Figure 4Percentage ( % ) of γ - H2AX positive cells in HeLa and CCNF K / O after transfection of the indicated siRNAs treated with Chk1i for 20 hours . Percent of γ - H2Ax positive cells were plotted as Mean % ± SD . Percentage ( % ) of hyper - RPA positive cells in HeLa and CCNF K / O after transfection of the indicated siRNAs treated with Chk1i for 20 hours . Percent of hyper RPA positive cells were plotted as Mean % ± SD . Percentage ( % ) of γ - H2AX positive cells in S phase cells by DAPI content after Chk1i ( LY2603618 ) Relative γ - H2AX intensity in cells transfected with either Empty Vector ( EV ) or HA - E2F1 treated with Chk1i for 20 hours . γ - H2AX fluorescence in HA positive cells was normalized to the empty vector transfected cells and compared to cells not expressing HA . HeLa and CCNF K / O cells transfected with the indicated siRNA and treated with 1μM Chk1i ( LY2603618 ) for 20 hours were harvested and lysed using SDS . Indicated protein were resolved by SDS page and detected by WB . TFIIH served as a loading control .

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 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 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 4Percentage (%) of γ-H2AX positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of γ-H2Ax positive cells were plotted as Mean % ± SD. Percentage (%) of hyper-RPA positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of hyper RPA positive cells were plotted as Mean % ± SD.Percentage (%) of γ-H2AX positive cells in S phase cells by DAPI content after Chk1i (LY2603618)Relative γ-H2AX intensity in cells transfected with either Empty Vector (EV) or HA-E2F1 treated with Chk1i for 20 hours. γ-H2AX fluorescence in HA positive cells was normalized to the empty vector transfected cells and compared to cells not expressing HA.HeLa and CCNF K/O cells transfected with the indicated siRNA and treated with 1μM Chk1i (LY2603618) for 20 hours were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. TFIIH served as a loading control.


Figure 5HeLa and CCNF K / O cells were harvested and lysed using SDS . Indicated protein were resolved by SDS page and detected by WB . Ponceau S staining was used as a loading control . U - 2 - OS cells transfected with non - targeting siRNA ( siNC ) or with an siRNA targeting Cyc F were lysed using SDS . Whole cell lysates were analyzed by immunoblotting 48h after transfection . H3 was used as a loading control . Heatmap showing gene set variance analysis ( GSVA ) score for gene sets related to E2F targets ( PID _ E2F _ PATHWAY , ISHIDA _ E2F _ TARGETS and HALLMARK _ E2F _ TARGETS ) . HEK293T cells transfected with empty vector ( EV ) , FLAG - cyclin F WT , FLAG - cyclin F ( ∆ F ) and FLAG - cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies . Immunoprecipitates were immunoblotted as indicated . HEK293T cells were cotransfected with MYC - tagged ubiquitin and HA - E2F1 in the presence of FLAG - cyclin F WT , FLAG - cyclin F ( ∆ F ) and FLAG - cyclin F M309A were indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions and immunoblotted . HeLa and CCNF K / O cells treated with 10 μM Chk1i ( LY2603618 ) for the indicated time points ( h = hours ) were harvested and lysed using SDS . Indicated protein were resolved by SDS page and detected by WB . H2B served as a loading control . HEK293 cells transfected with empty vector ( EV ) or HA - E2F1 were treated with Chk1i for 20 hours and harvested for immunoprecipitation using HA antibodies . Immunoprecipitates were immunoblotted as indicated . Quantification of immunoprecipitation results of Figure 5G from three independent experiments . Data from 3 independent experiments were plotted with Mean % ± SD . * < 0 . 05 p - value and calculated by two - tailed paired t - test . HEK293T cells were cotransfected with MYC - tagged ubiquitin and HA - E2F1 were treated with Chk1i for 20 hours and siRNA of cyclin F were indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions and immunoblotted .

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

Figure 5HeLa and CCNF K/O cells were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. Ponceau S staining was used as a loading control.U-2-OS cells transfected with non-targeting siRNA (siNC) or with an siRNA targeting Cyc F were lysed using SDS. Whole cell lysates were analyzed by immunoblotting 48h after transfection. H3 was used as a loading control.Heatmap showing gene set variance analysis (GSVA) score for gene sets related to E2F targets (PID_E2F_PATHWAY, ISHIDA_E2F_TARGETS and HALLMARK_E2F_TARGETS).HEK293T cells transfected with empty vector (EV), FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies. Immunoprecipitates were immunoblotted as indicated.HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 in the presence of FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.HeLa and CCNF K/O cells treated with 10 μM Chk1i (LY2603618) for the indicated time points (h=hours) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. H2B served as a loading control.HEK293 cells transfected with empty vector (EV) or HA-E2F1 were treated with Chk1i for 20 hours and harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.Quantification of immunoprecipitation results of Figure 5G from three independent experiments. Data from 3 independent experiments were plotted with Mean % ± SD. *< 0.05 p-value and calculated by two-tailed paired t-test.HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 were treated with Chk1i for 20 hours and siRNA of cyclin F were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


Figure 6HEK293T cells transfected with HA - E2F1 Wild Type ( WT ) and HA - E2F1 lacking a CY motif ( ∆ RxL ) harvested for immunoprecipitation using HA antibodies . Immunoprecipitates were immunoblotted as indicated . HEK293T cells were cotransfected with MYC - tagged ubiquitin and HA - E2F1 or HA - E2F1 ∆ RxL lacking the CY motif as indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions and immunoblotted . HeLa cells stably expressing HA - E2F1 WT and HA - E2F1 ∆ RxL under the control of a retroviral promoter were treated with Cycloheximide ( CHX ) at 50 μg / ml for the indicated minutes ( min ) Quantification of E2F1 half - lifeHeLa cells stably expressing an Empty Vector ( EV ) , HA - E2F1 WT and HA - E2F1 ∆ RxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours ( h ) . Indicated protein were resolved by SDS page and detected by WB . H3 was used as a loading control . Relative survival ( % ) of HeLa cells stably expressing an Empty Vector ( EV ) , HA - E2F1 WT and HA - E2F1 ∆ RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations .

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 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 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 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 6HEK293T cells transfected with HA-E2F1 Wild Type (WT) and HA-E2F1 lacking a CY motif (∆RxL) harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 or HA-E2F1 ∆RxL lacking the CY motif as indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min) Quantification of E2F1 half-lifeHeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours (h). Indicated protein were resolved by SDS page and detected by WB. H3 was used as a loading control.Relative survival (%) of HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations.


Figure 1A . Representative plots of splenocytes from CD45 . 1 mice adoptively transferred with CD45 . 2 OT - II WT or CCR5 - / - lymph node cell suspensions , five weeks after infection with rVACV - OVA virus . The gating strategy used to identify the memory CD4 + T cell subtypes is shown ( n = 5 ) . B . Absolute number of OT - II cells recovered in spleens of mice as in A ( n = 5 ) . C , Percentage of CD4 + TEM ( C ) in the OT - II WT and CCR5 - / - populations ( n = 5 ) . D . Percentage of TCM ( D ) in the OT - II WT and CCR5 - / - populations ( n = 5 ) . E . IFNγ - producing OT - II WT and CCR5 - / - memory cells isolated from mice as in A and restimulated ex vivo with OVA323 - 339 ( 1 μM ) ( n = 4 ) . G - I . Representative plots ( G ) and quantification of the frequency ( H ) and absolute number ( I ) of Tfh cells ( CD4 + CD44 + PD - 1 + CXCR5 + ) in the spleen after primary immunization ( day 7 ) with NIP - OVA ( n = 7 ) . J , K . ELISA analysis of high - ( J ) and low - affinity ( K ) isotype - specific anti - NIP antibodies in sera from OVA / OVA - and OVA / KLH - immunized mice ( day 15 post - challenge ; n = 5 mice / group ) . Data representative of one experiment of two .

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

Figure 1A. Representative plots of splenocytes from CD45.1 mice adoptively transferred with CD45.2 OT-II WT or CCR5-/- lymph node cell suspensions, five weeks after infection with rVACV-OVA virus. The gating strategy used to identify the memory CD4+ T cell subtypes is shown (n = 5).B. Absolute number of OT-II cells recovered in spleens of mice as in A (n = 5).C, Percentage of CD4+ TEM (C) in the OT-II WT and CCR5-/- populations (n = 5).D. Percentage of TCM (D) in the OT-II WT and CCR5-/- populations (n = 5).E. IFNγ-producing OT-II WT and CCR5-/- memory cells isolated from mice as in A and restimulated ex vivo with OVA323-339 (1 μM) (n = 4).G-I. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of Tfh cells (CD4+CD44+PD-1+CXCR5+) in the spleen after primary immunization (day 7) with NIP-OVA (n = 7).J, K. ELISA analysis of high- (J) and low-affinity (K) isotype-specific anti-NIP antibodies in sera from OVA/OVA- and OVA/KLH-immunized mice (day 15 post-challenge; n = 5 mice/group). Data representative of one experiment of two.


Figure 2A , Representative histograms and quantification of mean fluorescence intensity ( MFI ; A ) in OT - II WT and CCR5 ‑ / ‑ lymphoblasts expanded in IL - 2 or IL - 15 , as specified . Data shown as mean ± SEM ( n ≥ 3 ) . B . the percentage of cells positive for the indicated memory markers ( B ) in OT - II WT and CCR5 ‑ / ‑ lymphoblasts expanded in IL - 2 or IL - 15 , as specified . Data shown as mean ± SEM ( n ≥ 3 ) . C - F . IL - 2 - ( C , D ) and IL - 15 - expanded lymphoblasts ( E , F ) were restimulated with indicated concentrations of OVA323 - 339 ; cell proliferation ( thymidine incorporation into DNA ; C , E ) and IL - 2 production ( by ELISA ; D , F ) were measured after 72 h . Data are presented as mean ± SEM ( n = 5 ) .

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 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

Figure 2A, Representative histograms and quantification of mean fluorescence intensity (MFI; A) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3).B. the percentage of cells positive for the indicated memory markers (B) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3).C-F. IL-2- (C, D) and IL-15-expanded lymphoblasts (E, F) were restimulated with indicated concentrations of OVA323-339; cell proliferation (thymidine incorporation into DNA; C, E) and IL-2 production (by ELISA; D, F) were measured after 72 h. Data are presented as mean ± SEM (n = 5).


Figure 3A - C . Analysis of TCR nanoclustering by EM in OT - II WT and CCR5 - / - naïve cells ( A ; n = 6 cells / genotype ; WT : 3427 , CCR5 - / - : 3528 particles ) , and IL - 2 - ( B ; WT , n = 8 cells , 15419 particles ; CCR5 - / - , n = 6 cells , 5410 particles ) or IL - 15 - expanded lymphoblasts ( C ; WT , n = 8 cells , 27518 particles ; CCR5 - / - , n = 7 cells , 22696 particles ) . A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti - CD3ε - labeled cells ; at bottom , quantification ( mean ± SEM ) of gold particles in clusters of indicated size in WT ( gray bars ) and CCR5 - / - cells ( red ) . Insets show the distribution of clusters of one , two , three , four , or more than four particles , and statistical analysis . D , E . Posterior distribution in naïve ( D ) and IL - 2 - expanded lymphoblasts ( E ) of the clustering parameter b for WT ( gray ) and CCR5 - / - cells ( red ) ; randomly generated distributions of receptors are shown in blue . The mean value of the b parameter is indicated for each condition . The probability of a chance distribution similar to that determined in cells is nearly 0 % by the ROPE . F . Comparison of TCR oligomer size using BN - PAGE and anti - CD3ζ immunoblotting in day 10 , IL - 2 - expanded WT and CCR5 - / - OT - II lymphoblasts lysed in buffer containing digitonin or Brij - 96 . The marker protein is ferritin ( f1 , 440 and f2 , 880 kDa forms ) . The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry ( right ; n = 5 ) . G . Top , representative small field EM images showing gold particle distribution in the cell surface replicas of CD4 + T cells isolated from OVA / OVA - immunized WT and CCR5 - / - mice . Bottom , quantification ( mean ± SEM ) of gold particles in clusters of the indicated size ( WT , gray bars ; n = 5 cells , 14680 particles ; CCR5 - / - , red ; n = 7 cells , 15374 particles ) . Insets show the distribution between clusters of one , two , three , four or more than four particles , and statistical analysis .

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 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 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 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

Figure 3A-C. Analysis of TCR nanoclustering by EM in OT-II WT and CCR5-/- naïve cells (A; n = 6 cells/genotype; WT: 3427, CCR5-/-: 3528 particles), and IL-2- (B; WT, n = 8 cells, 15419 particles; CCR5-/-, n = 6 cells, 5410 particles) or IL-15-expanded lymphoblasts (C; WT, n = 8 cells, 27518 particles; CCR5-/-, n = 7 cells, 22696 particles). A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti-CD3ε-labeled cells; at bottom, quantification (mean ± SEM) of gold particles in clusters of indicated size in WT (gray bars) and CCR5-/- cells (red). Insets show the distribution of clusters of one, two, three, four, or more than four particles, and statistical analysis.D, E. Posterior distribution in naïve (D) and IL-2-expanded lymphoblasts (E) of the clustering parameter b for WT (gray) and CCR5-/- cells (red); randomly generated distributions of receptors are shown in blue. The mean value of the b parameter is indicated for each condition. The probability of a chance distribution similar to that determined in cells is nearly 0% by the ROPE.F. Comparison of TCR oligomer size using BN-PAGE and anti-CD3ζ immunoblotting in day 10, IL-2-expanded WT and CCR5-/- OT-II lymphoblasts lysed in buffer containing digitonin or Brij-96. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms). The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry (right; n = 5).G. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of CD4+ T cells isolated from OVA/OVA-immunized WT and CCR5-/- mice. Bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size (WT, gray bars; n = 5 cells, 14680 particles; CCR5-/-, red; n = 7 cells, 15374 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


Figure 4A . OT - II WT cells were activated with OVA323 - 339 , alone or with TAK - 779 . After three days , antigen and TAK - 779 were removed and lymphoblasts expanded in IL - 2 - containing medium . TCR nanoclustering was analyzed in anti - CD3ε - labeled surface replicas of day 10 lymphoblasts . Top , representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4 + T cells alone or with TAK - 779 . Bottom , quantification of gold particles in clusters of the indicated size . Inset , distribution of gold particles between clusters of one , two , three , four or more than four particles in vehicle - ( gray bars ; n = 5 cells , 11266 particles ) and TAK - 779 - treated cells ( black ; n = 6 cells , 5138 particles ) . B . OT - II WT cells were activated with OVA323 - 339 , and TAK - 779 was added at days 3 , 5 , and 7 after antigen removal . Analysis as above , untreated ( gray bars ; n = 5 cells , 6400 particles ) and TAK - 779 - treated cells ( black ; n = 6 cells , 7153 particles ) . Inset show the distribution between clusters of one , two , three , four or more than four particles , and statistical analysis . C . OT - II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100 . Left , representative EM images showing gold particle distribution in the cell surface replicas . Right , analysis of gold particles in clusters as above , vehicle - ( gray bars ; n = 6 cells , 12339 particles ) and AMD3100 - treated cells ( black ; n = 7 cells , 17059 particles ) . Inset show the distribution between clusters of one , two , three , four or more than four particles , and statistical analysis .

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 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

Figure 4A. OT-II WT cells were activated with OVA323-339, alone or with TAK-779. After three days, antigen and TAK-779 were removed and lymphoblasts expanded in IL-2-containing medium. TCR nanoclustering was analyzed in anti-CD3ε-labeled surface replicas of day 10 lymphoblasts. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4+ T cells alone or with TAK-779. Bottom, quantification of gold particles in clusters of the indicated size. Inset, distribution of gold particles between clusters of one, two, three, four or more than four particles in vehicle- (gray bars; n = 5 cells, 11266 particles) and TAK-779-treated cells (black; n = 6 cells, 5138 particles). B. OT-II WT cells were activated with OVA323-339, and TAK-779 was added at days 3, 5, and 7 after antigen removal. Analysis as above, untreated (gray bars; n = 5 cells, 6400 particles) and TAK-779-treated cells (black; n = 6 cells, 7153 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.C. OT-II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100. Left, representative EM images showing gold particle distribution in the cell surface replicas. Right, analysis of gold particles in clusters as above, vehicle- (gray bars; n = 6 cells, 12339 particles) and AMD3100-treated cells (black; n = 7 cells, 17059 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


Figure 5A . Total Chol levels in WT and CCR5 - / - OT - II lymphoblasts ( day 10 , IL - 2 - expanded ) as determined by a fluorometric assay ( n = 6 ) . B - D . SM ( B ) , Cer ( C ) and dhCer ( D ) levels in WT and CCR5 - / - OT - II 10 - day lymphoblasts , as determined by UPLC - TOF MS . Values , after normalization with C17 standards and cell number in each sample , are the mean of two independent experiments ( n = 6 ) . E . RT - qPCR determination of CerS mRNA levels in naïve and IL - 2 - expanded WT and CCR5 ‑ / - OT - II 10 - day lymphoblasts ( n = 3 - 5 ) . F . Representative immunoblot showing CerS2 protein levels in naïve and WT and CCR5 - / - OT - II 10 - day lymphoblasts , and densitometric quantification of blots as above ( n = 10 ) . G . ChIP analysis of the CerS2 promoter using an anti - H3K9Ac antibody . Scheme of the CerS2 promoter showing CpG islands and primers used for amplification . Relative ChIP of the CerS2 promoter in WT and CCR5 - / - OT - II 10 - day lymphoblasts ( n = 3 ) . H . Relative CerS2 mRNA level in CD4 T cells treated with PTx ( n = 3 ) . L . Representative immunofluorescence images showing pSer142 - GATA - 1 staining ( green ) of OT - II WT and CCR5 - / - lymphoblasts . The green channel ( top ) and the merge with nuclear DAPI staining ( blue ; bottom ) are shown . Scale bar , 10 μm . M . Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI - stained area ; n ≥ 50 cells / condition ) . N . Top , basic scheme of the CerS2 promoter , indicating the putative GATA - 1 binding site ( blue ) and location of the primers used for amplification in ChIP assays ( black arrows ) . Bottom , relative anti - GATA - 1 ChIP levels in OT - II WT and CCR5 - / - lymphoblasts ( n = 5 ) .

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

Figure 5A. Total Chol levels in WT and CCR5-/- OT-II lymphoblasts (day 10, IL-2-expanded) as determined by a fluorometric assay (n = 6).B-D. SM (B), Cer (C) and dhCer (D) levels in WT and CCR5-/- OT-II 10-day lymphoblasts, as determined by UPLC-TOF MS. Values, after normalization with C17 standards and cell number in each sample, are the mean of two independent experiments (n = 6).E. RT-qPCR determination of CerS mRNA levels in naïve and IL-2-expanded WT and CCR5‑/- OT-II 10-day lymphoblasts (n = 3-5).F. Representative immunoblot showing CerS2 protein levels in naïve and WT and CCR5-/- OT-II 10-day lymphoblasts, and densitometric quantification of blots as above (n = 10).G. ChIP analysis of the CerS2 promoter using an anti-H3K9Ac antibody. Scheme of the CerS2 promoter showing CpG islands and primers used for amplification. Relative ChIP of the CerS2 promoter in WT and CCR5-/- OT-II 10-day lymphoblasts (n = 3).H. Relative CerS2 mRNA level in CD4 T cells treated with PTx (n = 3).L. Representative immunofluorescence images showing pSer142-GATA-1 staining (green) of OT-II WT and CCR5-/- lymphoblasts. The green channel (top) and the merge with nuclear DAPI staining (blue; bottom) are shown. Scale bar, 10 μm. M. Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI-stained area; n ≥50 cells/condition).N. Top, basic scheme of the CerS2 promoter, indicating the putative GATA-1 binding site (blue) and location of the primers used for amplification in ChIP assays (black arrows). Bottom, relative anti-GATA-1 ChIP levels in OT-II WT and CCR5-/- lymphoblasts (n = 5).


Figure 6A . Scheme of the strategy used to form TCR proteoliposomes , and size of LUV generated at the indicated lipid molar ratio . Polydispersity index values are shown as black squares for each condition ( n = 3 ) . SBP , streptavidin - binding - peptide - tagged TCR . B . Representative immunoblots comparing TCR nanocluster sizes via BN - PAGE and anti - CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij - 96 or digitonin . The marker protein is ferritin ( f1 , 440 and f2 , 880 kDa forms ) . C . The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B ( n ≥ 4 ) . D . Cer levels in OT ‑ II 10 - day lymphoblasts , untreated or treated with SMase ( n = 4 ) . E . Representative small field images showing gold particle distribution , and quantification ( mean ± SEM ) of gold particles in clusters of the indicated size in cell surface replicas from untreated ( gray bars ; n = 5 cells , 8126 particles ) and SMase - treated ( 1h ) OT - II lymphoblasts ( black cyan ; n = 6 cells , 8457 particles ) after CD3ε labeling , as determined by EM . The inset shows distribution between clusters of one , two , three , four or more than four particles . F . GFP expression in shCtrl - ( black ) and shCerS2 ( orange ) - transduced 2B4 cells after puromycin selection , as determined by FACS . G . Relative CerS2 mRNA levels in TAK - 779 - treated 2B4 cells as in F . Values were normalized to those obtained in untransduced TAK - 779 - treated 2B4 cells ( n = 3 ) . H . Representative immunoblot with anti - CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2 - transduced 2B4 cells as in G . Filters were rehybridized with β - actin as loading control . I . TCR nanoclustering of shCtrl - and shCerS2 transduced 2B4 cells in the presence of TAK - 779 as determined by EM . Representative small field images and quantification ( mean ± SEM ) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl ( gray black bars ; n = 6 cells , 12337 particles ) and shCerS2 2B4 lymphoblasts ( orange ; n = 7 cells , 13456 particles ) . Inset , distribution between clusters of indicated size and statistical analysis . J . Percentage of CD69 + shCtrl ( black ) and shCerS2 ( orange ) 2B4 cells restimulated with plate - bound anti - CD3ε antibody in the presence of TAK - 779 ( n = 3 ) .

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 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 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 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 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 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 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 O O O O O O O O O O O

Figure 6A. Scheme of the strategy used to form TCR proteoliposomes, and size of LUV generated at the indicated lipid molar ratio. Polydispersity index values are shown as black squares for each condition (n = 3). SBP, streptavidin-binding-peptide-tagged TCR.B. Representative immunoblots comparing TCR nanocluster sizes via BN-PAGE and anti-CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij-96 or digitonin. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms).C. The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B (n ≥ 4).D. Cer levels in OT‑II 10-day lymphoblasts, untreated or treated with SMase (n = 4).E. Representative small field images showing gold particle distribution, and quantification (mean ± SEM) of gold particles in clusters of the indicated size in cell surface replicas from untreated (gray bars; n = 5 cells, 8126 particles) and SMase-treated (1h) OT-II lymphoblasts (black cyan; n = 6 cells, 8457 particles) after CD3ε labeling, as determined by EM. The inset shows distribution between clusters of one, two, three, four or more than four particles.F. GFP expression in shCtrl- (black) and shCerS2 (orange)-transduced 2B4 cells after puromycin selection, as determined by FACS.G. Relative CerS2 mRNA levels in TAK-779-treated 2B4 cells as in F. Values were normalized to those obtained in untransduced TAK-779-treated 2B4 cells (n = 3).H. Representative immunoblot with anti-CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2-transduced 2B4 cells as in G. Filters were rehybridized with β-actin as loading control.I. TCR nanoclustering of shCtrl- and shCerS2 transduced 2B4 cells in the presence of TAK-779 as determined by EM. Representative small field images and quantification (mean ± SEM) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl (gray black bars; n = 6 cells, 12337 particles) and shCerS2 2B4 lymphoblasts (orange; n = 7 cells, 13456 particles). Inset, distribution between clusters of indicated size and statistical analysis.J. Percentage of CD69+ shCtrl (black) and shCerS2 (orange) 2B4 cells restimulated with plate-bound anti-CD3ε antibody in the presence of TAK-779 (n = 3).


Figure 7A . Analysis of TCR nanoclustering in lymphoblasts from healthy WT and ccr5Δ32 homozygous donors by EM . Top , representative small field image showing gold particle distribution in cell surface replicas of anti - CD3ε - labeled cells ; bottom , quantification ( mean ± SEM ) of gold particles in clusters of the indicated size in the WT ( gray bars ; n = 5 cells , 17689 particles ) and Δ32 / Δ32 cells ( light red ; n = 4 cells , 16938 particles ) . Insets show the distribution between clusters of one , two , three , four or more than four particles , and statistical analysis . B . Normalized SM , Cer and dhCer levels in lymphoblasts obtained as in A . A representative experiment is shown ( n = 3 donors / genotype ; n = 2 independent experiments ) C . Relative CerS2 mRNA levels in day 8 WT and ccr5Δ32 lymphoblasts . Each data point is the average of a technical triplicate from 3 donors in two independent experiments ( n = 6 )

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 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 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

Figure 7A. Analysis of TCR nanoclustering in lymphoblasts from healthy WT and ccr5Δ32 homozygous donors by EM. Top, representative small field image showing gold particle distribution in cell surface replicas of anti-CD3ε-labeled cells; bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size in the WT (gray bars; n = 5 cells, 17689 particles) and Δ32/Δ32 cells (light red; n = 4 cells, 16938 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.B. Normalized SM, Cer and dhCer levels in lymphoblasts obtained as in A. A representative experiment is shown (n = 3 donors/genotype; n = 2 independent experiments)C. Relative CerS2 mRNA levels in day 8 WT and ccr5Δ32 lymphoblasts. Each data point is the average of a technical triplicate from 3 donors in two independent experiments (n = 6)


Figure 1OTULIN DNA sequence chromatograms showing the homozygous single base substitution ( c . 841G > A , p . Gly281Arg , arrowhead ) . Data are representative to two 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

Figure 1OTULIN DNA sequence chromatograms showing the homozygous single base substitution (c.841G>A, p.Gly281Arg, arrowhead). Data are representative to two independent experiments.


Figure 2Affinity measurements by fluorescence polarisation ( FP ) with recombinant catalytically inactive OTULINC129A or OTULINC129A / G281R and FlAsH - labelled M1 - linked diUb . Data represent mean ± SD of one experiment performed in technical triplicate . Data are representative of three independent experiments . a . u . , arbitrary units . K D , dissociation constant . M1 - linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver - stained SDS - PAGE gels . Data are representative of three independent experiments . First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R ( 1 . 0 mg / mL ) measured by nano differential scanning fluorimetry ( nanoDSF ) . Apparent melting temperatures ( Tm ) are indicated ( dashed lines ) . Data are representative to two 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 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 O O O O 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 2Affinity measurements by fluorescence polarisation (FP) with recombinant catalytically inactive OTULINC129A or OTULINC129A/G281R and FlAsH-labelled M1-linked diUb. Data represent mean ± SD of one experiment performed in technical triplicate. Data are representative of three independent experiments. a.u., arbitrary units. KD, dissociation constant.M1-linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver-stained SDS-PAGE gels. Data are representative of three independent experiments.First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R (1.0 mg/mL) measured by nano differential scanning fluorimetry (nanoDSF). Apparent melting temperatures (Tm) are indicated (dashed lines). Data are representative to two independent experiments.


Figure 3Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts . Data are representative of three independent experiments . Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 ( 10 μM ) for 24 h . Data are representative of two independent experiments . Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts . Data are representative of three independent experiments . Immunoblot ( left ) and densitometry ( right ) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of 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 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 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 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

Figure 3Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts. Data are representative of three independent experiments.Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 (10 μM) for 24 h. Data are representative of two independent experiments.Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts. Data are representative of three independent experiments.Immunoblot (left) and densitometry (right) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.


Figure 4Immunoblot analysis of IκBα phosphorylation and degradation as well as p65 / RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Densitometry analysis of p65 / RelA phosphorylation as presented in ( A ) from three independent experiments . ELISA analysis of IL - 8 secretion in response to TNF stimulation ( 10 ng / mL ) in healthy control or OTULING281R primary fibroblasts . Bars represent mean ± SEM ( n = 4 ) . Immunoblot analysis of the native TNF - RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts . Data are representative of 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 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 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

Figure 4Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments. Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.Densitometry analysis of p65/RelA phosphorylation as presented in (A) from three independent experiments.ELISA analysis of IL-8 secretion in response to TNF stimulation (10 ng/mL) in healthy control or OTULING281R primary fibroblasts. Bars represent mean ± SEM (n=4).Immunoblot analysis of the native TNF-RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts. Data are representative of three independent experiments.


Figure 5Immunoblot analysis of whole cell lysate from untreated human THP - 1 monocytes with stable expression of a non - targeting control shRNA or and shRNA targeting OTULIN . Data are representative of two independent experiments . Immunoblot ( left ) and densitometry ( right ) analysis of IκBα levels in shControl and shOTULIN THP - 1 cells treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of three independent experiments . ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP - 1 cells . Data represents mean ± SEM ( n = 4 ) and were analysed using the two - way ANOVA test of statistical significance with Sidak ' s correction for multiple comparisons . Immunoblot analysis of IκBα phosphorylation and degradation as well as p65 / RelA phosphorylation in shControl and shOTULIN THP - 1 cells in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Densitometry analysis of p65 / RelA phosphorylation as presented in ( D ) 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 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O

Figure 5Immunoblot analysis of whole cell lysate from untreated human THP-1 monocytes with stable expression of a non-targeting control shRNA or and shRNA targeting OTULIN. Data are representative of two independent experiments.Immunoblot (left) and densitometry (right) analysis of IκBα levels in shControl and shOTULIN THP-1 cells treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP-1 cells. Data represents mean ± SEM (n=4) and were analysed using the two-way ANOVA test of statistical significance with Sidak's correction for multiple comparisons.Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in shControl and shOTULIN THP-1 cells in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.Densitometry analysis of p65/RelA phosphorylation as presented in (D) from three independent experiments.


Figure 6Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , as indicated was analysed using MTT reduction assays . Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , Q - VD - OPh ( 10 μM ) , and Nec - 1 ( 10 μM ) as indicated was analysed using MTT reduction assays . Viability of shControl and shOTULIN THP - 1 cells ( C - D ) after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , shControl and shOTULIN THP - 1 cells ( C - D ) after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , Q - VD - OPh ( 10 μM ) , and Nec - 1 ( 10 μM ) as indicated was analysed using MTT reduction assays . Increased apoptotic cell death in the skin of ORAS patient III . 2 . Serial sections of normal skin ( top panels ) and a skin biopsy from patient III . 2 taken at an inflammatory flare ( bottom panels ) were immunostained for cleaved caspase - 3 ( centre panels ) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) assay ( right panels ) . Arrowheads indicate cleaved caspase - 3 - positive mesenchymal cells . None of these markers were present in the healthy control skin . H & E , haematoxylin and eosin . Boxes in H & E panels indicate the areas magnified in the cleaved caspase - 3 and TUNEL panels . Scale bars , 100 μm . Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III . 2 .

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

Figure 6Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), as indicated was analysed using MTT reduction assays.Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.Viability of shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL),shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.Increased apoptotic cell death in the skin of ORAS patient III.2. Serial sections of normal skin (top panels) and a skin biopsy from patient III.2 taken at an inflammatory flare (bottom panels) were immunostained for cleaved caspase-3 (centre panels) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (right panels). Arrowheads indicate cleaved caspase-3-positive mesenchymal cells. None
of these markers were present in the healthy control skin. H&E, haematoxylin and eosin. Boxes in H&E panels indicate the areas magnified in the cleaved caspase-3 and TUNEL panels. Scale bars, 100 μm.
Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III.2.


Figure 7Analysis of the blood cell chimerism in patient III . 2 after HSCT . Short terminal repeat ( STR ) analysis was used to determine the percentage of donor cells in peripheral blood . Arrow indicates point of relapse . Photograph of patient III . 2 at age ~ 10 years at an episode of inflammation caused by delayed etanercept administration . Arrows indicate the erythematous subcutaneous nodules ( panniculitis ) .

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

Figure 7Analysis of the blood cell chimerism in patient III.2 after HSCT. Short terminal repeat (STR) analysis was used to determine the percentage of donor cells in peripheral blood. Arrow indicates point of relapse.Photograph of patient III.2 at age ~10 years at an episode of inflammation caused by delayed etanercept administration. Arrows indicate the erythematous subcutaneous nodules (panniculitis).


Figure 1a ) Photomicrographs represent 2D optical sections from a Z stack acquisition of whole mounted skin displaying IFE harbouring multiple clones of different colours at 3 , 5 , 12 and 24 weeks ( bar represents 50 μm ) . In each image a clone is circled in white , illustrating the progression of clone size at different times . b ) Histogram charts showing the relative frequency of clones according to their size between 3 and 24 weeks in mice induced at P21 and P42 . The frequency of larger clones increases over time . Chi square test p < 0 . 0001 for P21 group and p = 0 . 0056 for P42 group . The number of clones evaluated at each time point is indicated above each histogram series . c ) and d ) Correction for contiguous clones harbouring a same colour . d ) Plots show the probability for an individual keratinocyte to belong to a clone of a given size . Experimental data are represented in green bars . Red curve represents the 5 - colour simulation and the blue curve the real size simulation . Although the frequency of large clones is overestimated in experimental data due to the limited 5 - color palette , it is apparent that real clone size progresses over time . e ) Histogram charts showing the distribution of clones at D1 - 3 following tamoxifen induction according to total number of cells and number of basal cells . f ) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining . The frequency of larger clones increases over time . Chi square test p = 0 . 0013 for P21 group and p = 0 . 0296 for P42 group . All data are represented as mean ± s . e . m .

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 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 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 O O 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) Photomicrographs represent 2D optical sections from a Z stack acquisition of whole mounted skin displaying IFE harbouring multiple clones of different colours at 3, 5, 12 and 24 weeks (bar represents 50 μm). In each image a clone is circled in white, illustrating the progression of clone size at different times. b) Histogram charts showing the relative frequency of clones according to their size between 3 and 24 weeks in mice induced at P21 and P42. The frequency of larger clones increases over time. Chi square test p<0.0001 for P21 group and p=0.0056 for P42 group. The number of clones evaluated at each time point is indicated above each histogram series. c) and d) Correction for contiguous clones harbouring a same colour.d) Plots show the probability for an individual keratinocyte to belong to a clone of a given size. Experimental data are represented in green bars. Red curve represents the 5-colour simulation and the blue curve the real size simulation. Although the frequency of large clones is overestimated in experimental data due to the limited 5-color palette, it is apparent that real clone size progresses over time.e) Histogram charts showing the distribution of clones at D1-3 following tamoxifen induction according to total number of cells and number of basal cells.f) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining. The frequency of larger clones increases over time. Chi square test p=0.0013 for P21 group and p=0.0296 for P42 group. All data are represented as mean ± s.e.m.


Figure 2a ) Clones were categorized as attached or not attached to hair follicles . Average clone size of attached ( circles ) and non - attached ( squares ) clones were plotted over time . T - test , * * * p < 0 . 0001 , * * p < 0 . 001 . ( Number of clones : 1 week n = 271 , 3 weeks n = 1847 , 5 weeks n = 517 , 12 weeks n = 706 , 24 weeks n = 258 ) . Data are represented as mean ± s . d . b ) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 weeks . The yellow and blue clone in the IFE immediately adjacent to a HF are considered as attached to the HF . The orange and pink clones are not attached to the HF . Limits of clones ( lines ) and cells ( dots ) within each clone were identified on this optical section . White dashed circles represent the projection of hair follicle openings ( scale bar = 50 µm ) . c ) Bar charts showing the relative frequency of clone size between 3 and 24 weeks after recombination . In the P42 group , clones attached to HF were more likely to be bigger than those not attached at 3 , 5 , 12 and 24 weeks . ǂǂǂǂ Chi square test p < 0 . 0001 . The number of clones analyzed at each time - point is indicated on the graph . Of note , whether attached or not clones continued to grow over time : * * * * Chi square test p < 0 . 0001 . All data are represented as mean ± s . e . m . d ) Plot showing the probability of a clone to be attached to a hair follicle as a function of its size ( dashed line : predicted curve based on theoretical probabilities according to spacing of hair follicles at different time - points ; squares : experimental data ) . Between different time points the distance between hair follicles varies slightly ; therefore each time point ( 3 , 5 , 12 and 24 weeks ) has a distinct theoretical curve . The observed frequency of clone attachment to hair follicles is at every time point above the random prediction .

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 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 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 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

Figure 2a) Clones were categorized as attached or not attached to hair follicles. Average clone size of attached (circles) and non-attached (squares) clones were plotted over time. T-test, *** p<0.0001, ** p<0.001. (Number of clones: 1 week n = 271, 3 weeks n = 1847, 5 weeks n = 517, 12 weeks n = 706, 24 weeks n = 258). Data are represented as mean ± s.d.b) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 weeks. The yellow and blue clone in the IFE immediately adjacent to a HF are considered as attached to the HF. The orange and pink clones are not attached to the HF. Limits of clones (lines) and cells (dots) within each clone were identified on this optical section. White dashed circles represent the projection of hair follicle openings (scale bar= 50 µm).c) Bar charts showing the relative frequency of clone size between 3 and 24 weeks after recombination. In the P42 group, clones attached to HF were more likely to be bigger than those not attached at 3, 5, 12 and 24 weeks. ǂǂǂǂ Chi square test p<0.0001.The number of clones analyzed at each time-point is indicated on the graph. Of note, whether attached or not clones continued to grow over time: ****Chi square test p<0.0001. All data are represented as mean ± s.e.m.d) Plot showing the probability of a clone to be attached to a hair follicle as a function of its size (dashed line: predicted curve based on theoretical probabilities according to spacing of hair follicles at different time-points; squares: experimental data). Between different time points the distance between hair follicles varies slightly; therefore each time point (3, 5, 12 and 24 weeks) has a distinct theoretical curve. The observed frequency of clone attachment to hair follicles is at every time point above the random prediction.


Figure 3a ) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points . Histogram charts represent the relative frequency of clone size from clones attached ( top row ) or not ( bottom row ) to HF in anagen and telogen phase at the time of tissue collection . Clones connected to hair follicles were more likely to be larger in anagen compared to telogen ( Chi square test , * * * p = 0 . 0009 at 3 weeks , * * * p < 0 . 0001 at 5 weeks , * p = 0 . 0193 at 12 weeks , * * * p = 0 . 0001 at 24 weeks ) . However no difference between the two phases was observed in clones that were not attached to HFs . Clone numbers are indicated on the graph . All data are represented as mean ± s . e . m . b ) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post - induction . Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling . Average clone size of attached ( circles ) and non - attached ( squares ) clones were plotted over the number of club hair . T - test , * * * * p < 0 . 0001 . c ) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days . Histogram charts represent the relative frequency of clone size from clones attached ( top row ) or not ( bottom row ) to HF in anagen and telogen phase at induction . Even at a rate of one cell division per 24h , 7 cycles of divisions results in 128 cells . Therefore the findings reported are not incompatible with our claims as suggested . Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe . Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones . Chi square test , * * * p = 0 . 001 ; * * p = 0 . 01 .

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 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 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

Figure 3a) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at the time of tissue collection. Clones connected to hair follicles were more likely to be larger in anagen compared to telogen (Chi square test, *** p=0.0009 at 3 weeks, *** p<0.0001 at 5 weeks, * p=0.0193 at 12 weeks, *** p=0.0001 at 24 weeks). However no difference between the two phases was observed in clones that were not attached to HFs. Clone numbers are indicated on the graph. All data are represented as mean ± s.e.m.b) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post-induction. Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling. Average clone size of attached (circles) and non-attached (squares) clones were plotted over the number of club hair. T-test, **** p<0.0001.c) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at induction. Even at a rate of one cell division per 24h, 7 cycles of divisions results in 128 cells. Therefore the findings reported are not incompatible with our claims as suggested. Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe. Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones. Chi square test, *** p=0.001; ** p=0.01.


Figure 4 ( a - b ) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway ( a ) and corresponding scatter plots quantification . ( Scale bar 100µm ) ( b ) . Analysis focused on the basal layer of the IFE . Each dot represents one image . Proliferation in the IFE is significantly increased during the anagen phase except in mid - anagen . Unpaired t test , * * * * p < 0 . 0001 , * * p = 0 . 0020 . EA n = 32 , EMA n = 13 , MA n = 15 , C n = 21 , T n = 19 from 6 mice . Black arrows : positive Ki67 cells . ( c ) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti - Ki67 antibody or upon BrdU incorporation . Ki67 + cells are labelled with Alexa 647 ( red ) . The density of Ki67 + cells increased in the anagen phase ( iii and iv ) compared to telogen ( i and ii ) . White arrows : ki67 + cells . ( Scale bar = 20µm ) . Similarly , short term BrdU pulse ( 2h ) resulted in significant staining of anagen epidermal areas ( vi , red nuclear stain ) reinforcing hair follicle openings as compared to telogen areas .

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 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

Figure 4(a-b) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway (a) and corresponding scatter plots quantification. (Scale bar 100µm) (b). Analysis focused on the basal layer of the IFE. Each dot represents one image. Proliferation in the IFE is significantly increased during the anagen phase except in mid-anagen. Unpaired t test, ****p<0.0001, **p=0.0020. EA n = 32, EMA n = 13, MA n = 15, C n = 21, T n = 19 from 6 mice. Black arrows: positive Ki67 cells.(c) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti-Ki67 antibody or upon BrdU incorporation. Ki67+ cells are labelled with Alexa 647 (red). The density of Ki67+ cells increased in the anagen phase (iii and iv) compared to telogen (i and ii). White arrows: ki67+ cells. (Scale bar = 20µm). Similarly, short term BrdU pulse (2h) resulted in significant staining of anagen epidermal areas (vi, red nuclear stain) reinforcing hair follicle openings as compared to telogen areas.


Figure 5a - b ) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase . a ) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti - BrdU antibody ( red ) . White dots represent hair follicles at different chase time points . a - b ) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase . b ) At 5 weeks of chase , BrdU staining ( green ) in skinbowskin revealed single cells distant from hair follicles . White dashed circles : hair follicles . White line : small clones . White arrows : BrdU + cells . ( scale bar = 20 µm ) . c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 weeks post - induction and animals were sacrificed at 48hours following a short pulse of BrdU ( 2h ) . c ) Photomicrographs show optical sections from a confocal image stack acquired on a 0 . 5 mm back skin section immunostained with anti - ki67 antibody . Ki67 + cells are labelled with Alexa 647 ( red ) . Back skin from treated and untreated areas was collected 48hrs after TPA topical application . c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 weeks post - induction and animals were sacrificed at 48hours following a short pulse of BrdU ( 2h ) . d ) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti - BrdU ( green ) . Small clones not attached to HF ( 2 examples figured by dashed lines ) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation . e ) ( i to iv ) Representative individual sections from Z stacks , show small clones ( lines ) identified in back skin treated ( i and ii ) or not with TPA ( iii and iv ) . ( scale bar = 100 µm ) . ( v to viii ) Maximal intensity projection from stack acquired on whole mounted skin . A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA ( v to vi ) compared to skin treated with vehicle ( vii and viii ) . ( scale bars = 100 µm ) . f ) Reduction in small clone frequency in the areas treated with TPA . Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse . Mann Whitney test , * * p = 0 . 0079 . Data pooled from 3 mice represented as median with interquartile range . The number of clones analyzed is indicated on the graph . * p = 0 . 0272 . Data are represented as mean ± s . e . m .

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 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 O O O O O O 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 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

Figure 5a-b) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. a) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti-BrdU antibody (red). White dots represent hair follicles at different chase time points.a-b) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. b) At 5 weeks of chase, BrdU staining (green) in skinbowskin revealed single cells distant from hair follicles. White dashed circles: hair follicles. White line: small clones. White arrows: BrdU+ cells. (scale bar = 20 µm).c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). c) Photomicrographs show optical sections from a confocal image stack acquired on a 0.5 mm back skin section immunostained with anti-ki67 antibody. Ki67+ cells are labelled with Alexa 647 (red). Back skin from treated and untreated areas was collected 48hrs after TPA topical application.c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). d) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti-BrdU (green). Small clones not attached to HF (2 examples figured by dashed lines) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation.e) (i to iv) Representative individual sections from Z stacks, show small clones (lines) identified in back skin treated (i and ii) or not with TPA (iii and iv). (scale bar = 100 µm). (v to viii) Maximal intensity projection from stack acquired on whole mounted skin. A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA (v to vi) compared to skin treated with vehicle (vii and viii). (scale bars = 100 µm). f) Reduction in small clone frequency in the areas treated with TPA. Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse. Mann Whitney test, ** p=0.0079. Data pooled from 3 mice represented as median with interquartile range. The number of clones analyzed is indicated on the graph. * p=0.0272. Data are represented as mean ± s.e.m.


Figure 6a ) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42 . Data are represented as mean ± s . e . m .

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

Figure 6a) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42. Data are represented as mean ± s.e.m.


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 .

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

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 .

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

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.