Figure 4 ( B - C ' ' ) Immunostaining of mosaic germaria with anti - GFP ( green ) , to identify clonal cells by the lack of GFP , and either 8C4 ( B - B ' ' ) or 10F1 ( C - C ' ' ) monoclonal anti - Cbl ( red ) . DAPI was used to visualize DNA . White arrowheads indicate aubHN2 / + control GSCs ; yellow arrowheads indicate clonal mutant aubHN2 GSCs . Scale bar : 10 μm . ( B - C ' ' ) Immunostaining of mosaic germaria with anti - GFP ( green ) , to identify clonal cells by the lack of GFP , and either 8C4 ( B - B ' ' ) or 10F1 ( C - C ' ' ) monoclonal anti - Cbl ( red ) . DAPI was used to visualize DNA . White arrowheads indicate aubHN2 / + control GSCs ; yellow arrowheads indicate clonal mutant aubHN2 GSCs . Scale bar : 10 μm . ( D - E ) Quantification of Cbl protein levels in aubHN2 / + and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1 . Fluorescence intensity was measured in arbitrary units using the ImageJ software . Horizontal bars correspond to the mean and standard deviation . * * * p - value < 0 . 001 using the two - tailed Student ' s t test . ( D - E ) Quantification of Cbl protein levels in aubHN2 / + and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1 . Fluorescence intensity was measured in arbitrary units using the ImageJ software . Horizontal bars correspond to the mean and standard deviation . * * * p - value < 0 . 001 using the two - tailed Student ' s t test . ( F ) RNA immunoprecitation ( IP ) with anti - GFP antibody in wild - type ( mock IP ) and nos - Gal4 / UASp - GFP - Aub ovarian extracts . Cbl mRNA was quantified using RT - qPCR . Normalization was with RpL32 mRNA . Mean of three biological replicates . The error bar represents standard error to the mean . * p - value < 0 . 05 using the two - tailed Student ' s t test . ( G ) ePAT assay of CblL mRNA . Ovaries from 1 - day - old ( germarium to stage 8 ) wild - type , aub and twin mutant females , and from 4 - to 7 - day - old bam mutant females were used .

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

Figure 4(B-C'') Immunostaining of mosaic germaria with anti-GFP (green), to identify clonal cells by the lack of GFP, and either 8C4 (B-B'') or 10F1 (C-C'') monoclonal anti-Cbl (red). DAPI was used to visualize DNA. White arrowheads indicate aubHN2/+ control GSCs; yellow arrowheads indicate clonal mutant aubHN2 GSCs. Scale bar: 10 μm.(B-C'') Immunostaining of mosaic germaria with anti-GFP (green), to identify clonal cells by the lack of GFP, and either 8C4 (B-B'') or 10F1 (C-C'') monoclonal anti-Cbl (red). DAPI was used to visualize DNA. White arrowheads indicate aubHN2/+ control GSCs; yellow arrowheads indicate clonal mutant aubHN2 GSCs. Scale bar: 10 μm.(D-E) Quantification of Cbl protein levels in aubHN2/+ and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1. Fluorescence intensity was measured in arbitrary units using the ImageJ software. Horizontal bars correspond to the mean and standard deviation. *** p-value <0.001 using the two-tailed Student's t test.(D-E) Quantification of Cbl protein levels in aubHN2/+ and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1. Fluorescence intensity was measured in arbitrary units using the ImageJ software. Horizontal bars correspond to the mean and standard deviation. *** p-value <0.001 using the two-tailed Student's t test.(F) RNA immunoprecitation (IP) with anti-GFP antibody in wild-type (mock IP) and nos-Gal4/UASp-GFP-Aub ovarian extracts. Cbl mRNA was quantified using RT-qPCR. Normalization was with RpL32 mRNA. Mean of three biological replicates. The error bar represents standard error to the mean. * p-value <0.05 using the two-tailed Student's t test.(G) ePAT assay of CblL mRNA. Ovaries from 1-day-old (germarium to stage 8) wild-type, aub and twin mutant females, and from 4- to 7-day-old bam mutant females were used.


Figure 5 ( D , E ) Coverage of CblL ( D ) and CblS ( E ) mRNAs with ovarian and embryonic piRNAs . Significant crosslink clusters are indicated in red at the top of the graphs . ( D , E ) Coverage of CblL ( D ) and CblS ( E ) mRNAs with ovarian and embryonic piRNAs . Significant crosslink clusters are indicated in red at the top of the graphs .

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

Figure 5(D, E) Coverage of CblL (D) and CblS (E) mRNAs with ovarian and embryonic piRNAs. Significant crosslink clusters are indicated in red at the top of the graphs.(D, E) Coverage of CblL (D) and CblS (E) mRNAs with ovarian and embryonic piRNAs. Significant crosslink clusters are indicated in red at the top of the graphs.


Figure 6 ( A - E ) Cbl expression in germaria . ( A ) Immunostaining of wild - type germaria with anti - Cbl 8C4 . ( A - E ) Cbl expression in germaria . ( B - B ' ' ) Immunostaining of bamP - GFP germaria that express GFP under the bam promoter , with anti - GFP ( green ) and anti - Cbl 8C4 ( red ) . ( A - E ) Immunostaining expression in germaria . ( C - C ' ) Immunostaining of germaria expressing GFP - Par1 to label spectrosomes with anti - GFP ( green ) and anti - Cbl 8C4 ( red ) . DAPI ( blue ) was used to visualize DNA . White arrowheads indicate GSCs ; yellow arrowheads indicate cystoblasts . ( F ) Western blots of protein extracts from wild - type and Cbl mutant ovaries showing CblL ( left panel ) and CblS ( right panel ) revealed with 8C4 and 10F1 antibodies , respectively . A low level of CblS remained , while no CblL was present in the CblEY11427 / MB05683 combination . The 10F1 antibody recognizes very poorly CblL in western blot . ( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes . ( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes . ( I ) Quantification of germaria with increased number of spectrosomes ( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes . ( J ) quantification of spectrosomes per germarium . The number of scored germaria ( n ) is indicated in ( I ) . * * * p - value < 0 . 001 using the χ2 test in ( I ) , and the two - tailed Student ' s t test in ( J ) . ( K - M ) Cbl induces GSC differentiation . Immunostaining of germaria overexpressing Cbl with Hsp83 - CblL ( K ) White arrows indicate a cyst in the GSC niche ( K , outlined ) ( K - M ) Cbl induces GSC differentiation . Immunostaining of germaria overexpressing Cbl with Hsp83 - CblS ( L ) with anti - Vasa ( green ) and anti - Hts ( red ) . DNA ( blue ) was revealed with DAPI . White arrows indicatethe loss of GSCs and germ cells ( L ) . ( K - M ) Cbl induces GSC differentiation . ( M ) Quantification of germaria with 0 to 1 GSC in the indicated genotypes . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the χ2 test . Scale bar : 10 μm in A - C ' , G , H , K , L .

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

Figure 6(A-E) Cbl expression in germaria. (A) Immunostaining of wild-type germaria with anti-Cbl 8C4.(A-E) Cbl expression in germaria. (B-B'') Immunostaining of bamP-GFP germaria that express GFP under the bam promoter, with anti-GFP (green) and anti-Cbl 8C4 (red).(A-E) Immunostaining expression in germaria. (C-C') Immunostaining of germaria expressing GFP-Par1 to label spectrosomes with anti-GFP (green) and anti-Cbl 8C4 (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs; yellow arrowheads indicate cystoblasts.(F) Western blots of protein extracts from wild-type and Cbl mutant ovaries showing CblL (left panel) and CblS (right panel) revealed with 8C4 and 10F1 antibodies, respectively. A low level of CblS remained, while no CblL was present in the CblEY11427/MB05683 combination. The 10F1 antibody recognizes very poorly CblL in western blot.(G-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes.(G-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes. (I) Quantification of germaria with increased number of spectrosomes(G-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes. (J) quantification of spectrosomes per germarium. The number of scored germaria (n) is indicated in (I). ***p-value <0.001 using the χ2 test in (I), and the two-tailed Student's t test in (J).(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblL (K)White arrows indicate a cyst in the GSC niche (K, outlined)(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblS (L) with anti-Vasa (green) and anti-Hts (red). DNA (blue) was revealed with DAPI. White arrows indicatethe loss of GSCs and germ cells (L).(K-M) Cbl induces GSC differentiation.(M) Quantification of germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test. Scale bar: 10 μm in A-C', G, H, K, L.


Figure 7 ( A - B ) Immunostaining of germaria from aubHN2 / QC42 ( A ) females with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . The white arrow indicates the lack of GSCs ; white arrowheads indicate GSCs . Scale bar : 10 μm . ( A - B ) Immunostaining of germaria from aubHN2 / QC42 ; CblMB / + ( B ) females with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . The white arrow indicates the lack of GSCs ; white arrowheads indicate GSCs . Scale bar : 10 μm . ( C ) Quantification of mutant germaria with 0 to 1 GSC in the indicated genotypes . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 , using the χ2 test .

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 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 7(A-B) Immunostaining of germaria from aubHN2/QC42 (A) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.(A-B) Immunostaining of germaria from aubHN2/QC42; CblMB/+ (B) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.(C) Quantification of mutant germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001, ** p-value <0.01, * p-value <0.05, using the χ2 test.


Figure 1A . Stills depicting actin flow in extracellular RH , myoA KO and adf KD parasites expressing Cb EmeraldFP . Continuous F - actin flow can be seen from the apical tip , to the Golgi region towards the posterior pole of the parasite . No difference can be observed between RH and myoA KO . For adf KD , the flow is completely abrogated . Red arrowhead marks the Golgi region , while the yellow arrowhead marks the apical nucleation centre . B . Skeletonisation processing of ( A ) depicting areas where F - actin dynamics / flow is prevalent . Individual signals for F - actin form a continuous , dynamic network that connects apical and posterior pole of the parasite . Red arrowhead marks the Golgi region nucleation centre , while the yellow arrowhead marks the apical nucleation centre . The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F - actin . C . Kymograph analysis of ( A ) and ( B ) . The kymograph was generated by tracing a line in the middle of the parasite ' s body ( red line ) . The colour - coded kymograph represents forward movement ( red ) , backwards movement ( green ) and static F - actin ( blue ) . The results demonstrate continuous exchange of F - actin from the apical to the posterior pole of the parasite within the cytosol of the parasite . This cytosolic exchange is similar in RH and myoA KO parasites , while completely abolished in the case of adf KD . Data information : Numbers indicate minutes : seconds ; and Scale bars represent 5 µm . D . Top three rows : Parasites expressing Cb EmeraldFP along with myoA - SNAP before and after Ca2 + - Ionophore ( A23187 ) or BIPPO treatment . The parasites were also treated with 2 µM of Cytochalasin D or DMSO ( as control ) for 30 min . After addition of A23187 , preferential relocalisation of actin can be observed at the apical tip , while addition of BIPPO caused F - actin accumulation at the basal end . 4th row : myoAKO parasites expressing Cb - EmeraldFP before and after treatment with A23187 and BIPPO . While no apical or basal accumulation of F - actin is observed , some peripheral location of F - actin occurs in presence of A23187 or BIPPO , preferentially in the apical half of the parasite . Bottom row : adf cKD parasites expressing Cb - EmeraldFP before and after treatment with Ca2 + Ionophore or BIPPO . No apparent change in F - actin localisation can be seen upon treatment . E . Skeletonization of movies shown in D . Before addition of A23187 or BIPPO , RH and myoA KO parasites behave similar with no accumulation of F - actin at the apical tip . After treatment with calcium ionophore A23187 , preferential accumulation of F - actin at the apical tip can be observed for RH ( red arrowhead ) , while BIPPO presented preferential accumulation in the basal end ( red arrowhead ) . In myoA KO , some relocalisation of actin is observed at the periphery of the parasite ( yellow arrowhead ) . In the case of adf cKD , no relocalisation occurs . The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F - actin . F . Quantification of actin accumulation before and after treatment with Ca2 + Ionophore or BIPPO . The parasites were counted for F - actin accumulation after adding Ca2 + Ionophore or BIPPO . Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip . A minimum of 200 parasites were counted upon 3 biological replicates . Two - way ANOVA was used for statistical analysis and Tukey ' s multiple comparisons test . * * * * p value < 0 . 0001 . Error bars represent standard deviation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A. Stills depicting actin flow in extracellular RH, myoA KO and adf KD parasites expressing Cb EmeraldFP. Continuous F-actin flow can be seen from the apical tip, to the Golgi region towards the posterior pole of the parasite. No difference can be observed between RH and myoA KO. For adf KD, the flow is completely abrogated. Red arrowhead marks the Golgi region, while the yellow arrowhead marks the apical nucleation centre. B. Skeletonisation processing of (A) depicting areas where F-actin dynamics/flow is prevalent. Individual signals for F-actin form a continuous, dynamic network that connects apical and posterior pole of the parasite. Red arrowhead marks the Golgi region nucleation centre, while the yellow arrowhead marks the apical nucleation centre. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin. C. Kymograph analysis of (A) and (B). The kymograph was generated by tracing a line in the middle of the parasite's body (red line). The colour-coded kymograph represents forward movement (red), backwards movement (green) and static F-actin (blue). The results demonstrate continuous exchange of F-actin from the apical to the posterior pole of the parasite within the cytosol of the parasite. This cytosolic exchange is similar in RH and myoA KO parasites, while completely abolished in the case of adf KD. Data information: Numbers indicate minutes:seconds; and Scale bars represent 5 µm.D. Top three rows: Parasites expressing Cb EmeraldFP along with myoA-SNAP before and after Ca2+-Ionophore (A23187) or BIPPO treatment. The parasites were also treated with 2 µM of Cytochalasin D or DMSO (as control) for 30 min. After addition of A23187, preferential relocalisation of actin can be observed at the apical tip, while addition of BIPPO caused F-actin accumulation at the basal end. 4th row: myoAKO parasites expressing Cb-EmeraldFP before and after treatment with A23187 and BIPPO. While no apical or basal accumulation of F-actin is observed, some peripheral location of F-actin occurs in presence of A23187 or BIPPO, preferentially in the apical half of the parasite. Bottom row: adf cKD parasites expressing Cb-EmeraldFP before and after treatment with Ca2+ Ionophore or BIPPO. No apparent change in F-actin localisation can be seen upon treatment. E. Skeletonization of movies shown in D. Before addition of A23187 or BIPPO, RH and myoA KO parasites behave similar with no accumulation of F-actin at the apical tip. After treatment with calcium ionophore A23187, preferential accumulation of F-actin at the apical tip can be observed for RH (red arrowhead), while BIPPO presented preferential accumulation in the basal end (red arrowhead). In myoA KO, some relocalisation of actin is observed at the periphery of the parasite (yellow arrowhead). In the case of adf cKD, no relocalisation occurs. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin.F. Quantification of actin accumulation before and after treatment with Ca2+ Ionophore or BIPPO. The parasites were counted for F-actin accumulation after adding Ca2+ Ionophore or BIPPO. Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip. A minimum of 200 parasites were counted upon 3 biological replicates. Two-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. Error bars represent standard deviation.


Figure 2A . Wild Type RH parasites mid - invasion . Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes , fixed with 4 % PFA before labelling the TJ with anti - Ron2 ( yellow ) and DAPI ( magenta ) for nuclear staining . White arrow points at invasion direction . B . Graph showing deformation of the whole parasite and its nucleus between invading and non - invading parasites . 100 parasites found mid - invasion were counted in triplicate and compared to extracellular , freshly egressed parasites . One - way ANOVA was used for statistical analysis . * * * * p value < 0 . 0001 . Data information : Error bars represent standard deviation . and Scale bars represent 5 µm . C . Graph showing deformation of the whole parasite and its nucleus between invading and non - invading parasites . Indicated parasites were analysed . For adf cKD induced and non - induced conditions were compared , as indicated . 30 parasites were counted for each condition in triplicate . One - way ANOVA was used for statistical analysis and Tukey ' s multiple comparisons test . * * * * p value < 0 . 0001 . * * * p value 0 . 0009 . Data information : Error bars represent standard deviation . ; and Scale bars represent 5 µm . D . Time - lapse microscopy analysis of indicated parasites invading HFFs cells . Time was determined from the onset of invasion . White arrow points at invasion direction ; yellow arrow points at tight junction ; red dot follows the apical pole of the parasite . Numbers indicate minutes : secondsE . Speed profiles of invading parasites The relative displacement of the apical tip over time allowed speed calculations . Individual stages were named in regard to the invasion step ( Penetration initiation , Speed decrease and finalisation ) . Invasion was analysed with Icy Image Processing Software ( Pasteur Institut ) using the Manual Tracking plugin by analysing each movie 10 times and averaging the measurementsF . Time - lapse analysis demonstrating abortive invasion of indicated parasites . Time was determined from the onset of invasion . White arrow points at invasion direction ; yellow arrow points at tight junction ; red dot follows the apical pole of the parasite . Numbers indicate minutes :

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

Figure 2A. Wild Type RH parasites mid-invasion. Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes, fixed with 4% PFA before labelling the TJ with anti-Ron2 (yellow) and DAPI (magenta) for nuclear staining. White arrow points at invasion direction.B. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. 100 parasites found mid-invasion were counted in triplicate and compared to extracellular, freshly egressed parasites. One-way ANOVA was used for statistical analysis. **** p value <0.0001. Data information: Error bars represent standard deviation. and Scale bars represent 5 µm.C. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. Indicated parasites were analysed. For adf cKD induced and non-induced conditions were compared, as indicated. 30 parasites were counted for each condition in triplicate. One-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. *** p value 0.0009. Data information: Error bars represent standard deviation. ; and Scale bars represent 5 µm.D. Time-lapse microscopy analysis of indicated parasites invading HFFs cells. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:secondsE. Speed profiles of invading parasites The relative displacement of the apical tip over time allowed speed calculations. Individual stages were named in regard to the invasion step (Penetration initiation, Speed decrease and finalisation). Invasion was analysed with Icy Image Processing Software (Pasteur Institut) using the Manual Tracking plugin by analysing each movie 10 times and averaging the measurementsF. Time-lapse analysis demonstrating abortive invasion of indicated parasites. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds


Figure 3A . Time - lapse analysis depicting invading RH adfcKD parasites expressing Cb - EmeraldFP . A F - actin ring can be observed at the TJ in the majority of cases ( red arrowheads , first panel ) . Invasion can also occur without formation of an F - actin ring ( second panel ) . During capped invasion , an F - actin ring is formed ( third panel ) . Invasion of adf cKD parasites occurs without formation of an F - actin ring ( fourth panel ) . F - actin accumulation is observed at the posterior pole of the parasite in all cases ( orange arrows ) . Green segmented lines depict the area that was measured for generation of intensity plot profiles shown in ( B ) . Yellow and purple dotted lines indicate the extra - and intracellular part of the parasite respectively . B . Intensity plot profiles of the movies shown in ( A ) . Depicted plots correlate to half - invaded parasites . In the case of F - actin ring formation , two distinct peaks are detected , correlating to the F - actin ring at the TJ ( red arrowhead ) and the posterior pole of the parasite ( orange arrow ) . In the case of no F - actin ring , no accumulation at the TJ can be observed ; only the peak correlating to posterior F - actin can be detected . For adf cKD , only one peak can be seen corresponding to the posterior pole of the parasite . C . Quantification of F - actin accumulation at the TJ for RH Cb - EmeraldFP parasites as detected in time lapse analysis . The values are expressed as percentage . 28 total parasite invasion events were captured across three different biological replicates . Two - tailed unpaired t - test analysis was performed . * * * * p value < 0 . 0001 . Data information : Error bars represent standard deviation . White arrows point to direction of invasion . Scale bars represent 5 µm . * * * * p value < 0 . 0001 . D . Representative immunofluorescence assays on indicated parasite strains during host cell penetration . Parasites were labelled with indicated antibodies . SAG1 staining was performed prior to permeabilisation to stain only the extracellular portion of the parasite . Antibodies against Ron2 were used to stain the TJ . Red arrowhead : position of the TJ . Orange arrow : posterior pole of the parasite . Data information : Error bars represent standard deviation . White arrows point to direction of invasion . Scale bars represent 5 µm . E . Tight Junction assay on fixed samples depicting invasion rates of indicated parasites . While in the majority of control parasites F - actin can be detected at the junction ( ~ 80 % ) , no junctional ring can be detected in ~ 20 % of all invasion events . In contrast , adf cKD , act1 cKO or myoA KO only show an overall invasion rate of ~ 20 % compared to controls . In these cases junctional F - actin accumulation could never be detected . Note that the number of total invasion events for adf cKD , act1cKO and myoAKO correlates to the number of invasion events , seen for RH without F - actin ring formation . Data information : Error bars represent standard deviation . One - way ANOVA analysis was performed for each graph * * * * p value < 0 . 0001 . F . Quantification of posterior F - actin accumulation during invasion of indicated parasite lines . One - way ANOVA analysis was performed for each graph * * * * p value < 0 . 0001 .

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

Figure 3A. Time-lapse analysis depicting invading RH adfcKD parasites expressing Cb-EmeraldFP. A F-actin ring can be observed at the TJ in the majority of cases (red arrowheads, first panel). Invasion can also occur without formation of an F-actin ring (second panel). During capped invasion, an F-actin ring is formed (third panel). Invasion of adf cKD parasites occurs without formation of an F-actin ring (fourth panel). F-actin accumulation is observed at the posterior pole of the parasite in all cases (orange arrows). Green segmented lines depict the area that was measured for generation of intensity plot profiles shown in (B). Yellow and purple dotted lines indicate the extra- and intracellular part of the parasite respectively. B. Intensity plot profiles of the movies shown in (A). Depicted plots correlate to half-invaded parasites. In the case of F-actin ring formation, two distinct peaks are detected, correlating to the F-actin ring at the TJ (red arrowhead) and the posterior pole of the parasite (orange arrow). In the case of no F-actin ring, no accumulation at the TJ can be observed; only the peak correlating to posterior F-actin can be detected. For adf cKD, only one peak can be seen corresponding to the posterior pole of the parasite. C. Quantification of F-actin accumulation at the TJ for RH Cb-EmeraldFP parasites as detected in time lapse analysis. The values are expressed as percentage. 28 total parasite invasion events were captured across three different biological replicates. Two-tailed unpaired t-test analysis was performed. **** p value <0.0001. Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm. ****p value <0.0001.D. Representative immunofluorescence assays on indicated parasite strains during host cell penetration. Parasites were labelled with indicated antibodies. SAG1 staining was performed prior to permeabilisation to stain only the extracellular portion of the parasite. Antibodies against Ron2 were used to stain the TJ. Red arrowhead: position of the TJ. Orange arrow: posterior pole of the parasite. Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm.E. Tight Junction assay on fixed samples depicting invasion rates of indicated parasites. While in the majority of control parasites F-actin can be detected at the junction (~80%), no junctional ring can be detected in ~20% of all invasion events. In contrast, adf cKD, act1 cKO or myoA KO only show an overall invasion rate of ~20% compared to controls. In these cases junctional F-actin accumulation could never be detected. Note that the number of total invasion events for adf cKD, act1cKO and myoAKO correlates to the number of invasion events, seen for RH without F-actin ring formation. Data information: Error bars represent standard deviation. One-way ANOVA analysis was performed for each graph ****p value <0.0001.F. Quantification of posterior F-actin accumulation during invasion of indicated parasite lines. One-way ANOVA analysis was performed for each graph ****p value <0.0001.


Figure 4A . Time - lapse analysis of invading RH Cb - EmeraldFP parasites . During penetration , an F - actin ring is formed at the TJ ( orange arrow ) . The nucleus ( purple ) is squeezed through the TJ ( red arrowhead ) and posterior F - actin appears to be directly connected to the nucleus and TJ ( blue arrow ) . B . Analysis of nucleus deformation during live imaging shown in A . The nucleus constricts while its passing through the TJ ( red arrowheads ) . C . The posterior end of the parasite deforms during the invasion process . F - actin is accumulated during invasion , with the posterior end contracting ( blue arrow ) . Data information : Numbers indicate minutes : seconds . Scale bar represents 5 µm . D . Time - lapse stills depicting RH Cb - EmeraldFP parasites invading in absence of detectable F - actin at the junction ( orange arrow ) . The nucleus ( purple ) is squeezed through the TJ ( red arrowhead ) during invasion . Actin accumulation at the posterior pole of the parasite is strongly detected in all cases ( blue arrow ) . E . Analysis of nucleus deformation during live imaging shown in ( D ) . The nucleus appears to not show constriction once its passing through the TJ when the F - actin ring is not present . F . The posterior end of the parasite also deforms during the invasion process . G . Z - stack gallery depicting mid - invading myoA KO parasites . Note that the nucleus is located located at the back during invasion events . Data information : Scale bar represents 5 µm . White arrow points to direction of invasion .

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

Figure 4A. Time-lapse analysis of invading RH Cb-EmeraldFP parasites. During penetration, an F-actin ring is formed at the TJ (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) and posterior F-actin appears to be directly connected to the nucleus and TJ (blue arrow). B. Analysis of nucleus deformation during live imaging shown in A. The nucleus constricts while its passing through the TJ (red arrowheads). C. The posterior end of the parasite deforms during the invasion process. F-actin is accumulated during invasion, with the posterior end contracting (blue arrow). Data information: Numbers indicate minutes:seconds. Scale bar represents 5 µm.D. Time-lapse stills depicting RH Cb-EmeraldFP parasites invading in absence of detectable F-actin at the junction (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) during invasion. Actin accumulation at the posterior pole of the parasite is strongly detected in all cases (blue arrow). E. Analysis of nucleus deformation during live imaging shown in (D). The nucleus appears to not show constriction once its passing through the TJ when the F-actin ring is not present. F. The posterior end of the parasite also deforms during the invasion process.G. Z-stack gallery depicting mid-invading myoA KO parasites. Note that the nucleus is located located at the back during invasion events. Data information: Scale bar represents 5 µm. White arrow points to direction of invasion.


Figure 5A . SR - SIM images depicting invading parasites with and without the presence of the F - actin ring at the tight junction and invading adf cKD Cb - EmeraldFP parasites . SAG1 staining ( in red ) was performed prior to permeabilisation to specifically label extracellular part of the parasite . Note the accumulation of actin ( blue arrow ) around the nucleus and the posterior pole of the parasite , irrespective of F - actin ring formation at the TJ ( orange arrowhead ) . 3D rendering is shown for each presented case ( inlet ) . Data information : Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion . B . SR image of an invading parasite before nuclear entry . F - actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole . C . F - actin localisation in relation to the TJ during invasion . F - actin ( white arrow ) is in close vicinity to the Ron2 ring ( light blue arrow ) during invasion . Data information : cale bar represents 5 µm for SR - SIM images hite arrow points to direction of invasion . D . Quantification of perinuclear actin in indicated parasites . Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus . Still images to the right represent what was considered as F - actin - nucleus association . The orange arrowhead points to F - actin surrounding the nucleus in the posterior end of the parasite , the red and yellow arrowhead shows F - actin in the vicinity of the nucleus , hinting at a close interaction between the two . Three biological replicates were analysed with a minimum of 25 parasites per replicate . Data information : Error bars represent standard deviation . Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion . E - G . SR - SIM images showing three stages of invading wt parasites . SiR - tubulin staining for microtubules ( in red ) was performed prior to fixation to specifically label microtubules in the parasite . During invasion , the microtubules ( MT ) are deformed at the TJ area ( white arrowhead ) . Accumulation of actin ( green arrow ) at the posterior forms a F - actin meshwork . The nucleus is deformed when it enters the TJ area ( orange arrowhead ) . H - J . In the case of the myoA KO strain , the MTs also deform when passing through the TJ ( white arrowhead ) . However , F - actin is not concentrated at the posterior pole or the TJ , but evenly distributed within the cytosol of the parasite , with some peripheral location . K - M . In case of adfKD , MTs are deformed at the TJ area ( white arrowhead ) . F - actin is accumulated at both ends of the parasite ( green arrowheads ) , with no accumulation at the TJ . Data information : Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion .

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

Figure 5A. SR-SIM images depicting invading parasites with and without the presence of the F-actin ring at the tight junction and invading adf cKD Cb-EmeraldFP parasites. SAG1 staining (in red) was performed prior to permeabilisation to specifically label extracellular part of the parasite. Note the accumulation of actin (blue arrow) around the nucleus and the posterior pole of the parasite, irrespective of F-actin ring formation at the TJ (orange arrowhead). 3D rendering is shown for each presented case (inlet). Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.B. SR image of an invading parasite before nuclear entry. F-actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole. C. F-actin localisation in relation to the TJ during invasion. F-actin (white arrow) is in close vicinity to the Ron2 ring (light blue arrow) during invasion. Data information: cale bar represents 5 µm for SR-SIM images hite arrow points to direction of invasion.D. Quantification of perinuclear actin in indicated parasites. Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus. Still images to the right represent what was considered as F-actin - nucleus association. The orange arrowhead points to F-actin surrounding the nucleus in the posterior end of the parasite, the red and yellow arrowhead shows F-actin in the vicinity of the nucleus, hinting at a close interaction between the two. Three biological replicates were analysed with a minimum of 25 parasites per replicate. Data information: Error bars represent standard deviation. Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.E-G. SR-SIM images showing three stages of invading wt parasites. SiR-tubulin staining for microtubules (in red) was performed prior to fixation to specifically label microtubules in the parasite. During invasion, the microtubules (MT) are deformed at the TJ area (white arrowhead). Accumulation of actin (green arrow) at the posterior forms a F-actin meshwork. The nucleus is deformed when it enters the TJ area (orange arrowhead). H-J. In the case of the myoA KO strain, the MTs also deform when passing through the TJ (white arrowhead). However, F-actin is not concentrated at the posterior pole or the TJ, but evenly distributed within the cytosol of the parasite, with some peripheral location. K-M. In case of adfKD, MTs are deformed at the TJ area (white arrowhead). F-actin is accumulated at both ends of the parasite (green arrowheads), with no accumulation at the TJ. Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


Figure 6A . Different invasion stages of P . falciparum merozoites expressing Cb - EmeraldFP . DAPI labels the nucleus ( DNA ) , Cb - EmeraldFP labels actin filaments ( CB - EME , green ) and anti - RON4 labels the junction ( red ) . Left panel : onset of invasion . Middle panel : A merozoite in the middle of invasion ; note the constriction of the nucleus ( blue ) as it passes through the RON4 junction and F - actin colocalising with it ( green ) . Right panel : A merozoite completing invasion ; note the F - actin ring beyond the RON4 - junction and actin filaments surrounding the nucleus . Data information : Scale bar represents 1 µm for Plasmodium falciparumB . IFA showing various stages of invasion of erythrocytes by P . knowlesi merozoites . DAPI labels the nucleus ( DNA ) , anti - actin Antibody labels actin filaments ( anti - actin , green ) and the invasion junction is marked with a C - terminally tagged PkRON2 mCherryHA ( red ) . Left panel : a merozoite beginning invasion ; note the formation of an F - actin ring ( green ) behind the PkRON2 junction ( red ) . Middle panel : A merozoite in the middle of the invasion event ; note the constriction of the nucleus ( blue ) as it passes through the PkRON2 junction and F - actin colocalising with it ( green ) . Right panel : A merozoite completing the process of invasion ; note the F - actin ring beyond the PkRON2 - junction and actin filaments colocalising with the nucleus . Data information : Scale bar represents 3 µm for Plasmodium knowlesi . C . Super - resolution images showing various stages of invasion of erythrocytes by P . falciparum merozoites expressing Cb - EmeraldFP . Left panel : DAPI labels the nucleus ( blue ) , Cb - EmeraldFP labels actin filaments ( CB - EME , green ) and the invasion junction is marked with an anti - RON4 antibody ( red ) . Brightfield images have been marked with red dots depicting the junction , black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell . Top panel shows an attached merozoite beginning the process of invasion , the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion . Right panel : Schematic depicting merozoite invasion into red blood cells . Letters depict the corresponding event in the model ( left ) and microscopy pictures ( right ) . White arrows depict actin filaments that colocalise with the nucleus . D . Quantification of Plasmodium falciparum invasion events categorising actin - TJ and actin - nucleus association events . 121 events were counted in total . Data information : Error bars represent standard deviation . Scale bar represents 1 µm for Plasmodium

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

Figure 6A. Different invasion stages of P. falciparum merozoites expressing Cb-EmeraldFP. DAPI labels the nucleus (DNA), Cb-EmeraldFP labels actin filaments (CB-EME, green) and anti-RON4 labels the junction (red). Left panel: onset of invasion. Middle panel: A merozoite in the middle of invasion; note the constriction of the nucleus (blue) as it passes through the RON4 junction and F-actin colocalising with it (green). Right panel: A merozoite completing invasion; note the F-actin ring beyond the RON4-junction and actin filaments surrounding the nucleus. Data information: Scale bar represents 1 µm for Plasmodium falciparumB. IFA showing various stages of invasion of erythrocytes by P. knowlesi merozoites. DAPI labels the nucleus (DNA), anti-actin Antibody labels actin filaments (anti-actin, green) and the invasion junction is marked with a C-terminally tagged PkRON2 mCherryHA (red). Left panel: a merozoite beginning invasion; note the formation of an F-actin ring (green) behind the PkRON2 junction (red). Middle panel: A merozoite in the middle of the invasion event; note the constriction of the nucleus (blue) as it passes through the PkRON2 junction and F-actin colocalising with it (green). Right panel: A merozoite completing the process of invasion; note the F-actin ring beyond the PkRON2-junction and actin filaments colocalising with the nucleus. Data information: Scale bar represents 3 µm for Plasmodium knowlesi.C. Super-resolution images showing various stages of invasion of erythrocytes by P. falciparum merozoites expressing Cb-EmeraldFP. Left panel: DAPI labels the nucleus (blue), Cb-EmeraldFP labels actin filaments (CB-EME, green) and the invasion junction is marked with an anti-RON4 antibody (red). Brightfield images have been marked with red dots depicting the junction, black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell. Top panel shows an attached merozoite beginning the process of invasion, the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion. Right panel: Schematic depicting merozoite invasion into red blood cells. Letters depict the corresponding event in the model (left) and microscopy pictures (right). White arrows depict actin filaments that colocalise with the nucleus. D. Quantification of Plasmodium falciparum invasion events categorising actin-TJ and actin-nucleus association events. 121 events were counted in total. Data information: Error bars represent standard deviation. Scale bar represents 1 µm for Plasmodium falciparum


Figure 4 ( H ) - ( J ) mRNA expression of ( H ) U2AF1 , ( I ) SMN2 - FL , or ( J ) SMN2 ∆ 7 in SMA patient - derived fibroblasts ( red bars ) 72 hours post - transfection with SMN2 SSO , scrambled siRNA , or U2AF1 siRNA at the indicated dose . Unaffected , SMA carrier fibroblasts ( blue bars ) treated with scrambled siRNA . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; empirical p - values are determined by a simulation and sampling method The error bars show the Standard Error of Mean ( SEM ) . Data represent 3 biological replicates . Three technical replicates were performed during qPCR for each biological replicate and averaged . Scrambled siRNA condition for each experiment was set to 1 . ( K ) Representative Western blot . ( L ) - ( M ) Quantification of protein expression of ( L ) U2AF1 or ( M ) SMN normalized to GAPDH ( n = 3 ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; empirical p - values are determined by a simulation and sampling method The error bars show the Standard Error of Mean ( SEM ) . Data represent 3 biological replicates .

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 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

Figure 4(H)-(J) mRNA expression of (H) U2AF1, (I) SMN2-FL, or (J) SMN2∆7 in SMA patient-derived fibroblasts (red bars) 72 hours post-transfection with SMN2 SSO, scrambled siRNA, or U2AF1 siRNA at the indicated dose. Unaffected, SMA carrier fibroblasts (blue bars) treated with scrambled siRNA. Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates. Three technical replicates were performed during qPCR for each biological replicate and averaged. Scrambled siRNA condition for each experiment was set to 1.(K) Representative Western blot. (L)-(M) Quantification of protein expression of (L) U2AF1 or (M) SMN normalized to GAPDH (n=3). Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates.


Figure 1Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ cells are dependent on Mec1 . These phosphoproteomic analyses were conducted in the presence of 0 . 02 % MMS for 2 hours .

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

Figure 1Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ cells are dependent on Mec1. These phosphoproteomic analyses were conducted in the presence of 0.02% MMS for 2 hours.


Figure 3Immunoblot analysis of Sgs1 N - terminus ( amino acids 1 - 647 ) from cells treated with increasing doses of the DNA alkylating drug MMS in the absence of RAD9 and / or MEC1 . This truncated Sgs1 protein was expressed from its native promoter in this and all subsequent experiments . Rmi1 , a component of the Sgs1 - Top3 - Rmi1 ( STR ) complex , is hyper - phosphorylated on S / T - Q motifs in rad9Δ cells .

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

Figure 3Immunoblot analysis of Sgs1 N-terminus (amino acids 1-647) from cells treated with increasing doses of the DNA alkylating drug MMS in the absence of RAD9 and/or MEC1. This truncated Sgs1 protein was expressed from its native promoter in this and all subsequent experiments.Rmi1, a component of the Sgs1-Top3-Rmi1 (STR) complex, is hyper-phosphorylated on S/T-Q motifs in rad9Δ cells.


Figure 4Immunoblot showing co - immunoprecipitation of Dpb11 with Sgs1 . Both DPB11 - 3xHA and SGS11 - 647 - 5xFLAG were expressed at their endogenous loci .

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 showing co-immunoprecipitation of Dpb11 with Sgs1. Both DPB11-3xHA and SGS11-647-5xFLAG were expressed at their endogenous loci.


Figure 5Immunoblots showing co - immunoprecipitation between Dpb11 and Sgs1 in the presence of 0 . 04 % MMS in either wildtype , mec1Δ , tel1Δ or mec1Δ tel1Δ cell lines . DPB11 - 3HA was tagged at its endogenous locus and co - immunoprecipitated with SGS11 - 647 - FLAG tagged and truncated at its endogenous locus .

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

Figure 5Immunoblots showing co-immunoprecipitation between Dpb11 and Sgs1 in the presence of 0.04% MMS in either wildtype, mec1Δ, tel1Δ or mec1Δ tel1Δ cell lines. DPB11-3HA was tagged at its endogenous locus and co-immunoprecipitated with SGS11-647-FLAG tagged and truncated at its endogenous locus.


Figure 6Dilution assay of wild - type or rev3Δ cells expressing BRCT3 / 4 - SGS1 in the presence of 0 . 0025 % MMS .

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

Figure 6Dilution assay of wild-type or rev3Δ cells expressing BRCT3/4-SGS1 in the presence of 0.0025% MMS.


Figure 7Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3 / 4 fused to wild - type or mutant versions of SGS1 . Mutants include a helicase deficient allele of SGS1 ( SGS1hd ) or an allele lacking the Top3 Interacting Motif ( TIM ; SGS1TIM ) . Error bars represent SEM of at least 3 replicate experiments . P value was calculated with a two - tailed , unpaired t - test . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 . C . Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3 / 4 fused to either SGS1 , TOP3 , or RMI1 . Error bars represent SEM of at least 3 replicate experiments . P value was calculated with a two - tailed , unpaired t - test . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 . D . Measurement of BIR efficiency in cells carrying an empty vector or DPB11BRCT3 / 4 fused to either SGS1 , hBLM , PIF1 , or RRM3 . Error bars represent SEM of at least 3 replicate experiments . P value was calculated with a two - tailed , unpaired t - test . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 .

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

Figure 7Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to wild-type or mutant versions of SGS1. Mutants include a helicase deficient allele of SGS1 (SGS1hd) or an allele lacking the Top3 Interacting Motif (TIM; SGS1TIM). Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001. C. Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to either SGS1, TOP3, or RMI1. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001. D. Measurement of BIR efficiency in cells carrying an empty vector or DPB11BRCT3/4 fused to either SGS1, hBLM, PIF1, or RRM3. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.


Figure 8Serial dilution assay of sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH . BIR assay in rad9Δ sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH . Error bars represent SEM of 3 independent experiments . Error bars represent SEM of at least 3 replicate experiments . P value was calculated with a two - tailed , unpaired t - test . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 .

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

Figure 8Serial dilution assay of sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH.BIR assay in rad9Δ sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH. Error bars represent SEM of 3 independent experiments. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.


Figure 1 ( H ) Ang - 2 dose and time - dependently activates p - EphrinB , p - TIE2 and p - AKT in HUVEC ; immunoblotting results ; NA3VO4 : sodium orthovanadate . ( I ) Tumor vessels in B16F10 tumors are mostly p - TIE2 ( Tyr992 ) + and pSHP2 ( Tyr542 ) + ; confocal images ( scale bars : 200μm ) .

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

Figure 1(H) Ang-2 dose and time-dependently activates p-EphrinB, p-TIE2 and p-AKT in HUVEC; immunoblotting results; NA3VO4: sodium orthovanadate.(I) Tumor vessels in B16F10 tumors are mostly p-TIE2 (Tyr992)+ and pSHP2(Tyr542)+; confocal images (scale bars: 200μm).


Figure 2 ( A ) Representative tissue immunostaining of human melanoma , colon carcinoma and colon adenoma ; confocal images from serial sections ( scale bars : 50μm tissues ) . Arrowheads point to CD31 + / p - EphrinB + ; CD31 + / p - SHP2 + ; CD31 + / p - Tie2 + and CD31 + / Ang2 + endothelial cells . ( B ) Gene expression in tumor ( black bar ) and control ( white bar ) ; top panel : melanoma ( GSE7553 , primary and metastatic ; tumor n = 54 ; control skin n = 5 ) ; bottom panel : colon cancer ( TCGA , primary and metastatic ; tumor n = 269 ; control n = 41 . * * P < 0 . 01 , * * * * P < 0 . 0001 ; P values from Mann Whitney U - test . ( C ) Impact of high and low ( 75 % quartile ) mRNA levels on the survival probability of patients with primary melanoma ; Kaplan - Meier analysis using TCGA primary melanoma datasets ( n = 103 ) ; P values from Mann Whitney U - test . ( D ) Impact of high and low ( median ) mRNA levels on the survival probability of patients with colon cancer ; Kaplan - Meier analysis using TCGA colon cancer dataset ( n = 262 ) .

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

Figure 2(A) Representative tissue immunostaining of human melanoma, colon carcinoma and colon adenoma; confocal images from serial sections (scale bars: 50μm tissues). Arrowheads point to CD31+/p-EphrinB+; CD31+/p-SHP2+; CD31+/p-Tie2+ and CD31+/Ang2+ endothelial cells.(B) Gene expression in tumor (black bar) and control (white bar); top panel: melanoma (GSE7553, primary and metastatic; tumor n=54; control skin n=5); bottom panel: colon cancer (TCGA, primary and metastatic; tumor n=269; control n=41. **P<0.01, ****P<0.0001; P values from Mann Whitney U-test.(C) Impact of high and low (75% quartile) mRNA levels on the survival probability of patients with primary melanoma; Kaplan-Meier analysis using TCGA primary melanoma datasets (n=103); P values from Mann Whitney U-test. (D) Impact of high and low (median) mRNA levels on the survival probability of patients with colon cancer; Kaplan-Meier analysis using TCGA colon cancer dataset (n=262).


Figure 3 ( C , D ) The JAK inhibitor Tofacitinib ( Tofa , 50nM ) enhances the proliferation of HUVEC and BMEC compared to control ( C ) and reduces endogenous p - STAT3 levels in HUVEC and BMEC compared to control ( D ) after 72 - hour culture . SHP099 ( 5μM HUVEC ; 20μM BMEC ) reduces cell proliferation and increases p - STAT3 in HUVEC and BMEC compared to control ; error bars : ± S . D . Results of immunoblotting are representative of 3 independent blots . ( E , F ) The MAP kinases inhibitor PD098059 ( PD0980 , 10μM ) and SHP099 ( 5μM HUVEC ; 20μM BMEC ) inhibit HUVEC and BMEC proliferation compared to control ( E ) and reduce p - ERK1 / 2 levels by immunoblotting after 72 - hour culture ( F ) . Results of proliferation in panels C and E ( % of control ) from triplicate cultures are representative of 3 - 4 experiments ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ; two - tailed Student ' s t - test ; error bars : ± S . D . Results of immunoblotting are representative of 3 independent blots . ( G , H ) The ERK1 / 2 inhibitors AZD6244 and Tamnitinib dose - dependently reduce B16F10 cell proliferation ( G ) ; representative experiment of 3 independent experiments ; the results reflect the means of 5 replicate / condition ; error bars : ± S . D . ; and levels of endogenous p - STAT3 and p - ERK in B16F10 cells ( representative of 3 blots ) ( H ) .

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

Figure 3(C,D) The JAK inhibitor Tofacitinib (Tofa, 50nM) enhances the proliferation of HUVEC and BMEC compared to control (C) and reduces endogenous p-STAT3 levels in HUVEC and BMEC compared to control (D) after 72-hour culture. SHP099 (5μM HUVEC; 20μM BMEC) reduces cell proliferation and increases p-STAT3 in HUVEC and BMEC compared to control; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.(E,F) The MAP kinases inhibitor PD098059 (PD0980, 10μM) and SHP099 (5μM HUVEC; 20μM BMEC) inhibit HUVEC and BMEC proliferation compared to control (E) and reduce p-ERK1/2 levels by immunoblotting after 72-hour culture (F). Results of proliferation in panels C and E (% of control) from triplicate cultures are representative of 3-4 experiments; *P<0.05, **P<0.01 ***P<0.001; two-tailed Student's t-test; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.(G,H) The ERK1/2 inhibitors AZD6244 and Tamnitinib dose-dependently reduce B16F10 cell proliferation (G); representative experiment of 3 independent experiments; the results reflect the means of 5 replicate/condition; error bars: ± S.D.; and levels of endogenous p-STAT3 and p-ERK in B16F10 cells (representative of 3 blots) (H).


Figure 4 ( A - C ) SHP099 ( A , 75mg / kg ; B , C : 100mg / kg ) reduces B16F10 tumor growth in syngeneic mice . Panels depict tumor growth from initiation of treatment to endpoint ( left ) and tumor weight ( right ) at endpoint ; no . mice / group : 6 - 11 control ; 7 - 9 SHP099 . ( E ) " Vascular sleeves " ( Collagen IV + CD31 - ) identified in a representative SHP099 - treated tumor . Representative confocal images from 6 tumors ( scale bar : 50μm ) . ( F ) Reduced CD31 + vascular area in SHP099 - treated tumors compared to controls ( n = 6 / group ) ; quantification by ImageJ . ( G ) The ratio of Collagen IV + / CD31 + area is increased in SHP099 - treated tumors compared to controls ( n = 6 / group ) ; quantification by ImageJ ; Coll : collagen . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . ( H ) Cleaved caspase - 3 detection in a CD31 + vessel ( representative of 6 SHP099 - treated tumors ) ; arrowheads point to cleaved caspase - 3 + CD31 + cells . Cleaved caspase - 3 + tumor cell is marked by * . Confocal images ( scale bar : 10μm ) . ( I ) Increased cleaved caspase - 3 fluorescence intensity in CD31 + cells from SHP099 - treated tumors compared to controls ( n = 3 / group ) ; at least 100 cells counted / sample . ( J ) Ki67 + proliferating cells in representative of 3 SHP099 - treated and 3 control tumors ; CD31 identifies the vessels ; DAPI identifies cell nuclei . Confocal images ( scale bar : 50μm ) . ( K ) Quantification of proliferating cells within SHP099 and control ( n = 4 / group ) tumors by ImageJ . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . ( L ) Hypoxic tumor identified Hypoxyprobe ( green ) representative of 3 SHP099 - treated tumors ; confocal images ( scale bar : 100μm ) , left ; quantification of hypoxic areas in control ( n = 3 ) and SHP099 - treated ( n = 3 ) tumors by ImageJ . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . ( M ) Representative control ( of 5 ) and SHP099 - treated ( of 5 ) B16F10 tumor sections through the maximum diameter ; H & E staining ( scale bar : 10mm ) . ( N ) Cleaved caspase - 3 + fluorescence intensity in SHP099 and control tumors ( n = 3 / group ) . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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

Figure 4(A-C) SHP099 (A, 75mg/kg; B, C: 100mg/kg) reduces B16F10 tumor growth in syngeneic mice. Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; no. mice/group: 6-11 control; 7-9 SHP099.(E) "Vascular sleeves" (Collagen IV+ CD31-) identified in a representative SHP099-treated tumor. Representative confocal images from 6 tumors (scale bar: 50μm). (F) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ. (G) The ratio of Collagen IV+/CD31+ area is increased in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ; Coll: collagen. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.(H) Cleaved caspase-3 detection in a CD31+ vessel (representative of 6 SHP099-treated tumors); arrowheads point to cleaved caspase-3+CD31+ cells. Cleaved caspase-3+ tumor cell is marked by *. Confocal images (scale bar: 10μm). (I) Increased cleaved caspase-3 fluorescence intensity in CD31+ cells from SHP099-treated tumors compared to controls (n=3/group); at least 100 cells counted/sample. (J) Ki67+ proliferating cells in representative of 3 SHP099-treated and 3 control tumors; CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm). (K) Quantification of proliferating cells within SHP099 and control (n=4/group) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.(L) Hypoxic tumor identified Hypoxyprobe (green) representative of 3 SHP099-treated tumors; confocal images (scale bar: 100μm), left; quantification of hypoxic areas in control (n=3) and SHP099-treated (n=3) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.(M) Representative control (of 5) and SHP099-treated (of 5) B16F10 tumor sections through the maximum diameter; H&E staining (scale bar: 10mm). (N) Cleaved caspase-3+ fluorescence intensity in SHP099 and control tumors (n=3/group). Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


Figure 5 ( B ) Extravasation of FITC - dextran ( green ) in tumors established in NSG mice treated with SHP099 ; scale bar ( 50μm ) . Representative images from 3 mice / group . ( C ) Reduced CD31 + vascular area in SHP099 - treated tumors compared to controls ( n = 5 / group ) ; ImageJ quantification . P values from two - tailed Student ' s t - test ; * P < 0 . 05 . Error bars : ± S . D . E ) SHP099 reduces weight of control ( p - LKO vector ) and SHP2 - depleted ( shSHP2 ) B16F10 tumors compared to untreated controls : p - LKO untreated ( n = 5 ) , p - LKO treated with SHP099 ( n = 9 ) , shSHP2 untreated ( n = 5 ) , and shSHP2 treated with SHP099 ( n = 13 ) . P values by analysis of variance with Dunnett ' s multiple comparison test ; * * * P < 0 . 001 . ( F ) Persistent depletion of SHP2 in tumors induced by shSHP2 - B16F10 cells ( n = 5 ) compared to control tumors induced by p - LKO - B16F10 ( n = 5 ) ; tumors removed from mice 18 days after cell inoculation . ( N ) Representative popliteal lymph nodes from control and SHP099 - treated mice with microscopic evidence of metastases ; H & E stain ; scale bars 500 μm ( low magnification ) 20 μm ( high magnification ) .

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 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 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

Figure 5(B) Extravasation of FITC-dextran (green) in tumors established in NSG mice treated with SHP099; scale bar (50μm). Representative images from 3 mice/group. (C) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=5/group); ImageJ quantification. P values from two-tailed Student's t-test; *P<0.05. Error bars: ± S.D. E) SHP099 reduces weight of control (p-LKO vector) and SHP2-depleted (shSHP2) B16F10 tumors compared to untreated controls: p-LKO untreated (n=5), p-LKO treated with SHP099 (n=9), shSHP2 untreated (n=5), and shSHP2 treated with SHP099 (n=13). P values by analysis of variance with Dunnett's multiple comparison test; ***P<0.001. (F) Persistent depletion of SHP2 in tumors induced by shSHP2-B16F10 cells (n=5) compared to control tumors induced by p-LKO-B16F10 (n=5); tumors removed from mice 18 days after cell inoculation. (N) Representative popliteal lymph nodes from control and SHP099-treated mice with microscopic evidence of metastases; H&E stain; scale bars 500 μm (low magnification) 20 μm (high magnification).


Figure 6 ( C , D ) p - TIE2 in tumor vessels from control and AMG386 - treated mice ; representative confocal images ; arrowheads point to CD31 + / p - TIE2 - endothelial cells ( scale bars : 50μm ) ( C ) , and quantification ( D ) of p - TIE - 2 / CD31 + in control ( n = 3 ) and AMG386 treated ( n = 3 ) tumors ( 1 , 200 CD31 + cells / group ) . P values from two - tailed Student ' s t - test ; * * * P < 0 . 001 . ( E , F ) p - EphrinB ( E ) in representative tumor vessels from control ( n = 3 ) and AMG386 - treated ( n = 3 ) tumors ; confocal images ( E ) , ( scale bar : 50μm ) and quantification of p - EphrinB / CD31 + ( F ) ; P values from two - tailed Student ' s t - test ; * * * P < 0 . 001 . ( G ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 ( red ) identifies the endothelium ; DAPI ( blue ) identifies the nuclei ; representative tumor confocal images ( scale bar : 50μm ) of 3 mice / group ; arrow heads point to FITC - dextran extravasation ; asterisks to extravasated erythrocytes . ( H ) Quantification of cleaved caspase - 3 in the tumor vasculature ( CD31 + cells ) ; n = 3 tumors / group ; number of CD31 + cells counted : control ( n = 311 ) ; SHP099 ( n = 425 ) ; AMG386 ( n = 451 ) ; combined SHP099 + AMG386 ( n = 484 ) . Error bars : ± S . D . ; P values by analysis of variance with Dunnett ' s multiple comparison test ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 6(C,D) p-TIE2 in tumor vessels from control and AMG386-treated mice; representative confocal images; arrowheads point to CD31+/p-TIE2- endothelial cells (scale bars: 50μm) (C), and quantification (D) of p-TIE-2/CD31+ in control (n=3) and AMG386 treated (n=3) tumors (1,200 CD31+ cells/group). P values from two-tailed Student's t-test; ***P<0.001. (E,F) p-EphrinB (E) in representative tumor vessels from control (n=3) and AMG386-treated (n=3) tumors; confocal images (E), (scale bar: 50μm) and quantification of p-EphrinB/CD31+ (F); P values from two-tailed Student's t-test; ***P<0.001. (G) Vessel perfusion visualized by FITC-dextran (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar:50μm) of 3 mice/group; arrow heads point to FITC-dextran extravasation; asterisks to extravasated erythrocytes.(H) Quantification of cleaved caspase-3 in the tumor vasculature (CD31+ cells); n=3 tumors/group; number of CD31+ cells counted: control (n=311); SHP099 (n=425); AMG386 (n=451); combined SHP099+AMG386 (n=484). Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.


Figure 7 ( A - D ) Nuclear p - STAT3 ( A , B ) and p - ERK1 / 2 ( C , D ) in tumor CD31 + endothelial cells of mice treated for 14 days with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or the combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) immunostaining ( A , C ) and quantification of staining intensity / CD31 + cell ( B , D ) ; horizontal line : mean intensity / group ; n = 3 tumors / group ; number of CD31 + cells counted in B : control ( n = 180 ) , SHP099 ( n = 308 ) , AMG386 ( n = 300 ) , combined SHP099 + AMG386 ( n = 322 ) ; number of CD31 + cells counted in D : control ( n = 1 , 733 ) , SHP099 ( n = 821 ) , AMG386 ( n = 1 , 399 ) , combined SHP099 + AMG386 ( n = 783 ) . Scale bars : A , 50μm ; C , 10μm . FOXO1 detection in tumor CD31 + endothelial cells after treatment with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or the combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) confocal

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7(A-D) Nuclear p-STAT3 (A,B) and p-ERK1/2 (C,D) in tumor CD31+ endothelial cells of mice treated for 14 days with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) immunostaining (A,C) and quantification of staining intensity/CD31+ cell (B, D); horizontal line: mean intensity/group; n=3 tumors/group; number of CD31+ cells counted in B: control (n=180), SHP099 (n=308), AMG386 (n=300), combined SHP099+AMG386 (n=322); number of CD31+ cells counted in D: control (n=1,733), SHP099 (n=821), AMG386 (n=1,399), combined SHP099+AMG386 (n=783). Scale bars: A, 50μm; C, 10μm.FOXO1 detection in tumor CD31+ endothelial cells after treatment with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) confocal images


Figure 8 ( C ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 identifies endothelial cells ( red ) ; DAPI ( blue ) identifies the nuclei ; representative ( of 3 mice / group ) confocal images ( scale bar : 50μm ) ; arrow heads point to FITC - dextran extravasation . ( H ) Representative images depicting Ki67 + cells in control and treated MC38 tumors ; representative images ( scale bar : 50μm ) of 4 tumors / group .

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 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

Figure 8(C) Vessel perfusion visualized by FITC-dextran (green); CD31 identifies endothelial cells (red); DAPI (blue) identifies the nuclei; representative (of 3 mice/group) confocal images (scale bar: 50μm); arrow heads point to FITC-dextran extravasation.(H) Representative images depicting Ki67+ cells in control and treated MC38 tumors; representative images (scale bar: 50μm) of 4 tumors/group.


Figure 1d ) representative images of Ki67 and p16Ink4a immunofluorescence and SA - β - Gal staining ; e ) % of Ki67 positive cells ( 28 days after co - culture ) ; f ) % of p16INK4A positive cells ( 28days after co - culture ) ; g ) % of SA - β - Gal positive cells ( 28 days after co - culture ) ; h ) Western blotting for p21 and quantification ; i ) MitoSOX fluorescence ;

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

Figure 1d) representative images of Ki67 and p16Ink4a immunofluorescence and SA-β-Gal staining; e) % of Ki67 positive cells (28 days after co-culture); f) % of p16INK4A positive cells (28days after co-culture); g) % of SA-β-Gal positive cells (28 days after co-culture); h) Western blotting for p21 and quantification; i) MitoSOX fluorescence;


Figure 2h ) Representative Immuno - FISH micrographs ( using telomere ‐ specific ( CCCTAA ) peptide nucleic acid probe and anti - 53BP1 antibody ) 20 days following co - culture ; i ) quantification of mean TAF and % TAF in MRC5 fibroblasts 8 and 20 days following neutrophil co - culture . Data are mean ± S . E . M . of 3 independent experiments ; J - L ) mRNA expression of p21 ( j ) , IL - 6 ( k ) and IL - 8 ( l ) 20 days after neutrophil co - culture ; Data are mean ± S . E . M . of 2 - 3 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 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

Figure 2h) Representative Immuno-FISH micrographs (using telomere‐specific (CCCTAA) peptide nucleic acid probe and anti-53BP1 antibody) 20 days following co-culture; i) quantification of mean TAF and % TAF in MRC5 fibroblasts 8 and 20 days following neutrophil co-culture. Data are mean±S.E.M. of 3 independent experiments; J-L) mRNA expression of p21 (j), IL-6 (k) and IL-8 (l) 20 days after neutrophil co-culture; Data are mean±S.E.M. of 2-3 independent experiments.


Figure 3f ) mean number of TAF in hepatocytes ; g ) % of TAF positive hepatocytes ; h ) number of p21 positive hepatocytes per field ; i ) number of p16Ink4a positive hepatocytes per field .

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

Figure 3f) mean number of TAF in hepatocytes; g) % of TAF positive hepatocytes; h) number of p21 positive hepatocytes per field; i) number of p16Ink4a positive hepatocytes per field.


Figure 4e ) Representative micrographs of neutrophil marker Ly6G and senescent - associated marker p21 ( 96h ) ; f ) number of Ly6G positive cells per field ( 96h ) ; g ) % of TAF positive hepatocytes ( 96h ) ; h ) mean number of TAF in hepatocytes ( 96h ) ; i ) total number of γH2A . X foci in hepatocytes ( 96h ) ; j ) Lactate dehydrogenase ( LD ) activity ( 96h ) ; k ) p21 positive cells per field ( 96h ) .

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

Figure 4e) Representative micrographs of neutrophil marker Ly6G and senescent-associated marker p21 (96h); f) number of Ly6G positive cells per field (96h); g) % of TAF positive hepatocytes (96h); h) mean number of TAF in hepatocytes (96h); i) total number of γH2A.X foci in hepatocytes (96h); j) Lactate dehydrogenase (LD) activity (96h); k) p21 positive cells per field (96h).


Figure 5m ) Representative immunoFISH ( Telomere - FISH and 8 - oxodG ) ; n ) mean number of 8 - oxodG co - localizing with telomeres in hepatocytes ; o ) Mean telomere FISH intensity in hepatocytes .

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 5m) Representative immunoFISH (Telomere-FISH and 8-oxodG); n) mean number of 8-oxodG co-localizing with telomeres in hepatocytes; o) Mean telomere FISH intensity in hepatocytes.


Figure 6a ) RNA - sequencing data showing expression of genes associated with neutrophil recruitment during liver aging ; i ) Comparison between p16Ink4a mRNA levels of young ( 3 months old ) and old INK - ATTAC mice ( 28 - 29 month old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 8 mice per age group ; j ) % of TAF positive hepatocytes in old INK - ATTAC mice ( 28 - 29 months old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 6 mice per group ; k ) Liver neutrophils ( Ly6G ) in old INK - ATTAC mice ( 28 - 29 months old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 7 mice per group ; p ) Quantification of liver neutrophils ( Ly6G ) in mice injected with vehicle or Cxcl1 . Data are mean ± SEM of n = 7 - 10 mice per group ; q ) Quantification of the % of TAF positive hepatocytes in mice injected with vehicle or Cxcl1 . Data are mean ± SEM of n = 6 - 7 mice per group ;

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6a) RNA-sequencing data showing expression of genes associated with neutrophil recruitment during liver aging;i) Comparison between p16Ink4a mRNA levels of young (3 months old) and old INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-8 mice per age group; j) % of TAF positive hepatocytes in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-6 mice per group; k) Liver neutrophils (Ly6G) in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-7 mice per group; p) Quantification of liver neutrophils (Ly6G) in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 7-10 mice per group ; q) Quantification of the % of TAF positive hepatocytes in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 6-7 mice per group;


Figure 1Primary BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with SMPrimary BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with TAK1i for 6 or 4 h respectively . Time - lapse confocal images ( hour : min ) of BMDMs stimulated with recombinant murine TNF ( 100 ng / ml ) and SM ( 250 nM ) stained with propidium iodide ( red ) for 6 h . Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies . immortalized BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with SMimmortalized BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with TAK1i for 6 or 4 h respectively .

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

Figure 1Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SMPrimary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.Time-lapse confocal images (hour:min) of BMDMs stimulated with recombinant murine TNF (100 ng/ml) and SM (250 nM) stained with propidium iodide (red) for 6 h. Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies.immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SMimmortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.


Figure 2BMDMs were stimulated with TNF ( 100 ng / ml ) in combination with TAK1i ( 125 nM ) for the indicated time points . LDH releaseBMDMs were stimulated with TNF ( 100 ng / ml ) in combination with TAK1i ( 125 nM ) for the indicated time points . mixed supernatant and cell extracts were analysed . BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h in the presence or absence of KCl ( 50 mM ) . Where indicated , cells were pre - incubated with MCC950 ( 10 μM ) 20 - 30 min prior to TNF / TAK1i stimulation . BMDMs were costimulated with TNF ( 100 ng / ml ) and SM ( 250 nM ; 6 h ) and mixed supernatant and cell extracts were analysed by immunoblotBMDMs were costimulated with TNF ( 100 ng / ml ) and SM LDH release in the cell culture supernatant were quantified at the indicated time points . BMDMs were costimulated with TNF ( 100 ng / ml ) and

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

Figure 2BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. LDH releaseBMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. mixed supernatant and cell extracts were analysed.BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h in the presence or absence of KCl (50 mM). Where indicated, cells were pre-incubated with MCC950 (10 μM) 20-30 min prior to TNF/TAK1i stimulation.BMDMs were costimulated with TNF (100 ng/ml) and SM (250 nM; 6 h) and mixed supernatant and cell extracts were analysed by immunoblotBMDMs were costimulated with TNF (100 ng/ml) and SM LDH release in the cell culture supernatant were quantified at the indicated time points.BMDMs were costimulated with TNF (100 ng/ml) and SM


Figure 3HEK 293T cells were transfected with doxycycline - inducible DmrB - caspase - 8 and the indicated GSDMD constructs . Cells were stimulated with doxycycline ( 10 μg / ml ) for 18 h to induce DmrB - caspase - 8 expression and exposed to B / B homodimerizer ( 12 . 5 nM ) for another 2 h to activate caspase - 8 . Mixed supernatant and extracts were analysed by immunoblot . Immortalized Gsdmd - / - BDMDs expressing GSDMDWT and GSDMDD88A were costimulated with TNF ( 100 ng / ml ) and SM for 6 h and LDH release was quantified . Immortalized Gsdmd - / - BDMDs expressing GSDMDWT and GSDMDD88A were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) and stimulated with LCL161 ( 1 μM ) for 24 h and LDH release was quantified . BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i for 4 h and ( D ) mixed supernatant and extracts were analysed by immunoblotBMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i for 4 h and LDH release was quantified at the indicated time points .

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

Figure 3HEK 293T cells were transfected with doxycycline-inducible DmrB-caspase-8 and the indicated GSDMD constructs. Cells were stimulated with doxycycline (10 μg/ml) for 18 h to induce DmrB-caspase-8 expression and exposed to B/B homodimerizer (12.5 nM) for another 2 h to activate caspase-8. Mixed supernatant and extracts were analysed by immunoblot.Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were costimulated with TNF (100 ng/ml) and SM for 6 h and LDH release was quantified.Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with LCL161 (1 μM) for 24 h and LDH release was quantified.BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and (D) mixed supernatant and extracts were analysed by immunoblotBMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and LDH release was quantified at the indicated time points.