Figure 5A . A luciferase reporting system was used to detect the regulatory effects of gga - mir - 12211 on the activity of BMP4 - 3 ' UTR ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * * * p < 0 . 0001 , one - way ANOVA ) . B . qRT - PCR was used to detect the regulatory effects of gga - mir - 12211 on LncBMP4 expression ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) . Enrichment levels of m6A in LncBMP4 were detected by RIP - qPCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) . H and I . The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

Figure 5A. A luciferase reporting system was used to detect the regulatory effects of gga-mir-12211 on the activity of BMP4-3'UTR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA).B. qRT-PCR was used to detect the regulatory effects of gga-mir-12211 on LncBMP4 expression (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001, **** p < 0.0001, one-way ANOVA).Enrichment levels of m6A in LncBMP4 were detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).H and I. The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA.).


Figure 6A . The influence of m6A on the stability of LncBMP4 was detected by qRT - PCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) . D . The enrichment level of m6A , for LncBMP4 variants with the progressive accumulation of RRACH site mutations , was detected by RIP - qPCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA ) . E . The ability of LncBMP4 , modified with different levels of m6A , to bind gga - mir - 12211 was detected by the luciferase reporting system ( Data are shown as mean ± SEM , n = 3 independent experiments , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

Figure 6A. The influence of m6A on the stability of LncBMP4 was detected by qRT-PCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).D. The enrichment level of m6A, for LncBMP4 variants with the progressive accumulation of RRACH site mutations, was detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, one-way ANOVA).E. The ability of LncBMP4, modified with different levels of m6A, to bind gga-mir-12211 was detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).


Figure 7B . qRT - PCR was used to detect the expression of related genes in each group , during induction ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one - way ANOVA ) . D . The number of CVH - positive cells was determined by flow cytometry ( Data are shown as mean ± SEM , n = 3 independent experiments , * * p < 0 . 01 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

Figure 7B. qRT-PCR was used to detect the expression of related genes in each group, during induction (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA).D. The number of CVH-positive cells was determined by flow cytometry (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).


Figure 8C . Effects of m6A on small peptide expression , assessed by western blot ( Up ) , Image J software was used for gray level analysis ( Down ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) . D and E . Western blot ( Up ) was used to detect gga - mir - 12211 and m6A affect the ability of LncBMP4 to translate EPC5 . Image J software was used for gray level analysis ( Down ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

Figure 8C. Effects of m6A on small peptide expression, assessed by western blot (Up), Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).D and E. Western blot(Up) was used to detect gga-mir-12211 and m6A affect the ability of LncBMP4 to translate EPC5. Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001,**** p < 0.0001, one-way ANOVA.).


Figure 1A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNF - α - stimulated VE - cadherin WT ( WT ) or VE - cadherin Y731F ( Y731F ) primary endothelial cells , each transfected with control or SHP2 - specific siRNA . The transmigration rate is presented relative to that of WT cells transfected with control siRNA , set as 100 % . On the right , Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α - tubulin . B Transmigration of human neutrophils towards the chemokine IL - 8 through TNFα - stimulated human endothelial cells ( HUVEC ) , transfected with control or SHP2 - specific siRNA and were either not transduced ( - ) or transduced ( + ) with adenoviruses expressing human SHP2 ( Adv - SHP2 ) . The transmigration rate is presented relative to that of cells transfected with control siRNA , set as 100 % . On the right , Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α - tubulin . C Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST , GST ‐ SHP2 ‐ WT , or GST ‐ SHP2 ‐ DA . Pulldowns and total cell lysates were analyzed by Western blotting for VE - cadherin and GST . Graph below represents quantification of two independent experiments , presented as fold change of bound VE - cadherin . TNFα - activated bEnd . 5 cells were incubated with mouse T cells for 0 - 30 min . SHP2 ( above ) or VE - cadherin ( below ) were immunoprecipitated ( IP ) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens . Molecular weight markers are indicated in kDa . Graph below represents quantification of five independent experiments , presented as fold change of bound VE - cadherin .

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

Figure 1A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNF-α-stimulated VE-cadherin WT (WT) or VE-cadherin Y731F (Y731F) primary endothelial cells, each transfected with control or SHP2-specific siRNA. The transmigration rate is presented relative to that of WT cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.B Transmigration of human neutrophils towards the chemokine IL-8 through TNFα-stimulated human endothelial cells (HUVEC), transfected with control or SHP2-specific siRNA and were either not transduced (-) or transduced (+) with adenoviruses expressing human SHP2 (Adv-SHP2). The transmigration rate is presented relative to that of cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.C Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST, GST‐SHP2‐WT, or GST‐SHP2‐DA. Pulldowns and total cell lysates were analyzed by Western blotting for VE-cadherin and GST. Graph below represents quantification of two independent experiments, presented as fold change of bound VE-cadherin.TNFα-activated bEnd.5 cells were incubated with mouse T cells for 0-30 min. SHP2 (above) or VE-cadherin (below) were immunoprecipitated (IP) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. Graph below represents quantification of five independent experiments, presented as fold change of bound VE-cadherin.


Figure 2endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( A ) bEnd . 5 cells were incubated with antigen - stimulated mouse T cells for 0 - 15 min . endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( B ) luEnd cells devoid of PECAM - 1 ( KO ) or re - transfected with PECAM - 1 ( RT ) were incubated with T cells for 30 min . endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( C ) HUVEC transfected with control or PECAM - 1 - specific siRNA were incubated with differentiated HL60 cells for 20 min . endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( D ) bEnd . 5 cells were pre - treated with anti - PECAM - 1 ( Mec13 . 3 ) or anti - endomucin antibodies for 30 min prior to incubation with T cells for 30 min . endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above ) and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( E ) HUVEC , siRNA depleted for endogenous PECAM - 1 , were transduced with wild - type PECAM - 1 ( PECAM - 1 WT ) or Y663 , 686F PECAM - 1 double mutant ( PECAM - 1 YF ) and incubated with HL60 - derived neutrophils for 20 min . On the right , immunoblot analysis of lysates from siRNA treated cells before PECAM - 1 re - expression . F Transmigration of human neutrophils towards IL - 8 through TNFα - stimulated HUVEC expressing PECAM - 1 WT or PECAM - 1 - Y663 , 686F . The transmigration rate is presented relative to that of PECAM - 1 WT expressing cells , set as 100 % .

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

Figure 2endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (A) bEnd.5 cells were incubated with antigen-stimulated mouse T cells for 0-15 min.endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (B) luEnd cells devoid of PECAM-1 (KO) or re-transfected with PECAM-1 (RT) were incubated with T cells for 30 min.endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (C) HUVEC transfected with control or PECAM-1-specific siRNA were incubated with differentiated HL60 cells for 20 min.endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (D) bEnd.5 cells were pre-treated with anti-PECAM-1 (Mec13.3) or anti-endomucin antibodies for 30 min prior to incubation with T cells for 30 min.endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above) and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (E) HUVEC, siRNA depleted for endogenous PECAM-1, were transduced with wild-type PECAM-1 (PECAM-1 WT) or Y663,686F PECAM-1 double mutant (PECAM-1 YF) and incubated with HL60-derived neutrophils for 20 min. On the right, immunoblot analysis of lysates from siRNA treated cells before PECAM-1 re-expression.F Transmigration of human neutrophils towards IL-8 through TNFα-stimulated HUVEC expressing PECAM-1 WT or PECAM-1-Y663,686F. The transmigration rate is presented relative to that of PECAM-1 WT expressing cells, set as 100%.


Figure 3Endocytosis of VE - cadherin in 17 h TNF - α treated HUVEC was induced by adding HL60 - derived neutrophils for 20 - 30 min at 37 ° C and monitored by the internalization of preincubated mAb BV6 , which was visualized in fixed cells with a secondary fluorescent antibody ( white ) . Total VE - cadherin was stained with another mAb ( red ) , nuclei stained with Hoechst ( white ) . ( A ) HUVEC had been transfected prior to the experiment with control or PECAM - 1 - specific siRNA . Cell lysates were analyzed for expression levels of PECAM - 1 and tubulin ( below ) . Relative VE - cadherin internalization efficiency is given on the right . Endocytosis of VE - cadherin in 17 h TNF - α treated HUVEC was induced by adding HL60 - derived neutrophils for 20 - 30 min at 37 ° C and monitored by the internalization of preincubated mAb BV6 , which was visualized in fixed cells with a secondary fluorescent antibody ( white ) . Total VE - cadherin was stained with another mAb ( red ) , nuclei stained with Hoechst ( white ) . ( B ) PECAM - 1 siRNA treated HUVEC were transduced with WT PECAM - 1 - EGFP or PECAM - 1 - Y663 , 686F - EGFP . Expression levels of transduced PECAM - 1 forms and of EGFP and VE - cadherin are shown in immunoblots ( on the right ) . Relative VE - cadherin internalization efficiency is given on the right .

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

Figure 3Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (A) HUVEC had been transfected prior to the experiment with control or PECAM-1-specific siRNA. Cell lysates were analyzed for expression levels of PECAM-1 and tubulin (below). Relative VE-cadherin internalization efficiency is given on the right.Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (B) PECAM-1 siRNA treated HUVEC were transduced with WT PECAM-1-EGFP or PECAM-1-Y663,686F-EGFP. Expression levels of transduced PECAM-1 forms and of EGFP and VE-cadherin are shown in immunoblots (on the right). Relative VE-cadherin internalization efficiency is given on the right.


Figure 4A Transmigration of mouse neutrophils towards CXCL - 1 through TNF - α - stimulated bEnd . 5 cells transfected with control or SHP2 - specific siRNA and pre - treated with anti - endomucin ( 7C7 . 1 ) or anti - PECAM - 1 ( Mec13 . 3 ) antibodies for 30 min at 37 ° C before addition of neutrophils . The transmigration rate is presented relative to that of control cells , set as 100 % . B Transmigration of mouse neutrophils towards CXCL - 1 through TNF - α - stimulated WT or Y731F primary mouse endothelial cells pre - treated with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to that of control cells , set as 100 % . Extravasated leukocytes ( C ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy . adherent leukocytes ( D ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy . ( D ) and rolling flux fraction of leukocytes ( E ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

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

Figure 4A Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated bEnd.5 cells transfected with control or SHP2-specific siRNA and pre-treated with anti-endomucin (7C7.1) or anti-PECAM-1 (Mec13.3) antibodies for 30 min at 37°C before addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.B Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated WT or Y731F primary mouse endothelial cells pre-treated with isotype-control or anti-PECAM-1 (2H8) antibodies for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.Extravasated leukocytes (C) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.adherent leukocytes (D) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.(D) and rolling flux fraction of leukocytes (E) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


Figure 5A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2 . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens . B bEnd . 3 cells were lysed and VE - cadherin or immature VE - cadherin was immunoprecipitated respectively with C5 or VD47 antibodies . Precipitates were blotted against pY731 - VE - cadherin , β - catenin , plakoglobin , p120 , α - catenin and VE - cadherin . C VE - cadherin - catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated ( no SDS ) or treated with 0 . 2 % SDS for 30 min at room temperature , followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30 ° C . Precipitates were analyzed in immunoblots for pY731 - VE - cadherin , plakoglobin , β - catenin and VE - cadherin . D Quantification of pY731 blot signals of three independent experiments ( from C ) , with the signal intensity for untreated VE - cadherin set as 1 . Molecular weight markers are indicated in kDa .

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Figure 5A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens.B bEnd.3 cells were lysed and VE-cadherin or immature VE-cadherin was immunoprecipitated respectively with C5 or VD47 antibodies. Precipitates were blotted against pY731-VE-cadherin, β -catenin, plakoglobin, p120, α -catenin and VE-cadherin.C VE-cadherin-catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated (no SDS) or treated with 0.2% SDS for 30 min at room temperature, followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30°C. Precipitates were analyzed in immunoblots for pY731-VE-cadherin, plakoglobin, β-catenin and VE-cadherin. D Quantification of pY731 blot signals of three independent experiments (from C), with the signal intensity for untreated VE-cadherin set as 1. Molecular weight markers are indicated in kDa.


Figure 6A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNFα - stimulated WT or Y731F primary endothelial cells , either pre - treated with vehicle control ( DMSO ) or Ca2 + chelator ( MAPTAM ) for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to DMSO - treated endothelial cells expressing WT VE - cadherin . B TNFα - activated bEnd . 5 cells were treated with DMSO or MAPTAM , prior to incubation with antigen - stimulated mouse T cells for 30 min . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens . Quantification of pY731 blot signals of five independent experiments are shown below , with the signal intensity for the control sample ( no T cells , vehicle - control ) set as 100 % . A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNFα - stimulated WT or Y731F primary endothelial cells , either pre - treated with vehicle control ( DMSO ) or Ca2 + chelator for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to DMSO - treated endothelial cells expressing WT VE - cadherin . C Transmigration assays treatment with Blebbistatin . B TNFα - activated bEnd . 5 cells were treated with DMSO prior to incubation with antigen - stimulated mouse T cells for 30 min . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens . Quantification of pY731 blot signals of five independent experiments are shown below , with the signal intensity for the control sample ( no T cells , vehicle - control ) set as 100 % . treatment with Blebbistatin . E HUVEC transduced with lacZ or with SHP2 were either untreated ( - ) or treated ( + ) with thrombin for 30 min followed by immunoblotting either VE - cadherin immunoprecipitates or cell lysates for the indicated antigens on the right .

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Figure 6A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator (MAPTAM) for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin.B TNFα-activated bEnd.5 cells were treated with DMSO or MAPTAM, prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%.A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin. C Transmigration assays treatment with Blebbistatin.B TNFα-activated bEnd.5 cells were treated with DMSO prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%. treatment with Blebbistatin.E HUVEC transduced with lacZ or with SHP2 were either untreated (-) or treated (+) with thrombin for 30 min followed by immunoblotting either VE-cadherin immunoprecipitates or cell lysates for the indicated antigens on the right.


Figure 7HUVEC were transfected with control or VE - cadherin - specific siRNA and transduced with VE - cad FL or VE - cad FL - TS . Cell lysates were immunoblotted for VE - cadherin and actin . C Immunofluorescence staining for VE - cadherin of HUVEC transfected with control or VE ‑ cadherin specific siRNA ( VE - cad kd ) and transduced with VE - cad FL or VE - cad FL - TS . D HUVEC were either transfected with control or VE - cadherin targeting siRNA and transduced with VE ‑ cad FL or VE - cad FL ‑ TS . The tension sensor was immunoprecipitated from cell lysates using an anti - GFP antibody . Precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens . Molecular weight markers are indicated in kDa . E , Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL or VE - cad FL - TS , after thrombin stimulation ( 1 U / ml ) or under control conditions .

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Figure 7HUVEC were transfected with control or VE-cadherin-specific siRNA and transduced with VE-cad FL or VE-cad FL-TS. Cell lysates were immunoblotted for VE-cadherin and actin.C Immunofluorescence staining for VE-cadherin of HUVEC transfected with control or VE‑cadherin specific siRNA (VE-cad kd) and transduced with VE-cad FL or VE-cad FL-TS. D HUVEC were either transfected with control or VE-cadherin targeting siRNA and transduced with VE‑cad FL or VE-cad FL‑TS. The tension sensor was immunoprecipitated from cell lysates using an anti-GFP antibody. Precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. E, Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS, after thrombin stimulation (1 U/ml) or under control conditions.


Figure 8A - C Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL ( A , B ) or VE - cad FL - TS ( C ) . HUVEC were stimulated 4h with TNFα prior to adding IL - 8 for 4 min , followed by flow with buffer alone ( A ) or together with PMNs ( B , C ) . The connected data points represent FRET efficiency of same junctions compared before and after addition of flow / PMNs . D Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL or VE - cad FL - TS . Cells were either exposed to flow and PMNs or to flow alone , fixed using 4 % PFA and washed with PBS . FLIM measurements were performed at sites of transmigration ( PMNs ) or at junctions without PMNs ( Flow ) . E Representative images of HUVEC expressing VE - cad FL at a site of PMN transmigration ( left micrographs ) or without PMN ( right micrographs ) . Maximum intensity projection of a Z - stack of YPet fluorescence ( VE - cadherin in green ) and CellTracker DeepRed ( PMN in red ) is shown for the upper micrographs . The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue . The amplitude averaged lifetime of YPet per pixel ( FLIM measurement ) of the same cells is shown in the bottom micrographs . F HUVEC were grown on collagen - coated polyacrylamide gels of varying physiologic stiffness of 0 . 2 kPa and 20 kPa , and stimulated with TNFα for 17 h prior to adding HL60 - derived neutrophils for 20 min . VE - cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens . Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE - cadherin are shown on the

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

Figure 8A-C Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL (A, B) or VE-cad FL-TS (C). HUVEC were stimulated 4h with TNFα prior to adding IL-8 for 4 min, followed by flow with buffer alone (A) or together with PMNs (B, C). The connected data points represent FRET efficiency of same junctions compared before and after addition of flow/PMNs.D Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS. Cells were either exposed to flow and PMNs or to flow alone, fixed using 4 % PFA and washed with PBS. FLIM measurements were performed at sites of transmigration (PMNs) or at junctions without PMNs (Flow).E Representative images of HUVEC expressing VE-cad FL at a site of PMN transmigration (left micrographs) or without PMN (right micrographs). Maximum intensity projection of a Z-stack of YPet fluorescence (VE-cadherin in green) and CellTracker DeepRed (PMN in red) is shown for the upper micrographs. The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue. The amplitude averaged lifetime of YPet per pixel (FLIM measurement) of the same cells is shown in the bottom micrographs.F HUVEC were grown on collagen-coated polyacrylamide gels of varying physiologic stiffness of 0.2 kPa and 20 kPa, and stimulated with TNFα for 17 h prior to adding HL60-derived neutrophils for 20 min. VE-cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens. Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE-cadherin are shown on the right


Figure 1D , Scoring of the PI3Kα activation transcriptomic signature was used to cluster patients with high , medium ( both groups were combined , high + medium : red ) and low ( black ) scoring levels in the primary tumours of confirmed PDAC patients from PAAD ( TCGA ) or PACA - AU ( BH corrected p - values ) . The survival curves of each cluster were then plotted , and the statistical significance was calculated using the logrank test .

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Figure 1D, Scoring of the PI3Kα activation transcriptomic signature was used to cluster patients with high, medium (both groups were combined, high+medium: red) and low (black) scoring levels in the primary tumours of confirmed PDAC patients from PAAD (TCGA) or PACA-AU (BH corrected p-values). The survival curves of each cluster were then plotted, and the statistical significance was calculated using the logrank test.


Figure 2E , Murine pancreatic tumour cells R211 were treated with the vehicle , α , β , β / δ , γ - specific or pan - PI3K inhibitors at 0 . 1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . Representative image of filter after Crystal violet staining is shown . Scale = 200µm . n = 3 in each group . F , Murine pancreatic tumour cells R211 were treated with vehicle , BYL - 719 1 µM , AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 4 in each group . G L , PANC - 1 - transduced cells were subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 5 in each group .

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Figure 2E, Murine pancreatic tumour cells R211 were treated with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors at 0.1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. Representative image of filter after Crystal violet staining is shown. Scale=200µm. n = 3 in each group. F, Murine pancreatic tumour cells R211 were treated with vehicle, BYL-719 1 µM, AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 4 in each group. G L, PANC-1-transduced cells were subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 5 in each group.


Figure 3A - B A , Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle , α - specific A66 or pan - PI3K inhibitor BKM120 at 0 . 01 , 0 . 05 , 0 . 1 , 0 . 5 , 1 or 5 µM in the presence of 10 % FBS and protein levels of P - Akt ( Ser473 ) , total Akt and β Actin were observed by western blot . B , P - Akt on Ser473 was quantified and normalised with β Actin . n = 3 in each group . R211 cells were treated with the vehicle , α - specific inhibitor A66 or pan - PI3K inhibitor BKM120 at 1 µM D , migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle ( DMSO ) . n = 3 in each group . E , Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle ( 0 . 01 % DMSO ) or α - specific A66 or pan - PI3K inhibitor BKM120 at 1µM . Phospholipids were extracted , total PIP , PIP2 and PIP3 were quantified and compared to the vehicle ( DMSO ) . n = 3 in each group .

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Figure 3A-B A, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle, α-specific A66 or pan-PI3K inhibitor BKM120 at 0.01, 0.05, 0.1, 0.5, 1 or 5 µM in the presence of 10% FBS and protein levels of P-Akt (Ser473), total Akt and β Actin were observed by western blot. B, P-Akt on Ser473 was quantified and normalised with β Actin. n = 3 in each group.R211 cells were treated with the vehicle, α-specific inhibitor A66 or pan-PI3K inhibitor BKM120 at 1 µM D, migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle (DMSO). n = 3 in each group.E, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle (0.01% DMSO) or α-specific A66 or pan-PI3K inhibitor BKM120 at 1µM. Phospholipids were extracted, total PIP, PIP2 and PIP3 were quantified and compared to the vehicle (DMSO). n = 3 in each group.


Figure 4A , Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells ( R211 , PDAC8661 , PANC - 1 , 10593 , 10158 , R6344 , R6430 , R6065 , R6141 ) . The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor . IC50 is determined on recombinant proteins . Pearson correlation tests were performed and its p - values were presented separately for each class I PI3K isoform .

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Figure 4A, Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells (R211, PDAC8661, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141). The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor. IC50 is determined on recombinant proteins. Pearson correlation tests were performed and its p-values were presented separately for each class I PI3K isoform.


Figure 5A , Representative images of PI3K activation ( as shown by positive pAkt substrate staining ) on the primary tumours of PDAC patients , with values of quantification of cfDNA and mutated KRAS allele frequency . C , Survival curve of KPC mice presenting different levels of cfDNA . n in each group is indicated . Log rank test , * * * p < 0 .

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Figure 5A, Representative images of PI3K activation (as shown by positive pAkt substrate staining) on the primary tumours of PDAC patients, with values of quantification of cfDNA and mutated KRAS allele frequency.C, Survival curve of KPC mice presenting different levels of cfDNA. n in each group is indicated. Log rank test, *** p<0.001


Figure 6G , Survival curve of KPC mice treated with the vehicle or BYL - 719 . Log rank test , * p < 0 . 05 . H - J H , Representative images and i , quantification of Ki67 positive cells in pancreatic tumours and in J , metastasized liver sections after treatment with oral doses of vehicle ( 0 . 5 % methyl cellulose with 0 . 2 % Tween - 80 ) or of BYL - 719 ( 50mg / kg ) . K - L K , Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle ( n is indicated ) , quantification at two time points and L , representative images of luminescence via IVIS ® Spectrum in vivo imaging system . N , Tail vein injection experiment in C57 / B6 mice of murine syngeneic pancreatic cancer cells ( Kras mutated A338 , Kras mutated and half PI3Kα inactive , A260 ; n > 8 in each group ) , quantification of micro - and macro - metastasis at final time point .

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Figure 6G, Survival curve of KPC mice treated with the vehicle or BYL-719. Log rank test, * p<0.05.H-J H, Representative images and i, quantification of Ki67 positive cells in pancreatic tumours and in J, metastasized liver sections after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).K-L K, Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle (n is indicated), quantification at two time points and L, representative images of luminescence via IVIS® Spectrum in vivo imaging system.N, Tail vein injection experiment in C57/B6 mice of murine syngeneic pancreatic cancer cells (Kras mutated A338, Kras mutated and half PI3Kα inactive, A260; n>8 in each group), quantification of micro- and macro-metastasis at final time point.


Figure 7A - D Blood count of A , white blood cells , B , monocytes , C , granulocytes and D , lymphocytes in KPC mice , with cohort size in each group . h - jH - J H , Representative images and quantification of I , F4 / 80 positive cells and J , CD206 positive cells after the pharmacological inhibition of PI3Kα . Black arrowheads show positive immune cells . S , Murine pancreatic tumour cells R211 were treated with vehicle , TNF - α 20 ng / mL , BYL - 719 1 µM or both treatments and subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 4 independent values .

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Figure 7A-D Blood count of A, white blood cells, B, monocytes, C, granulocytes and D, lymphocytes in KPC mice, with cohort size in each group.h-jH-J H, Representative images and quantification of I, F4/80 positive cells and J, CD206 positive cells after the pharmacological inhibition of PI3Kα. Black arrowheads show positive immune cells.S, Murine pancreatic tumour cells R211 were treated with vehicle, TNF-α 20 ng/mL, BYL-719 1 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n= 4 independent values.


Figure 1B . Size - exclusion chromatography ( SEC ) experiments of Mdm12 , tMmm1 , and the Mdm12 - tMmm1 complex comparing the molecular size of these proteins in solution . The proteins indicated were injected into a Superdex 200 column ( GE Healthcare ) with a buffer containing 25 mM Tris - HCl ( pH 7 . 5 ) , 150 mM NaCl , and 5 mM DTT . The standard molecular masses for the SEC experiments ( top ) are shown for relative molecular weight comparison ( blue dextran , void ; ferritin , 440 kDa ; aldolase , 158 kDa ; cobalbumin , 75 kDa ; ovalbumin , 44 kDa ; and carbonic anhydrase , 29 kDa ) . C . Graph indicating the molecular weights of Mdm12 , His6 - Mdm12 , and the Mdm12 - tMmm1 complex in solution as measured by analytical ultracentrifugation .

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Figure 1B. Size-exclusion chromatography (SEC) experiments of Mdm12, tMmm1, and the Mdm12-tMmm1 complex comparing the molecular size of these proteins in solution. The proteins indicated were injected into a Superdex 200 column (GE Healthcare) with a buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM DTT. The standard molecular masses for the SEC experiments (top) are shown for relative molecular weight comparison (blue dextran, void; ferritin, 440 kDa; aldolase, 158 kDa; cobalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa).C. Graph indicating the molecular weights of Mdm12, His6-Mdm12, and the Mdm12-tMmm1 complex in solution as measured by analytical ultracentrifugation.


Figure 2A . Ribbon diagram of the yeastMdm12 dimer . The crystal structure of full - length Mdm12 was determined by SAD and refined with native data to 3 . 1 Å resolution . Lipids bound to Mdm12 are drawn with black stick models . B . Schematic diagram indicating the secondary structure elements and their organization in Mdm12 . Three structural elements of Mdm12 are highlighted in different colored boxes . C , D . Surface representations of the Mdm12 dimer are shown in different orientations .

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Figure 2A. Ribbon diagram of the yeastMdm12 dimer. The crystal structure of full-length Mdm12 was determined by SAD and refined with native data to 3.1 Å resolution. Lipids bound to Mdm12 are drawn with black stick models.B. Schematic diagram indicating the secondary structure elements and their organization in Mdm12. Three structural elements of Mdm12 are highlighted in different colored boxes.C, D. Surface representations of the Mdm12 dimer are shown in different orientations.


Figure 3A . Ribbon diagram showing the 2 - fold dimerization interface of Mdm12 . Oxygen and nitrogen atoms are shown in red and blue , respectively . The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12 . The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N - terminus β1 - strand . Ten orthologs are aligned from residues 1 to 11 . Absolute and highly conserved residues are indicated in red and orange , respectively . B . The molecular weight of the Mdm12 ( I5P ) mutant was measured by size - exclusion chromatography ( below ) and ultracentrifugation ( top ) as in Figure 1B and C . C . Ribbon diagram showing the structures of the SMP domain in E - SYT2 , CETP , and BPI for the comparison of dimeric interfaces among SMP domains . Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains .

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Figure 3A. Ribbon diagram showing the 2-fold dimerization interface of Mdm12. Oxygen and nitrogen atoms are shown in red and blue, respectively. The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12. The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N-terminus β1-strand. Ten orthologs are aligned from residues 1 to 11. Absolute and highly conserved residues are indicated in red and orange, respectively.B. The molecular weight of the Mdm12 (I5P) mutant was measured by size-exclusion chromatography (below) and ultracentrifugation (top) as in Figure 1B and C.C. Ribbon diagram showing the structures of the SMP domain in E-SYT2, CETP, and BPI for the comparison of dimeric interfaces among SMP domains. Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains.


Figure 4A . Simulated annealing omit map ( Fo - Fc , contoured at 1 . 5σ ) showing the molecule bound to Mdm12 ( left ) . The final model for the bound PE is shown as in stick representation . The electron density ( 2Fo - Fc ) calculated in the final model is shown with the stick model of PE in the right ( 3 . 1 Å resolution , contoured at 0 . 8σ ) . B . Surface representation of the Mdm12 dimer . Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh . Lipids built in Mdm12 are in space - filling representation . C . Ribbon diagram showing lipid coordination by Mdm12 . Mdm12 residues and lipid fatty acids are colored in green and yellow , respectively . D . Mdm12 binds NBD - PE . Wild - type and monomeric ( I5P mutant ) Mdm12 were incubated with NBD - PE and separated from free NBD - PE in native PAGE . Coomassie staining ( left ) and fluorescent ( right ) detection indicates that Mdm12 directly interacts with NBD - PEin vitro . E . Quantitative data showing binding affinities for NBD - PE by Mdm12 . The binding affinities of Mdm12 ( monomer / dimer shown in Native PAGE and I5P mutant ) for NBD - PE was measured with a NBD - PE concentration - dependent manner . All experiments were carried out three times and the means ± SD are given . F . Mdm12 mutants ( L256W , I262W , and L256W / I262W double mutant ) were incubated with NBD - PE , and subjected to native PAGE . Because wild - type Mdm12 separates as both monomer and dimer on native PAGE , the purely monomeric form ( I5P ) of Mdm12 was used as the wild type for clarity . The graph in the right indicates the quantities measured in the experiments . The bar shows the relative amounts of the band ratio ( fluorescence / Coomassie ) . Values represent the means and SD from three independent experiments .

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

Figure 4A. Simulated annealing omit map (Fo-Fc, contoured at 1.5σ) showing the molecule bound to Mdm12 (left). The final model for the bound PE is shown as in stick representation. The electron density (2Fo-Fc) calculated in the final model is shown with the stick model of PE in the right (3.1 Å resolution, contoured at 0.8σ).B. Surface representation of the Mdm12 dimer. Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh. Lipids built in Mdm12 are in space-filling representation.C. Ribbon diagram showing lipid coordination by Mdm12. Mdm12 residues and lipid fatty acids are colored in green and yellow, respectively.D. Mdm12 binds NBD-PE. Wild-type and monomeric (I5P mutant) Mdm12 were incubated with NBD-PE and separated from free NBD-PE in native PAGE. Coomassie staining (left) and fluorescent (right) detection indicates that Mdm12 directly interacts with NBD-PEin vitro.E. Quantitative data showing binding affinities for NBD-PE by Mdm12. The binding affinities of Mdm12 (monomer/dimer shown in Native PAGE and I5P mutant) for NBD-PE was measured with a NBD-PE concentration-dependent manner. All experiments were carried out three times and the means ± SD are given.F. Mdm12 mutants (L256W, I262W, and L256W/I262W double mutant) were incubated with NBD-PE, and subjected to native PAGE. Because wild-type Mdm12 separates as both monomer and dimer on native PAGE, the purely monomeric form (I5P) of Mdm12 was used as the wild type for clarity. The graph in the right indicates the quantities measured in the experiments. The bar shows the relative amounts of the band ratio (fluorescence/Coomassie). Values represent the means and SD from three independent experiments.


Figure 5A . Structural views suggesting that Mdm12 might have a preference for certain phospholipids . The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution ( left ) and the sequence conservation ( right ) in the same orientation . Surface electrostatics and sequence conservation were calculated using an APBS program [ 31 ] with the nonlinear Poisson - Boltzmann equation and contoured at ± 3kT / e , and ConSurf website ( consurf . tau . ac . il ) [ 32 ] with 34 different yeast orthologs , respectively . The ribbon diagram shown in the middle indicates the overall orientation of Mdm12 . B . Lipid displacement experiments to identity the natural ligands of Mdm12in vitro ( see material and methods section for details ) . NBD - PE - preloaded Mdm12 - GST was mixed with a series of glycerophospholipids at different concentrations , and displacement of NBD - PE by non - labeled ligand was estimated from the decrease in fluorescence . Means ± SD are shown ( n = 3 independently performed experiments ) .

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

Figure 5A. Structural views suggesting that Mdm12 might have a preference for certain phospholipids. The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution (left) and the sequence conservation (right) in the same orientation. Surface electrostatics and sequence conservation were calculated using an APBS program [31] with the nonlinear Poisson-Boltzmann equation and contoured at ±3kT/e, and ConSurf website (consurf.tau.ac.il) [32] with 34 different yeast orthologs, respectively. The ribbon diagram shown in the middle indicates the overall orientation of Mdm12.B. Lipid displacement experiments to identity the natural ligands of Mdm12in vitro (see material and methods section for details). NBD-PE-preloaded Mdm12-GST was mixed with a series of glycerophospholipids at different concentrations, and displacement of NBD-PE by non-labeled ligand was estimated from the decrease in fluorescence. Means ± SD are shown (n=3 independently performed experiments).


Figure 6A . Overall structure ( left ) and electron density ( right ) of Mdm12 in the asymmetric unit . Four molecules ( two Mdm12 dimers ) are organized with 2 - fold rotation symmetry . The 2 - fold axes are indicated with a black dotted line . B . Overall structure ( left ) and electron density ( right ) of ΔMdm12 in the asymmetric unit . Six ΔMdm12 molecules ( three Mdm12 dimers ) are arranged with 2 - fold rotation symmetry as shown above . C . Ribbon diagram showing a 2 - fold interface ( tail - to - tail ) in two crystal structures ( See text for details ) . D . Size - exclusion chromatography revealing that the N - terminus - truncated version of Mdm12 ( residues 11 - 271 ) retains the ability to interact with Mmm1 . The experiment was performed as in Figure 1B . Eluted fractions indicated by shading were subjected to SDS - PAGE followed by Coomassie Blue staining .

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

Figure 6A. Overall structure (left) and electron density (right) of Mdm12 in the asymmetric unit. Four molecules (two Mdm12 dimers) are organized with 2-fold rotation symmetry. The 2-fold axes are indicated with a black dotted line.B. Overall structure (left) and electron density (right) of ΔMdm12 in the asymmetric unit. Six ΔMdm12 molecules (three Mdm12 dimers) are arranged with 2-fold rotation symmetry as shown above.C. Ribbon diagram showing a 2-fold interface (tail-to-tail) in two crystal structures (See text for details).D. Size-exclusion chromatography revealing that the N-terminus-truncated version of Mdm12 (residues 11-271) retains the ability to interact with Mmm1. The experiment was performed as in Figure 1B. Eluted fractions indicated by shading were subjected to SDS-PAGE followed by Coomassie Blue staining.


Figure 1A - F ' Upper panels ( A - F ) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage , lower panels ( A ' - F ' ) show c4da neuron morphology at 18 h after puparium formation ( APF ) . Neurons were labeled by CD8 : : GFP expression under ppk - GAL4 or by tdtomato expression in MARCM clones . ( A , A ' ) C4da neurons expressing control Orco dsRNA . Scale bars are 100 μm in A ( for larval images ) and 50 μm in A ' ( for pupal images ) . ( B , B ' ) C4da neurons expressing PP2A - 29B dsRNA . ( C , C ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones . ( D , D ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones expressing UAS - HAPP2A - 29B . ( E , E ' ) C4da neurons expressing dominant - negative Mts . ( F , F ' ) Homozygous mtss5286 mutant c4da neuron MARCM clones . G Penetrance of pruning defects at 18 h APF in A ' - E ' . * * P < 0 . 005 , * * * P < 0 . 0005 , Fisher ' s exact test . Number of neurons for each genotype are given in the figure , the number of animals is given below in parentheses . H Severity of pruning defects at 18 h APF in A ' - E ' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

Figure 1A - F' Upper panels (A - F) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage, lower panels (A' - F') show c4da neuron morphology at 18 h after puparium formation (APF). Neurons were labeled by CD8::GFP expression under ppk-GAL4 or by tdtomato expression in MARCM clones. (A, A') C4da neurons expressing control Orco dsRNA. Scale bars are 100 μm in A (for larval images) and 50 μm in A' (for pupal images). (B, B') C4da neurons expressing PP2A-29B dsRNA. (C, C') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones. (D, D') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones expressing UAS-HAPP2A-29B. (E, E') C4da neurons expressing dominant-negative Mts. (F, F') Homozygous mtss5286 mutant c4da neuron MARCM clones. G Penetrance of pruning defects at 18 h APF in A' - E'. ** P<0.005, *** P<0.0005, Fisher's exact test. Number of neurons for each genotype are given in the figure, the number of animals is given below in parentheses. H Severity of pruning defects at 18 h APF in A' - E' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


Figure 2A - J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and dendrite pruning defects were assessed at 18 h APF . Panels A - E show effects of Sox14 / Mical pathway manipulations alone , panels F - J show neurons coexpressing PP2A - 29B dsRNA . The scale bar in A is 50 μm . ( A , F ) C4da neurons ( co - ) expressing lacZ . ( B , G ) C4da neurons ( co - ) overexpressing Sox14 . ( C , H ) C4da neurons ( co - ) overexpressing Mical . ( D , I ) C4da neurons ( co - ) expressing Mical ∆ redox . ( E , J ) C4da neurons ( co - ) expressing Mical ∆ CH . K Penetrance of pruning defects in panels A - J . * P < 0 . 05 , * * * P < 0 . 0005 , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure the numbers of animals are given below in parentheses . L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

Figure 2A-J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and dendrite pruning defects were assessed at 18 h APF. Panels A - E show effects of Sox14/Mical pathway manipulations alone, panels F - J show neurons coexpressing PP2A-29B dsRNA. The scale bar in A is 50 μm. (A, F) C4da neurons (co-)expressing lacZ. (B, G) C4da neurons (co-)overexpressing Sox14. (C, H) C4da neurons (co-)overexpressing Mical. (D, I) C4da neurons (co-)expressing Mical∆redox. (E, J) C4da neurons (co-)expressing Mical∆CH. K Penetrance of pruning defects in panels A - J. * P<0.05, *** P<0.0005, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure the numbers of animals are given below in parentheses. L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


Figure 3C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and expression of Sox14 was assessed by immunofluorescence . A - C Sox14 stainings at 2 h APF . ( A ) Control c4da neurons . ( B ) C4da neurons expressing PP2A - 29B dsRNA . ( C ) C4da neuron expressing dominant - negative Mts . D Quantification of Sox14 expression in ( A - C ) . C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons ( identified by characteristic cell body shape ) . Data are mean + / - s . d . . The numbers of neurons ( animals ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * * P < 0 . 0005 , Wilcoxon ' s test . C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and expression of Mical was assessed by immunofluorescence . E - H Mical stainings at the indicated time points . ( E ) Mical expression in control c4da neurons at 2 h APF . ( F ) Mical expression in c4da neurons expressing dominant - negative Mts at 2 h APF . ( G ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 2 h APF . ( H ) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF . ( I ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 0 h APF . J Quantification of Mical expression in ( E - I ) . C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons ( marked by asterisks ) . Data are mean + / - s . d . . The numbers of neurons ( animals ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Data information : Scale bars in A and E are 10 μm .

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

Figure 3C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Sox14 was assessed by immunofluorescence. A - C Sox14 stainings at 2 h APF. (A) Control c4da neurons. (B) C4da neurons expressing PP2A-29B dsRNA. (C) C4da neuron expressing dominant-negative Mts. D Quantification of Sox14 expression in (A - C). C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons (identified by characteristic cell body shape). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, *** P<0.0005, Wilcoxon's test. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Mical was assessed by immunofluorescence. E - H Mical stainings at the indicated time points. (E) Mical expression in control c4da neurons at 2 h APF. (F) Mical expression in c4da neurons expressing dominant-negative Mts at 2 h APF. (G) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 2 h APF. (H) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF. (I) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 0 h APF. J Quantification of Mical expression in (E - I). C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons (marked by asterisks). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, ** P<0.005, *** P<0.0005, Wilcoxon's test. Data information: Scale bars in A and E are 10 μm.


Figure 4 A - G The indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA and lacZ as a UAS titration control . H Penetrance of pruning defects in panels A - G . * P < 0 . 05 , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure . I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure . J - L GFP - tagged Drosophila cofilin ( GFP : : tsr ) was expressed in c4da neurons under ppk - GAL4 , and its localization was assessed at the indicated developmental stages by live imaging . Panels J - L show GFP : : tsr localization in c4da neurons at the third instar stage , panels J ' - L ' show GFP : : tsr localization in c4da neurons at 6 h APF . ( J , J ' ) Control c4da neurons expressing Orco dsRNA . ( K , K ' ) C4da neurons expressing Mical dsRNA . ( L , L ' ) C4da neurons expressing PP2A - 29B dsRNA . M Quantification of GFP : : tsr punctae in samples J - L ' . Data are mean + / - s . d . , the numbers of neurons ( animals ) analyzed for each genotype are given in the figure ( data were not derived from independent experiments ) . * * P < 0 . 005 , * * * P < 0 . 0005 , n . s . , not significant , Wilcoxon ' s test .

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

Figure 4 A - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control. H Penetrance of pruning defects in panels A - G. * P<0.05, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure. J - L GFP-tagged Drosophila cofilin (GFP::tsr) was expressed in c4da neurons under ppk-GAL4, and its localization was assessed at the indicated developmental stages by live imaging. Panels J - L show GFP::tsr localization in c4da neurons at the third instar stage, panels J' - L' show GFP::tsr localization in c4da neurons at 6 h APF. (J, J') Control c4da neurons expressing Orco dsRNA. (K, K') C4da neurons expressing Mical dsRNA. (L, L') C4da neurons expressing PP2A-29B dsRNA. M Quantification of GFP::tsr punctae in samples J - L'. Data are mean +/- s. d., the numbers of neurons (animals) analyzed for each genotype are given in the figure (data were not derived from independent experiments). ** P<0.005, *** P<0.0005, n. s., not significant, Wilcoxon's test.


Figure 5A - H Genetic interactions between PP2A - 29B and actin regulators during dendrite pruning . The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . ( A ) C4da neuron coexpressing PP2A - 29B dsRNA and tdtomato as suppression control . ( B ) C4da neuron coexpressing PP2A - 29B dsRNA and GFP : : tsr . ( C ) C4da neuron coexpressing PP2A - 29B dsRNA and RacN17 . ( D ) C4da neuron coexpressing PP2A - 29B dsRNA and RhoN19 . ( E ) C4da neuron expressing PP2A - 29B dsRNA in ena210 / + background . ( F ) C4da neuron coexpressing PP2A - 29B dsRNA and Orco dsRNA as suppression control . ( G ) C4da neuron coexpressing PP2A - 29B dsRNA and Chicadee dsRNA . ( H ) C4da neuron coexpressing PP2A - 29B dsRNA and moesin dsRNA . I Penetrance of pruning defects in panels E - L . n . s . , not significant , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure . J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

Figure 5A - H Genetic interactions between PP2A-29B and actin regulators during dendrite pruning. The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. (A) C4da neuron coexpressing PP2A-29B dsRNA and tdtomato as suppression control. (B) C4da neuron coexpressing PP2A-29B dsRNA and GFP::tsr. (C) C4da neuron coexpressing PP2A-29B dsRNA and RacN17. (D) C4da neuron coexpressing PP2A-29B dsRNA and RhoN19. (E) C4da neuron expressing PP2A-29B dsRNA in ena210/+ background. (F) C4da neuron coexpressing PP2A-29B dsRNA and Orco dsRNA as suppression control. (G) C4da neuron coexpressing PP2A-29B dsRNA and Chicadee dsRNA. (H) C4da neuron coexpressing PP2A-29B dsRNA and moesin dsRNA. I Penetrance of pruning defects in panels E - L. n. s., not significant, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


Figure 2 ( A ) Sequential COX - SDH histochemical staining in muscle biopsies of Pa2 , Pa3 - 1 , Pa4 and Pa6 ( as indicated ) , indicating a mosaic pattern of COX deficiency . Scale bar represents 100 µm . ( C ) Long - range PCR assay to detect mtDNA deletions in TOP3A patient muscle ( lanes 2 - 5 ) . Samples from unaffected individuals ( lanes 6 and 7 ) and a single - deletion mitochondrial disease patient ( lane 8 ) are used as negative and positive controls , respectively . ' M ' indicates marker .

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Figure 2(A) Sequential COX-SDH histochemical staining in muscle biopsies of Pa2, Pa3-1, Pa4 and Pa6 (as indicated), indicating a mosaic pattern of COX deficiency. Scale bar represents 100 µm.(C) Long-range PCR assay to detect mtDNA deletions in TOP3A patient muscle (lanes 2-5). Samples from unaffected individuals (lanes 6 and 7) and a single-deletion mitochondrial disease patient (lane 8) are used as negative and positive controls, respectively. 'M' indicates marker.


Figure 3 ( A - B ) Analysis of mtDNA structure in muscle from individuals with TOP3A - related mitochondrial disease using Southern blotting following restriction with BamHI ( A ) or PvuII ( B ) . The black bar indicates the probe ( nt . 16 , 262 - 128 ) . mtDNA phenotypes in U2OS Flp - In cells treated with TOP3A siRNA , rescued using variant - containing siRNA - resistant forms of TOP3A . ( C ) Western blot of TOP3A protein expression . Cells were untransfected ( lane 1 ) or transfected with TOP3A siRNA ( lanes 2 - 11 ) , then induced to express WT TOP3A ( lane 3 ) or pathological variants of TOP3A ( lanes 4 - 11 ) . Note that TOP3A Ser810 * does not carry a C - terminal HA tag . β - actin is used as a loading control . mtDNA phenotypes in U2OS Flp - In cells treated with TOP3A siRNA , rescued using variant - containing siRNA - resistant forms of TOP3A . ( D ) mtDNA copy number for TOP3A rescue cells as in ( C ) determined using qPCR . Error bars represent SEM , n = 3 ( Ala95Val ) or n = 4 ( all other samples ) , where each data point represents the mean of 3 technical triplicates . Significance values are shown for one - way ANOVA compared to untreated wild - type cells , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . mtDNA phenotypes in U2OS Flp - In cells treated with TOP3A siRNA , rescued using variant - containing siRNA - resistant forms of TOP3A . ( E ) Southern blot of mtDNA structure from cells as in ( C ) . The probe ( black bar , nt . 16 , 262 - 128 ) detects both full - length ( FL ) mtDNA and 7S DNA . The presence of hemicatenated mtDNAs is indicated . ( F ) Southern blot of uncut mtDNA to visualise different topological forms , from cells as in ( C ) . Control DNA treated with BamHI ( lane 1 ) or E . coli topoisomerase I ( lane 2 ) act as markers for linear and relaxed monomeric mtDNA , respectively . Diagrams indicate the migration of different mtDNA forms .

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

Figure 3(A-B) Analysis of mtDNA structure in muscle from individuals with TOP3A-related mitochondrial disease using Southern blotting following restriction with BamHI (A) or PvuII (B). The black bar indicates the probe (nt. 16,262 - 128).mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (C) Western blot of TOP3A protein expression. Cells were untransfected (lane 1) or transfected with TOP3A siRNA (lanes 2-11), then induced to express WT TOP3A (lane 3) or pathological variants of TOP3A (lanes 4-11). Note that TOP3A Ser810* does not carry a C-terminal HA tag. β-actin is used as a loading control.mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (D) mtDNA copy number for TOP3A rescue cells as in (C) determined using qPCR. Error bars represent SEM, n = 3 (Ala95Val) or n = 4 (all other samples), where each data point represents the mean of 3 technical triplicates. Significance values are shown for one-way ANOVA compared to untreated wild-type cells, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (E) Southern blot of mtDNA structure from cells as in (C). The probe (black bar, nt. 16,262 - 128) detects both full-length (FL) mtDNA and 7S DNA. The presence of hemicatenated mtDNAs is indicated. (F) Southern blot of uncut mtDNA to visualise different topological forms, from cells as in (C). Control DNA treated with BamHI (lane 1) or E. coli topoisomerase I (lane 2) act as markers for linear and relaxed monomeric mtDNA, respectively. Diagrams indicate the migration of different mtDNA forms.


Figure 4 ( A ) Purification of TOP3A variants . Each variant ( 3 pmol ) was separated on a 4 - 20 % stain - free Criterion TGX SDS - PAGE gel ( Bio - Rad ) and imaged using a stain - free imager . Electrophoretic mobility shift assay ( EMSA ) to assess DNA binding activity of TOP3A variants to a 5 ' radiolabelled 80 nt - long ssDNA oligonucleotide . Electrophoretic mobility shift assay ( EMSA ) to assess DNA binding activity of TOP3A variants to a 5 ' radiolabelled 80 nt - long ssDNA oligonucleotide . Electrophoretic mobility shift assay ( EMSA ) to assess DNA binding activity of TOP3A variants to a 5 ' radiolabelled 80 nt - long ssDNA oligonucleotide . ( K ) Quantification of DNA binding activity as in ( B - J ) , expressed as the proportion of bound DNA . Data represents mean values with at least 3 independent experiments per data point . Error bars represent SEM . Symbols represent significance values ( one - way ANOVA ) , colour - coded to the data points . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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

Figure 4(A) Purification of TOP3A variants. Each variant (3 pmol) was separated on a 4-20% stain-free Criterion TGX SDS-PAGE gel (Bio-Rad) and imaged using a stain-free imager.Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.(K) Quantification of DNA binding activity as in (B-J), expressed as the proportion of bound DNA. Data represents mean values with at least 3 independent experiments per data point. Error bars represent SEM. Symbols represent significance values (one-way ANOVA), colour-coded to the data points. *p<0.05, **p<0.01, ****p<0.0001.


Figure 5 ( A - I ) Relaxation of negatively supercoiled pUC19 substrate by recombinant TOP3A variants . ' M ' indicates marker . ( J ) Quantification of DNA relaxation data as in ( A - I ) . Data represents the percentage of substrate released from negatively supercoiled form , expressed as the mean of 3 independent experiments ( except WT , for which n = 4 ) . Error bars represent SEM . Symbols represent significance values ( one - way ANOVA ) , colour - coded to the data points . * * * * p < 0 . 0001 .

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Figure 5(A-I) Relaxation of negatively supercoiled pUC19 substrate by recombinant TOP3A variants. 'M' indicates marker.(J) Quantification of DNA relaxation data as in (A-I). Data represents the percentage of substrate released from negatively supercoiled form, expressed as the mean of 3 independent experiments (except WT, for which n = 4). Error bars represent SEM. Symbols represent significance values (one-way ANOVA), colour-coded to the data points. ****p<0.0001.


Figure 6 ( B - J ) ssDNA decatenation assays using TOP3A variants , using substrates constructed as in ( A ) . ( K ) Quantification of ssDNA decatenation activity as in ( B - J ) . Data represents the percentage of decatenated substrate expressed as mean values , ± SEM , from independent experiments , n = 3 ( WT , p . Ala95Val , p . Met100Val , p . Arg558Trp ) , n = 4 ( p . Ala176Val ) , n = 5 ( p . Met575Val ) , n = 2 ( p . Leu37Val ) . Symbols represent significance values ( one - way ANOVA ) , colour - coded to the data points . * p < 0 . 05 , * * p < 0 . 01 . ( M ) Quantifications of ssDNA decatenation assays as in ( L ) . Data represents the mean percentage of decatenated substrate from 3 independent experiments . Significance values are shown for one - way ANOVA compared to the WT protein . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 6(B-J) ssDNA decatenation assays using TOP3A variants, using substrates constructed as in (A).(K) Quantification of ssDNA decatenation activity as in (B-J). Data represents the percentage of decatenated substrate expressed as mean values, ±SEM, from independent experiments, n = 3 (WT, p.Ala95Val, p.Met100Val, p.Arg558Trp), n = 4 (p.Ala176Val), n = 5 (p.Met575Val), n = 2 (p.Leu37Val). Symbols represent significance values (one-way ANOVA), colour-coded to the data points. *p<0.05, **p<0.01.(M) Quantifications of ssDNA decatenation assays as in (L). Data represents the mean percentage of decatenated substrate from 3 independent experiments. Significance values are shown for one-way ANOVA compared to the WT protein. *p<0.05, **p<0.01, ***p<0.001.


Figure 1Analysis of DNA content by flow cytometry . Analysis of BrdU incorporation by BrdU - IP - chip 45 minutes after release into S phase in cells treated with 0 . 1 % MMS as described in ( A ) . A representative region on chromosome VII is shown . The position of replication origins is indicated above the map . Asterisks : late origins firing in mrc1 ∆ cells . Quantification of the BrdU incorporation ( signal log ratio ) at 384 early and late replication origins in wild type , rad9 ∆ and mrc1 ∆ strains exposed to 0 . 1 or 0 . 033 % MMS . Red lines indicate median values . Median replication times ( Trep ) are indicated for bins of 96 origins .

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Figure 1Analysis of DNA content by flow cytometry.Analysis of BrdU incorporation by BrdU-IP-chip 45 minutes after release into S phase in cells treated with 0.1% MMS as described in (A). A representative region on chromosome VII is shown. The position of replication origins is indicated above the map. Asterisks: late origins firing in mrc1∆ cells.Quantification of the BrdU incorporation (signal log ratio) at 384 early and late replication origins in wild type, rad9∆ and mrc1∆ strains exposed to 0.1 or 0.033% MMS. Red lines indicate median values. Median replication times (Trep) are indicated for bins of 96 origins.


Figure 2Wild type , mrc1 ∆ and rad9 ∆ cells were released into S phase in the presence of BrdU and 0 . 033 % MMS , as described in Fig . 1A . DNA combing analysis was performed for samples collected 45 and 60 min after release into S phase . Representative DNA fibers are shown . Red : DNA , green : BrdU . Bar is 20 kb . Distribution of center - to - center distances in cells released for 45 minutes into S phase . Distribution of BrdU track lengths in cells released for 45 or 60 minutes into S phase , respectively . Wild type , mrc1 ∆ and rad9 ∆ cells were synchronized in G1 with α - factor prior to release with pronase in the presence of 0 . 033 % MMS . Cells were collected at the indicated times and Rad53 phosphorylation was monitored by western blot The asterisk indicates a non - specific band detected by the anti - Rad53 antibody . Wild type , mrc1 ∆ and rad9 ∆ cells were synchronized in G1 with α - factor prior to release with pronase in the presence of 0 . 033 % MMS . Cells were collected at the indicated times and Rad53 phosphorylation was monitored with an in situ phosphorylation assay - ISA ( F ) .

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

Figure 2Wild type, mrc1∆ and rad9∆ cells were released into S phase in the presence of BrdU and 0.033% MMS, as described in Fig. 1A. DNA combing analysis was performed for samples collected 45 and 60 min after release into S phase. Representative DNA fibers are shown. Red: DNA, green: BrdU. Bar is 20 kb.Distribution of center-to-center distances in cells released for 45 minutes into S phase.Distribution of BrdU track lengths in cells released for 45 or 60 minutes into S phase, respectively.Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored by western blot The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored with an in situ phosphorylation assay - ISA (F).


Figure 3Analysis of Rad53 phosphorylation by Western blot . Analysis of DNA content by flow cytometry . Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg / ml Zeocin . IdU tracks ( red ) correspond to initiation events occurring during the HU treatment . Elongation events after HU removal appear as CldU tracks ( yellow ) flanking IdU tracks . New initiation events as detected as isolated CldU tracks ( asterisks ) . Bar is 20 kb . Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in ( D ) . For each sample , at least 65 individual DNA fiber larger than 200 kb were analyzed , for a total of > 17 Mb . 2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212 . The experiment was performed as in ( A ) for the indicated times . Arrows indicate late origin firing . Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in ( D ) . Median values are indicated in red . For each sample , at least 250 tracks were analyzed in two biological replicates .

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

Figure 3Analysis of Rad53 phosphorylation by Western blot.Analysis of DNA content by flow cytometry.Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg/ml Zeocin. IdU tracks (red) correspond to initiation events occurring during the HU treatment. Elongation events after HU removal appear as CldU tracks (yellow) flanking IdU tracks. New initiation events as detected as isolated CldU tracks (asterisks). Bar is 20 kb.Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in (D). For each sample, at least 65 individual DNA fiber larger than 200 kb were analyzed, for a total of >17 Mb.2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212. The experiment was performed as in (A) for the indicated times. Arrows indicate late origin firing.Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in (D). Median values are indicated in red. For each sample, at least 250 tracks were analyzed in two biological replicates.