary human pPMNs with NET formation that were induced by PMA.


( D , E ) Representative and summary immunoblot ( IB ) detection of phospho - lamin B and total lamin B with the lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from human dPMNs that were treated either by PMA ( D , E ) for 0 or 3 h . Data represent mean ± SD ( n = 3 - 5 biological replicates ) for E . P * < 0 . 05 between groups as indicated . Comparison between two groups was analyzed by student t test .

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(D,E) Representative and summary immunoblot (IB) detection of phospho-lamin B and total lamin B with the lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were treated either by PMA (D,E) for 0 or 3 h. Data represent mean ± SD (n=3-5 biological replicates) for E. P*<0.05 between groups as indicated. Comparison between two groups was analyzed by student t test.


( A ) Summary and representative immunoblots of total PKCα and p - PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h , and then treated without or with PMA ( A ) for 3 h . Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit ( A )

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(A) Summary and representative immunoblots of total PKCα and p-PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated without or with PMA (A) for 3 h. Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit (A)


( B ) Immunoblot ( IB ) detection of the phospho - lamin B and total lamin B with lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h , and then treated by PMA ( B ) for 3 h .

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(B) Immunoblot (IB) detection of the phospho-lamin B and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated by PMA (B) for 3 h.


( C ) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h , and then treated without or with PMA for 3 h , followed by staining of lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , and phosphorylated PKCα ( primary anti - human p - PKCαS657 , and PE - labeled secondary antibody ) . The green / white arrows indicate the site of nuclear envelope rupture . Scale bars , 10 μm ( C ) .

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 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

(C) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h, and then treated without or with PMA for 3 h, followed by staining of lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and phosphorylated PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody). The green/white arrows indicate the site of nuclear envelope rupture. Scale bars, 10 μm (C).


( E ) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B ( WT control ) , or mutants with single or multiple defined point mutations at PKCα - consensus - phosphorylation sites ( S395A , S405A , S408A ) of lamin B , and treated for 3 h by 10 μM PAF in medium containing cell - permeable dye SYTO Red ( 500 nM ) for the total cell count , while the cells with NET formation were stained by cell impermeable dye SYTOX Blue ( 5 μM ) . Then the images were taken with Olympus confocal microscopy , followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation . percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ ( E )

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(E) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B (WT control), or mutants with single or multiple defined point mutations at PKCα-consensus-phosphorylation sites (S395A, S405A, S408A) of lamin B, and treated for 3 h by 10 μM PAF in medium containing cell-permeable dye SYTO Red (500 nM) for the total cell count, while the cells with NET formation were stained by cell impermeable dye SYTOX Blue (5 μM). Then the images were taken with Olympus confocal microscopy, followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation. percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ (E)


( A ) Summary and representative immunoblots of PKCα expressions in mouse bone marrow neutrophils from PKCα - / - , PKCα + / - , and their littermate WT ( PKCα + / + ) mice . Vinculin served as loading control .

O O O O O O O O O O O O O O O O O O O O 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) Summary and representative immunoblots of PKCα expressions in mouse bone marrow neutrophils from PKCα-/-, PKCα+/-, and their littermate WT (PKCα+/+) mice. Vinculin served as loading control.


( B ) Representative immunoblot ( IB ) detection of the phospho - lamin B ( Ser ) and total lamin B with lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from mouse peritoneal mPMNs from WT vs PKCα - / - mice , stimulated without or with PAF for 3 h .

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

(B) Representative immunoblot (IB) detection of the phospho-lamin B (Ser) and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from mouse peritoneal mPMNs from WT vs PKCα-/- mice, stimulated without or with PAF for 3 h.


( C ) Summary analyses of NET formation in peritoneal mPMNs , from WT , PKCα + / - , or PKCα - / - mice , which were treated without or with either 5 µM or 10 µM PAF for 3 h following by 2 % PFA fixation and fluorometric microplate analysis .

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

(C) Summary analyses of NET formation in peritoneal mPMNs, from WT, PKCα+/-, or PKCα-/- mice, which were treated without or with either 5 µM or 10 µM PAF for 3 h following by 2% PFA fixation and fluorometric microplate analysis.


( D ) The endpoint analysis of NET - DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and PKCα - / - mice without ( control ) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the end of the 3 h time point . The NET - DNA release index was reported in comparison to an assigned value of 100 % for the total DNA released by neutrophils lysed by 0 . 5 % ( v / v ) Triton - X - 100 .

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-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 B-MEASURED_VAR 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

(D) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and PKCα-/- mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the end of the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.


( E , F ) ( E ) Representative and ( F ) summary analysis of NET formation of mouse peritoneal mPMNs from WT vs PKCα - / - mice stimulated without or with 10 µM PAF for 3 h , and stained with both cell permeable SYTO Red dye and cell impermeable Sytox Green dye , without fixation . Images were taken by Olympus confocal microscopy , followed by automated quantification of NETs on 5 - 6 non - overlapping area per well using ImageJ for calculation of % cells with NET formation . ( E ) Scale bars , 50 μm . % cells with NET formation by image analysis using ImageJ ( F )

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

(E, F) (E) Representative and (F) summary analysis of NET formation of mouse peritoneal mPMNs from WT vs PKCα-/- mice stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red dye and cell impermeable Sytox Green dye, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (E) Scale bars, 50 μm. % cells with NET formation by image analysis using ImageJ (F)


( A , B ) representative analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT ( A ) vs Lmnb1TG ( B ) mice with UVB exposure , in which DNA was stained by DAPI , citrullinated histone H3 was probed by rabbit anti - mouse citrullinated Histone H3 , following stained by Alexa Fluor - 488 labeled donkey anti - rabbit secondary antibody , while neutrophil surface marker Ly6B was probed by rat anti - mouse Ly6B Ab following stained by Alexa Fluor - 647 conjugated goat anti - rat secondary antibody . The images of different staining and their overlays are shown ( A , B ) . The parental neutrophils ( stained by neutrophil membrane surface marker Ly6B ) and their released extracellular NET structures ( co - stained by anti - cit H3 and DAPI ) are indicated by red arrows and light blue arrows respectively . The white arrows indicated neutrophils without NET release . Scale bar , 50 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 O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(A,B) representative analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.


( C ) summary analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT ( A ) vs Lmnb1TG ( B ) mice with UVB exposure , in which DNA was stained by DAPI , citrullinated histone H3 was probed by rabbit anti - mouse citrullinated Histone H3 , following stained by Alexa Fluor - 488 labeled donkey anti - rabbit secondary antibody , while neutrophil surface marker Ly6B was probed by rat anti - mouse Ly6B Ab following stained by Alexa Fluor - 647 conjugated goat anti - rat secondary antibody . The images of different staining and their overlays are shown ( A , B ) . The parental neutrophils ( stained by neutrophil membrane surface marker Ly6B ) and their released extracellular NET structures ( co - stained by anti - cit H3 and DAPI ) are indicated by red arrows and light blue arrows respectively . The white arrows indicated neutrophils without NET release . Scale bar , 50 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 O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(C) summary analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.


( D , E ) Summary analysis for staining area of neutrophils with NET formation and their exhibition of IL - 17A ( D ) or TNFα ( E ) , in the skin tissue of WT vs Lmnb1TG mice without ( sham ) or with UVB exposure , by co - staining of Ly6B ( stained as described in panels A - C ) with IL - 17A ( D ) or TNFα ( E ) using FITC - conjugated rat anti - mouse IL - 17A , or FITC conjugated rat anti - mouse TNFα Abs .

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(D,E) Summary analysis for staining area of neutrophils with NET formation and their exhibition of IL-17A (D) or TNFα (E), in the skin tissue of WT vs Lmnb1TG mice without (sham) or with UVB exposure, by co-staining of Ly6B (stained as described in panels A-C) with IL-17A (D) or TNFα (E) using FITC-conjugated rat anti-mouse IL-17A, or FITC conjugated rat anti-mouse TNFα Abs.


( A , B ) Representative images of the expression of NEDD4L in the skins from IMQ - treated mice ( A ) or psoriasis patients ( B ) as detected by IHC on the left . The NEDD4L expression level was calculated by multiplying the staining intensity score and the extent score and is shown on the right . ( A , n = 5 , B , normal , n = 15 , psoriasis , n = 36 ) . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of the expression of NEDD4L in the skins from IMQ-treated mice (A) or psoriasis patients (B) as detected by IHC on the left. The NEDD4L expression level was calculated by multiplying the staining intensity score and the extent score and is shown on the right. (A, n=5, B, normal, n=15, psoriasis, n=36). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.


( C , D ) Representative images of the expression of EZH2 in the skin epidermis from the IMQ - treated mice ( C ) and the psoriasis patients ( D ) . The number of EZH2 - positive cells per HPF ( high power field ) in each mouse and patient is shown on the right . ( C , control , n = 3 , IMQ , n = 6 . D , normal , n = 13 , psoriasis , n = 27 ) . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Representative images of the expression of EZH2 in the skin epidermis from the IMQ-treated mice (C) and the psoriasis patients (D). The number of EZH2-positive cells per HPF (high power field) in each mouse and patient is shown on the right. (C, control, n=3, IMQ, n=6. D, normal, n=13, psoriasis, n=27). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.


( E ) WB analysis of NEDD4L , EZH2 and EZH1 expression in the EZH2 - specific siRNA ( siEZH2 ) - or negative control siRNA ( siNC ) - transfected NHEKs with IMQ stimulation . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) .

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(E) WB analysis of NEDD4L, EZH2 and EZH1 expression in the EZH2-specific siRNA (siEZH2)- or negative control siRNA (siNC)-transfected NHEKs with IMQ stimulation. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×).


( D ) Representative images of HE staining of the back tissues are shown on the left . The epidermal thickness measured using the scale in the microscope is shown on the right . ( E ) Representative images of Ki67 staining in back tissues are shown on the left . The number of Ki67 - positive cells per HPF in each mouse is shown on the right . ( F ) Representative images of HE staining of the ear tissues are shown on the left . The epidermis thickness measured using the scale in the microscope is shown on the right . ( G ) Representative images of Ki67 staining in ear tissues ( same specimen as in F ) are shown on the left . The number of Ki67 positive cells per HPF in each mouse is shown on the right . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments . For back tissues , scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . For ear tissues , scale bar , 200 μm ( 100 × ) or 200 μm ( 200 × ) . Significant differences were tested using a two - tailed Student ' s t test Control , n = 3 , IMQ , n = 6 . * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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

(D) Representative images of HE staining of the back tissues are shown on the left. The epidermal thickness measured using the scale in the microscope is shown on the right. (E) Representative images of Ki67 staining in back tissues are shown on the left. The number of Ki67-positive cells per HPF in each mouse is shown on the right. (F) Representative images of HE staining of the ear tissues are shown on the left. The epidermis thickness measured using the scale in the microscope is shown on the right. (G) Representative images of Ki67 staining in ear tissues (same specimen as in F) are shown on the left. The number of Ki67 positive cells per HPF in each mouse is shown on the right. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Student's t test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance.


NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs . WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L - WT or NEDD4L - C943S mutants . cell signaling pathway activation induced by IL - 6 were detected by WB analyses Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent

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

NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L-WT or NEDD4L-C943S mutants. cell signaling pathway activation induced by IL-6 were detected by WB analyses Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments


( G , H ) Primary keratinocytes were separated from the WT and Nedd4l KO mice . ( G ) WB analysis of the protein expression in IL - 6 - induced cell signaling pathway activation . ( H ) WB analysis of the proteins in the IL - 6 signaling pathway . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent

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

(G, H) Primary keratinocytes were separated from the WT and Nedd4l KO mice. (G) WB analysis of the protein expression in IL-6-induced cell signaling pathway activation. (H) WB analysis of the proteins in the IL-6 signaling pathway. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments


Six - to eight - weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment , the mice were injected intra - epidermally with 10 μg anti - GP130 antibody or with same dosage of isotype on every other day . Representative images of Ki67 staining in back tissues are on the left . The number of Ki67 - positive cells per HPF is shown on the right . Data information : Data are shown as mean ± s . e . m . , and are representative of two independent experiments . Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test C , control , n = 5 , IMQ , n = 6 . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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

Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Representative images of Ki67 staining in back tissues are on the left. The number of Ki67-positive cells per HPF is shown on the right. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test C, control, n=5, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.


Six - to eight - weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment , the mice were injected intra - epidermally with 10 μg anti - GP130 antibody or with same dosage of isotype on every other day . Keratinocyte proliferation associated genes in the skin tissues are detected using real - time PCR analysis . Data information : Data are shown as mean ± s . e . m . , and are representative of two independent experiments . Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test D , control , n = 3 , IMQ , n = 6 . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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

Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Keratinocyte proliferation associated genes in the skin tissues are detected using real-time PCR analysis. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test D, control, n=3, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.


( A ) Representative pictures of the expression of NEDD4L , GP130 and p - STAT3 in the epidermis of a normal skin section ( same specimen ) and sections from psoriasis patients . Isotype are IgG antibodies from same species served as negative control . ( B ) Semiquantitative scoring of NEDD4L , GP130 and p - STAT3 in the epidermis detected by IHC . Data information : Each symbol represents one patient Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . All data are shown as the mean ± s . e . m . , and significant differences were analyzed using two - tailed Student ' s t test ( normal , n = 15 , psoriasis , n = 36 ) . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 .

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

(A) Representative pictures of the expression of NEDD4L, GP130 and p-STAT3 in the epidermis of a normal skin section (same specimen) and sections from psoriasis patients. Isotype are IgG antibodies from same species served as negative control. (B) Semiquantitative scoring of NEDD4L, GP130 and p-STAT3 in the epidermis detected by IHC. Data information: Each symbol represents one patient Scale bar, 200 μm (100×) or 50 μm (400×). All data are shown as the mean ± s.e.m., and significant differences were analyzed using two-tailed Student's t test (normal, n=15, psoriasis, n=36). * P＜0.05, ** P＜0.01, *** P＜0.001.


( F ) Full length Flag - GP130 , GP130 - aa 616 - 918 , GP130 - aa 616 - 889 , GP130 - aa 616 - 764 , GP130 - aa 616 - 734 were transfected with Myc - NEDD4L into HEK293T cells , and whole cell lysates were co - IP with ANTI - FLAG M2 magnetic beads , followed by WB with anti - Flag or anti - Myc antibodies .

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(F) Full length Flag-GP130, GP130-aa 616-918, GP130-aa 616-889, GP130-aa 616-764, GP130-aa 616-734 were transfected with Myc-NEDD4L into HEK293T cells, and whole cell lysates were co-IP with ANTI-FLAG M2 magnetic beads, followed by WB with anti-Flag or anti-Myc antibodies.


( C ) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L - WT , NEDD4L WW domain truncated mutant ( NEDD4L - ΔWW ) or its enzymatic activity mutant ( NEDD4L - C943S ) , the cells were followed by denaturation - IP with anti - GP130 antibody and WB analysis with anti - Ub antibodies .

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(C) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L-WT, NEDD4L WW domain truncated mutant (NEDD4L-ΔWW) or its enzymatic activity mutant (NEDD4L-C943S), the cells were followed by denaturation-IP with anti-GP130 antibody and WB analysis with anti-Ub antibodies.


( E ) WB analysis of the polyubiquitination of GP130 in HEK293T cells co - transfected with full length Myc - NEDD4L , HA - Ub and Flag - GP130 or its lysine mutants ( K - R ) in the intracellular domain , followed by denaturation - IP with ANTI - FLAG M2 magnetic beads and WB analysis with anti - HA antibodies .

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(E) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with full length Myc-NEDD4L, HA-Ub and Flag-GP130 or its lysine mutants (K-R) in the intracellular domain, followed by denaturation-IP with ANTI-FLAG M2 magnetic beads and WB analysis with anti-HA antibodies.


( F ) Comparison of protein degradation efficiency using different degrons . Cells were incubated for indicated time periods with Asv or DOX / IAA or both ( DIA , see methods ) then collected for western blot analysis . Doxycycline was added 2 hours before addition of IAA or Asv / IAA . To determine protein degradation , either cyclin A2 or cyclin B1 were probed as indicated . Anti - myc antibody was used to check OsTIR1 - myc expression . Asterisks indicate non - specific protein crossreacting with the cyclin B1 antibody .

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(F) Comparison of protein degradation efficiency using different degrons. Cells were incubated for indicated time periods with Asv or DOX/IAA or both (DIA, see methods) then collected for western blot analysis. Doxycycline was added 2 hours before addition of IAA or Asv/IAA. To determine protein degradation, either cyclin A2 or cyclin B1 were probed as indicated. Anti-myc antibody was used to check OsTIR1-myc expression. Asterisks indicate non-specific protein crossreacting with the cyclin B1 antibody.


( G ) Timecourse of DIA induced cyclin A and B degradation , cross checking levels of each mitotic cyclin . Treatments were performed as for DIA treated Cell extracts were checked for both cyclin A2 and B1 levels following DIA

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(G) Timecourse of DIA induced cyclin A and B degradation, cross checking levels of each mitotic cyclin. Treatments were performed as for DIA treated Cell extracts were checked for both cyclin A2 and B1 levels following DIA addition


A ) Cyclin B2 knock - out and induced degradation by immunoblotting . Indicated cell lines were analysed 24 hours after mock or Dox / IAA / Asv ( DIA ) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation . (

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A) Cyclin B2 knock-out and induced degradation by immunoblotting. Indicated cell lines were analysed 24 hours after mock or Dox/IAA/Asv (DIA) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation. (


( D ) Cell proliferation of A2dd , CycB1dd and B1dd / B2ko following mock or DIA treatment . 1000 cells were plated in each well ( diameter 3 . 5cm ) and incubated for 10 days before methanol fixation and Crystal Violet staining .

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(D) Cell proliferation of A2dd, CycB1dd and B1dd/B2ko following mock or DIA treatment. 1000 cells were plated in each well (diameter 3.5cm) and incubated for 10 days before methanol fixation and Crystal Violet staining.


( E ) Kinetics of mitotic entry as measured by time lapse microscopy in A2dd and B1dd / B2ko cells following mock or four - hour DIA treatment of asynchronous cells . The cells were imaged for 16h with 5min intervals using widefield DIC , mitotic entry was manually scored by detecting cell rounding . Curves display the cumulative mitotic index ( data from three repeats , n > 500 cells per condition , s . d . indicated by shaded area ) .

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(E) Kinetics of mitotic entry as measured by time lapse microscopy in A2dd and B1dd/B2ko cells following mock or four-hour DIA treatment of asynchronous cells. The cells were imaged for 16h with 5min intervals using widefield DIC, mitotic entry was manually scored by detecting cell rounding. Curves display the cumulative mitotic index (data from three repeats, n>500 cells per condition, s. d. indicated by shaded area).


( I ) Cyclin A2 siRNA depletion causes endoreplication . Following 72 hours of siRNA transfection MCF7 and MCF10A cells were analysed by PI staining and FACS . The histograms show the changes in DNA content ( PI Int . ) towards > 4N following cyclin A2 depletion .

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(I) Cyclin A2 siRNA depletion causes endoreplication. Following 72 hours of siRNA transfection MCF7 and MCF10A cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards >4N following cyclin A2 depletion.


( J ) Cyclin A2 siRNA and degron depletion in RPE - 1 cells . RPE - 1 OsTIR1 and RPE - 1 A2dd cells were subjected to 72 hours of cyclin A2 siRNA depletion and / or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting . The longer exposure ( L . E . ) reveals incomplete depletion of cyclin 2 by siRNA .

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(J) Cyclin A2 siRNA and degron depletion in RPE-1 cells. RPE-1 OsTIR1 and RPE-1 A2dd cells were subjected to 72 hours of cyclin A2 siRNA depletion and/or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting. The longer exposure (L.E.) reveals incomplete depletion of cyclin 2 by siRNA.


( C ) Representative images from live - cell imaging of SiR - Tubulin labelled ( red ) mitotic B1dd / B2ko cells expressing FusionRed - H2B ( green ) , Mis12 - GFP ( white ) ; time in mins , scale bar = 5µm . Cells were imaged four hours after DIA treatment .

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(C) Representative images from live-cell imaging of SiR-Tubulin labelled (red) mitotic B1dd/B2ko cells expressing FusionRed-H2B (green), Mis12-GFP (white); time in mins, scale bar = 5µm. Cells were imaged four hours after DIA treatment.


( F ) Cyclin A2 levels were assessed by immunoblotting after 18 hours in 100ng / ml Nocodazole and / or 5µM STLC . 6µM proTAME was added to the indicated samples for the final six hours .

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(F) Cyclin A2 levels were assessed by immunoblotting after 18 hours in 100ng/ml Nocodazole and/or 5µM STLC. 6µM proTAME was added to the indicated samples for the final six hours.


( G ) Upper panel , confirmation of Cdc27 depletion by immunoblotting 72 hours after siRNA transfection in B1dd / B2ko cells . Lower panel , following 40 - hour siRNA transfection B1dd / B2ko cells were treated for 24 hours with Thymidine , released for 10 hours and treated with proTAME and Apcin for additional four hours . At this point , the cells were fixed and stained with tubulin and pericentrin for immunofluorescence analysis .

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(G) Upper panel, confirmation of Cdc27 depletion by immunoblotting 72 hours after siRNA transfection in B1dd/B2ko cells. Lower panel, following 40-hour siRNA transfection B1dd/B2ko cells were treated for 24 hours with Thymidine, released for 10 hours and treated with proTAME and Apcin for additional four hours. At this point, the cells were fixed and stained with tubulin and pericentrin for immunofluorescence analysis.


( H ) Representative images of mitotic spindles in control - and DIA - treated B1dd / B2ko cells following arrest in mitosis by proTAME / Apcin treatment Tubulin staining is shown in white , pericentrin in green and DAPI in red , scale bar = 5µm .

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(H) Representative images of mitotic spindles in control- and DIA-treated B1dd/B2ko cells following arrest in mitosis by proTAME/Apcin treatment Tubulin staining is shown in white, pericentrin in green and DAPI in red, scale bar = 5µm.


( L ) Immunofluorescence images from mitotic Ctr and DIA treated B1dd / B2ko cells after P / A synchronisation stained with anti - alpha - tubulin ( green ) , anti - gamma - tubulin ( red ) antibodies and DAPI ( blue ) , scale bar = 5µm . Cells were exposed to ice - cold medium and either fixed immediately after cold exposure , or incubated for 5 , 10 , or 20 minutes at 37 ° C before fixation .

(L) Immunofluorescence images from mitotic Ctr and DIA treated B1dd/B2ko cells after P/A synchronisation stained with anti-alpha-tubulin (green), anti-gamma-tubulin (red) antibodies and DAPI (blue), scale bar = 5µm. Cells were exposed to ice-cold medium and either fixed immediately after cold exposure, or incubated for 5,10, or 20 minutes at 37° C before fixation.


( A ) Time - lapse images of PCNA - mRuby tagged A2dd cells . The imaging sequence was started at the time of Doxycycline addition , and degron activity was triggered three hours later by addition of IAA and Asv or PBS ( indicated by dashed line ) . Time is shown as hh : min , scale bar equals 10µm . For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase .

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(A) Time-lapse images of PCNA-mRuby tagged A2dd cells. The imaging sequence was started at the time of Doxycycline addition, and degron activity was triggered three hours later by addition of IAA and Asv or PBS (indicated by dashed line). Time is shown as hh:min, scale bar equals 10µm. For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase.


( D ) Images from time - lapse sequence of mitosis showing cell division in controls and after addition of 0 . 5µM PD - 166285 in DIA treated A2dd labelled with SiR - DNA ( time is indicated as hh : min , the scale bars represent 20µm ) .

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(D) Images from time-lapse sequence of mitosis showing cell division in controls and after addition of 0.5µM PD-166285 in DIA treated A2dd labelled with SiR-DNA (time is indicated as hh:min, the scale bars represent 20µm).


( D ) Cyclin A2dd cells with inducible CycB1 - YFP ( B1 - WT ) and CycB1 - YFP - NLS ( B1 - NLS ) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion . Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and cyclin A2

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(D) Cyclin A2dd cells with inducible CycB1-YFP (B1-WT) and CycB1-YFP-NLS (B1-NLS) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion. Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and cyclin A2 antibodies


( I ) Immunoprecipitation of YFP from extract from A2dd following induction of YFP ( Y ) , CycB1 - YFP ( BY ) and CycB1 - YFP - NLS ( BYN ) . The samples in the odd lanes ( IAA / Asv - ) were treated with 1µg / ml doxycycline alone , while samples in even lanes ( IAA / Asv + ) were treated with Auxin and Asv to induce cyclin A2 depletion . Total cell extract and immune - precipitates were probed by immunoblotting with the indicated antisera .

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(I) Immunoprecipitation of YFP from extract from A2dd following induction of YFP (Y), CycB1-YFP (BY) and CycB1-YFP-NLS (BYN). The samples in the odd lanes (IAA/Asv-) were treated with 1µg/ml doxycycline alone, while samples in even lanes (IAA/Asv+) were treated with Auxin and Asv to induce cyclin A2 depletion. Total cell extract and immune-precipitates were probed by immunoblotting with the indicated antisera.


( E ) Quantification of changes in selected cyclin B - dependent mitotic phosphorylations including Ki - 67 ( left panel ) , INCENP / Borealin ( middle panel ) and Top2B , CDCA2 and Tpx2 .

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(E) Quantification of changes in selected cyclin B-dependent mitotic phosphorylations including Ki-67 (left panel), INCENP/Borealin (middle panel) and Top2B, CDCA2 and Tpx2.


( F ) Representative images of Ki - 67 immunofluorescent staining of chromosome spreads from control ( Ctr ) and DIA treated P / A synchronised B1dd / B2ko cells and quantification of Ki - 67 staining intensity using cross - sections of

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(F) Representative images of Ki-67 immunofluorescent staining of chromosome spreads from control (Ctr) and DIA treated P/A synchronised B1dd/B2ko cells and quantification of Ki-67 staining intensity using cross-sections of chromosomes


( G ) Representative images from live - cell imaging of DIA - treated SiR - Tubulin labelled ( red ) B1dd / B2ko cells expressing FusionRed - H2B ( green ) and AurB - GFP ( white ) ; time is indicated in minutes , scale bar = 5µm . Bottom panel shows frequencies of aberrant AurB localisation in P / A synchronised B1dd / B2ko cells .

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(G) Representative images from live-cell imaging of DIA-treated SiR-Tubulin labelled (red) B1dd/B2ko cells expressing FusionRed-H2B (green) and AurB-GFP (white); time is indicated in minutes, scale bar = 5µm. Bottom panel shows frequencies of aberrant AurB localisation in P/A synchronised B1dd/B2ko cells.


( J ) Tpx2 shows reduced spread on mitotic chromosomes in DIA treated B1dd / B2ko cells . The top panel shows immunofluorescence images , for quantification we measured the mean intensity of Tpx2 staining on the centrosomes and spindle and plotted the ratio of these values per cell . We also analysed the centrosome / spindle distribution of AurA that does not change following DIA treatment .

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(J) Tpx2 shows reduced spread on mitotic chromosomes in DIA treated B1dd/B2ko cells. The top panel shows immunofluorescence images, for quantification we measured the mean intensity of Tpx2 staining on the centrosomes and spindle and plotted the ratio of these values per cell. We also analysed the centrosome/spindle distribution of AurA that does not change following DIA treatment.


( B ) Confirmation of ENSA / ARPP19 S67 phosphorylation in mitotic extracts from Ctr and DIA treated cells , before and after ENSA / ARPP19 siRNA depletion .

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(B) Confirmation of ENSA/ARPP19 S67 phosphorylation in mitotic extracts from Ctr and DIA treated cells, before and after ENSA/ARPP19 siRNA depletion.


( E ) Dynamics of Greatwall , PP1 , and Cdc27 dephosphorylation following Cdk1 inhibition in proTAME / Apcin treated mitotic cell extracts from Ctr or DIA treated cells . Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol and analysed by

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(E) Dynamics of Greatwall, PP1, and Cdc27 dephosphorylation following Cdk1 inhibition in proTAME/Apcin treated mitotic cell extracts from Ctr or DIA treated cells. Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol and analysed by immunoblotting


A ) Representative images from live - cell imaging of siRNA transfected B1dd / B2ko cells ( FusionRed - H2B in red , SiR - Tubulin in white , scale bar = 10µm ) . Bar - plot panels on the right show single - cell analysis of 10 cells manually scored for length mitosis pre - NEBD and post - NEBD . Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus .

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A) Representative images from live-cell imaging of siRNA transfected B1dd/B2ko cells (FusionRed-H2B in red, SiR-Tubulin in white, scale bar = 10µm). Bar-plot panels on the right show single-cell analysis of 10 cells manually scored for length mitosis pre-NEBD and post-NEBD. Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus.


( B ) Widefield Imaging . DIC ( Grey ) , SiR - DNA ( b / w ) , of ENSA / ARPP19 siRNA transfected DIA treated B1dd / B2ko cells . Time is indicated in hh : min , scale bar = 10µm .

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(B) Widefield Imaging. DIC (Grey), SiR-DNA (b/w), of ENSA/ARPP19 siRNA transfected DIA treated B1dd/B2ko cells. Time is indicated in hh:min, scale bar = 10µm.


( C ) Panels of representative immunofluorescence images of siRNA transfected and P / A treated cells treated as indicated , scale bar = 10µm .

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(C) Panels of representative immunofluorescence images of siRNA transfected and P/A treated cells treated as indicated, scale bar = 10µm.


The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : Spinning disc confocal live cell imaging of Alexa 555 - EGF ( 555 - EGF ) shown as vesicle tracking with time represented as a colour spectrum . Tracking started 5 min after addition of 20 ng / mL 555 - EGF for the indicated durations . Scale bar : 10 μm .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Spinning disc confocal live cell imaging of Alexa 555-EGF (555-EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/mL 555-EGF for the indicated durations. Scale bar: 10 μm.


The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . B : Immunofluorescence staining of EGFR following 5 or 15 min stimulation with 20 ng / mL EGF . Scale bar : 20 μm .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). B: Immunofluorescence staining of EGFR following 5 or 15 min stimulation with 20 ng/mL EGF. Scale bar: 20 μm.