Figure 1 ( C ) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin - II probe Sqh : : GFP and the plasma membrane marker GAP43 : : mCherry . Scale bar , 5 μm ( D ) Movie ( still frames ) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus : : βH - spectrin imaged in a cross section using two - photon microscopy . βH - spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination ( arrowheads ) . Scale bar , 50 μm . ( E ) Z - Projections of confocal image stacks showing the apical cell surface of a Drosophila embryo expressing endogenously tagged mVenus : : βH - spectrin ( green ) and the plasma membrane marker GAP43 : : mCherry at 5 min ( top ) , 7 min ( middle ) and 9 min ( bottom ) after initial medio - apical accumulation of βH - spectrin . Scale bar , 20 μm ( F , G ) Immunostaining of βH - spectrin ( green ) visualized by STED nanoscopy revealed medio - apical βH - spectrin supra - cellular fibers in mesodermal cells ( F ) . In ectodermal cells , βH - spectrin localizes to apical cell junctions ( G ) . Note , in ( F ) the junctional βH - spectrin signal is shown in magenta as a proxy for cell membranes . Scale bars , 2 . 5 μm . ( H - J ) Confocal images of the ventral surface of a Drosophila embryo expressing endogenously tagged mVenus : : βH - spectrin ( H ) , the myosin - II marker Sqh : : mCherry ( I ) and a merge of the two ( J ) with βH - spectrin in green and Sqh : : mCherry in magenta . Scale bars : 20 μm . ( K - L ) Co - staining of phalloidin ( F - actin reporter ; magenta ) and βH - spectrin ( green ) of the apical surface of a mesodermal cell at the onset of ventral furrow formation ( K ) and at a later time point ( L ) visualized by confocal microscopy . White dashed lines indicate the cell boundaries segmented based on the phalloidin - staining of sub - apical confocal sections . Scale bars : 5 μm .

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

Figure 1(C) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin-II probe Sqh::GFP and the plasma membrane marker GAP43::mCherry. Scale bar, 5 μm(D) Movie (still frames) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin imaged in a cross section using two-photon microscopy. βH-spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination (arrowheads). Scale bar, 50 μm.(E) Z-Projections of confocal image stacks showing the apical cell surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry at 5 min (top), 7 min (middle) and 9 min (bottom) after initial medio-apical accumulation of βH-spectrin. Scale bar, 20 μm(F, G) Immunostaining of βH-spectrin (green) visualized by STED nanoscopy revealed medio-apical βH-spectrin supra-cellular fibers in mesodermal cells (F). In ectodermal cells, βH-spectrin localizes to apical cell junctions (G). Note, in (F) the junctional βH-spectrin signal is shown in magenta as a proxy for cell membranes. Scale bars, 2.5 μm.(H-J) Confocal images of the ventral surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (H), the myosin-II marker Sqh::mCherry (I) and a merge of the two (J) with βH-spectrin in green and Sqh::mCherry in magenta. Scale bars: 20 μm.(K-L) Co-staining of phalloidin (F-actin reporter; magenta) and βH-spectrin (green) of the apical surface of a mesodermal cell at the onset of ventral furrow formation (K) and at a later time point (L) visualized by confocal microscopy. White dashed lines indicate the cell boundaries segmented based on the phalloidin-staining of sub-apical confocal sections. Scale bars: 5 μm.


Figure 2 ( A ) Z - Projection of confocal image stacks spanning 5 μm of a control embryo ( left ) co - expressing endogenously tagged mVenus : : βH - spectrin ( green ) and the plasma membrane marker GAP43 : : mCherry ( magenta ) and of an embryo additionally expressing a maternally driven anti - GFP nanobody KD module to knock - down βH - spectrin ( right ) . Co - expression of the anti - GFP nanobody KD system resulted in a drastic decrease of βH - spectrin protein levels as assessed by the fluorescent signal intensity which dropped below the detection limit . ( B ) Pie diagrams quantifying the percentage of control embryos ( top , n = 50 ) and βH - spectrin knock - down embryos ( bottom , n = 40 ) that formed ventral furrow either normally or with minor phenotypes ( green ) and the percentage of embryos that displayed severe abnormalities or did not at all undergo tissue invagination ( red ) . The assessment of phenotypes was done in an unbiased randomized approach . ( C - J ) Confocal images of a control embryo ( C , D ) and βH - spectrin knock - down embryos with a minor phenotype ( E , F ) , with a severe phenotype that underwent tissue invagination ( G , H ) and with a severe phenotype that did not undergo tissue invagination ( I , J ) . Surface view is shown in ( C , E , G , I ) and the cross section through the tissue at two different time points in ( D , F , H , J ) . All embryos expressed the plasma membrane marker GAP43 : : mCherry . Scale bars , 20 μm . ( K , L ) Quantification of apical surface area ( K ) and anisotropy ( L ) in control embryos ( green , nControl : 6 embryos ) and in βH - spectrin knock - down embryos ( red , nβH - spectrin KD : 7 embryos ) . Solid lines indicate the mean values and the semi - transparent regions the corresponding standard deviation .

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

Figure 2(A) Z-Projection of confocal image stacks spanning 5 μm of a control embryo (left) co-expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry (magenta) and of an embryo additionally expressing a maternally driven anti-GFP nanobody KD module to knock-down βH-spectrin (right). Co-expression of the anti-GFP nanobody KD system resulted in a drastic decrease of βH-spectrin protein levels as assessed by the fluorescent signal intensity which dropped below the detection limit.(B) Pie diagrams quantifying the percentage of control embryos (top, n=50) and βH-spectrin knock-down embryos (bottom, n=40) that formed ventral furrow either normally or with minor phenotypes (green) and the percentage of embryos that displayed severe abnormalities or did not at all undergo tissue invagination (red). The assessment of phenotypes was done in an unbiased randomized approach.(C-J) Confocal images of a control embryo (C,D) and βH-spectrin knock-down embryos with a minor phenotype (E,F), with a severe phenotype that underwent tissue invagination (G, H) and with a severe phenotype that did not undergo tissue invagination (I, J). Surface view is shown in (C,E,G,I) and the cross section through the tissue at two different time points in (D,F,H,J). All embryos expressed the plasma membrane marker GAP43::mCherry. Scale bars, 20 μm.(K, L) Quantification of apical surface area (K) and anisotropy (L) in control embryos (green, nControl: 6 embryos) and in βH-spectrin knock-down embryos (red, nβH-spectrin KD: 7 embryos). Solid lines indicate the mean values and the semi-transparent regions the corresponding standard deviation.


Figure 3 ( D , E ) Still frames of a movie showing endogenously tagged mVenus : : βH - spectrin in the cross section of a gastrulating wild type ( D ) and twist mutant ( E ) embryo imaged using two - photon microscopy . The top frame corresponds to the time point where an initial bending of the apical surface was observed , the middle frame was taken 2 min and the lower frame 3 min afterwards . Scale bars , 30 µm . ( F , G ) Confocal images showing apical βH - spectrin ( endogenously tagged mVenus : : βH - spectrin ) in a wild type ( F ) and twist mutant ( G ) embryo . Scale bars , 20 μm . Medio - apical βH - spectrin fibers observed in wild type embryos were absent in twist mutant embryos . ( H - Q ) Confocal or two - photon microscopy images of the apical surface of an embryo mounted with the dorsal side facing the objective and co - expressing endogenously tagged mVenus : : βH - spectrin ( green , H , K , M , P ) , the myosin - II marker Sqh : : mCherry ( magenta , I , N ) , and the optogenetic modules as explained in ( C ) . The apical surface is shown before photoactivation ( H - L ) and 1 min after photoactivation ( M - Q ) . Panels ( J , O ) show a merge of βH - spectrin and myosin - II and the dashed rectangle indicates regions shown in ( L and Q ) at higher magnification . Panels ( K , L ) and ( P , Q ) are zoom - ins into the region indicated in ( J ) an ( O ) , respectively . Scale bars , 20 μm ( H - J , M - O ) . 2 . 5 μm ( K , L , P , Q ) . ( R ) Quantification of apical βH - spectrin upon optogenetic stimulation of Rho signaling . The graph shows the mean intensity of βH - spectrin ( green ) at the apical surface over time as the fold change relative to the initial time point ( before photo - activation ) . The semi - transparent region indicates the standard deviation and dots indicate individual data points ( n = 5 embryos ) . ( S - T ) Two - photon microscopy images of apical βH - spectrin ( green ) 1 . 5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water ( S ) or the ROCK - inhibitor Y - 27634 ( T ) . Dashed boxes indicate photo - activated cells . Y - 27634 - injected embryos did not undergo apical constriction and did not accumulate βH - spectrin upon photo - activation , while water - injected embryos constricted and showed increased βH - spectrin levels . Scale bars : 10 µm .

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

Figure 3(D, E) Still frames of a movie showing endogenously tagged mVenus::βH-spectrin in the cross section of a gastrulating wild type (D) and twist mutant (E) embryo imaged using two-photon microscopy. The top frame corresponds to the time point where an initial bending of the apical surface was observed, the middle frame was taken 2 min and the lower frame 3 min afterwards. Scale bars, 30 µm.(F, G) Confocal images showing apical βH-spectrin (endogenously tagged mVenus::βH-spectrin) in a wild type (F) and twist mutant (G) embryo. Scale bars, 20 μm. Medio-apical βH-spectrin fibers observed in wild type embryos were absent in twist mutant embryos.(H-Q) Confocal or two-photon microscopy images of the apical surface of an embryo mounted with the dorsal side facing the objective and co-expressing endogenously tagged mVenus::βH-spectrin (green, H, K, M, P), the myosin-II marker Sqh::mCherry (magenta, I, N), and the optogenetic modules as explained in (C). The apical surface is shown before photoactivation (H-L) and 1 min after photoactivation (M-Q). Panels (J, O) show a merge of βH-spectrin and myosin-II and the dashed rectangle indicates regions shown in (L and Q) at higher magnification. Panels (K, L) and (P, Q) are zoom-ins into the region indicated in (J) an (O), respectively. Scale bars, 20 μm (H-J, M-O). 2.5 μm (K, L, P, Q).(R) Quantification of apical βH-spectrin upon optogenetic stimulation of Rho signaling. The graph shows the mean intensity of βH-spectrin (green) at the apical surface over time as the fold change relative to the initial time point (before photo-activation). The semi-transparent region indicates the standard deviation and dots indicate individual data points (n = 5 embryos).(S-T) Two-photon microscopy images of apical βH-spectrin (green) 1.5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water (S) or the ROCK-inhibitor Y-27634 (T). Dashed boxes indicate photo-activated cells. Y-27634-injected embryos did not undergo apical constriction and did not accumulate βH-spectrin upon photo-activation, while water-injected embryos constricted and showed increased βH-spectrin levels. Scale bars: 10 µm.


Figure 4 ( A - C ) Confocal images of the apical cell surface of a βH - spectrin knock - down embryo showing the myosin - II marker Sqh : : mCherry ( green ) and E - cadherin : : mNeonGreen ( magenta ) during ventral furrow formation . ( A ) myosin - II accumulates normally at the apical surface and cells undergo an initial phase of constriction . The dashed rectangle indicates the region shown in ( B and C ) at higher magnification . Scale bar , 30 μm . Panels ( B , C ) show a sub - region within the ventral tissue at different time points . Initially a supracellular myosin - II network formed ( 0 s ) , before intercellular connections broke ( 13 s ) resulting in membrane tears ( dashed line ) with tissue retractions ( small arrow heads ) along the anterior - posterior axis . The intercellular network re - established ( 39 s , 65 s ) as new actomyosin fibers formed ( big arrow heads ) to reconnect neighboring cells . The intercellular network broke again causing tears ( dashed lines ) at different positions ( 104 s ) . Scale bars , 5 μm . ( D , E ) Confocal images showing the myosin - II marker Sqh : : mCherry ( green ) , E - cadherin : : mNeonGreen ( magenta ) and the segmented membrane ( red ) of a single cell at different time points in a control embryo undergoing ratcheted constriction ( D ) and in a βH - spectrin knock - down embryo undergoing non - ratcheted pulsations ( E ) . Scale bars , 2 . 5 μm . ( F ) Heatmaps showing the apical surface of individual cells over time of control embryos ( left ) and βH - spectrin knock - down embryos ( right ) normalized to the initial time point ( nControl = 233 ; nβH - spectrinKD = 368 ; of 5 different embryos each ) . The color - coded scale bar is depicted on the left . Dark blue indicates a cell area bigger than the initial value ( > 1 ) and yellow indicates an area constricted to less than 1 / 5 of the initial area ( < 0 . 2 ) . Three different cell behaviors in βH - spectrin knock - down embryos were identified : cells that constricted , cells that showed impaired constriction and cells that expanded . ( G ) Quantification of the percentage of cells that constricted ( to an area < 50 % , yellow ) , cells that showed impaired constriction ( to an area > 50 and < 100 % , green ) and cells that expanded ( to an area > 100 % , blue ) after 7 min in control embryos and in βH - spectrin knock - down embryos . ( H , I ) Graph showing the average area over time of control cells ( H , n = 235 cells ) and cells in βH - spectrin knock - down embryos ( I ) that constricted ( yelow , n = 274 ) , of cells that showed impaired constriction ( green , n = 232 ) and of cells that expanded ( blue , n = 32 ) . The lines indicate the mean value and the semi - transparent region the standard deviation . ( J ) Boxplots showing the distribution of apical cell area values 7 . 5 min after the onset of ventral furrow formation of ventral cells in control embryos , of ventral cells in βH - spectrin knock - down embryos that constricted , and ventral cells in βH - spectrin knock - down embryos that displayed impaired constriction and expanded . Sample numbers are equal to I . The values are presented as percentage of initial cell area . ANOVA result ( all data points ) : F ( 3 , 717 ) = 763 . 9 , p = 9 . 6e - 223 ; Cohen ' s d compared to the control : dconstricted = 0 . 1 , dimpaired = 1 . 5 , dexpanded = 3 . 7 . Due to a big effect size ( high sample number ) , statistical significance was assessed based on Cohen ' s d . A Cohen ' s d < 0 . 5 was considered not significant ( n . s . ) . ( K ) Quantification of the peak constriction rate and expansion rate relative to control cells . Control cells and cells in βH - spectrin knock - down embryos that showed impaired constriction or expanded were compared . The mean value and standard deviation are shown . Please note the logarithmic scale . While in βH - spectrin knock - down cells the constriction rate was ~ 24 % lower ( mean value of 0 . 8 ) compared to control cells , the expansion rate increased to ~ 60 % ( mean value of 1 . 6 ) . The absolute change of the expansion rate was 2 . 5 times higher than the change of the constriction rate . ANOVA result ( all data points ) : F ( 3 , 717 ) = 349 . 2 , p = 9 . 7e - 140 ; pairwise comparison of βH - spectrin knock - down cells to control using Cohen ' s d > 1 . 4 .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of the apical cell surface of a βH-spectrin knock-down embryo showing the myosin-II marker Sqh::mCherry (green) and E-cadherin::mNeonGreen (magenta) during ventral furrow formation. (A) myosin-II accumulates normally at the apical surface and cells undergo an initial phase of constriction. The dashed rectangle indicates the region shown in (B and C) at higher magnification. Scale bar, 30 μm. Panels (B,C) show a sub-region within the ventral tissue at different time points. Initially a supracellular myosin-II network formed (0 s), before intercellular connections broke (13 s) resulting in membrane tears (dashed line) with tissue retractions (small arrow heads) along the anterior-posterior axis. The intercellular network re-established (39 s, 65 s) as new actomyosin fibers formed (big arrow heads) to reconnect neighboring cells. The intercellular network broke again causing tears (dashed lines) at different positions (104 s). Scale bars, 5 μm.(D,E) Confocal images showing the myosin-II marker Sqh::mCherry (green), E-cadherin::mNeonGreen (magenta) and the segmented membrane (red) of a single cell at different time points in a control embryo undergoing ratcheted constriction (D) and in a βH-spectrin knock-down embryo undergoing non-ratcheted pulsations (E). Scale bars, 2.5 μm.(F) Heatmaps showing the apical surface of individual cells over time of control embryos (left) and βH-spectrin knock-down embryos (right) normalized to the initial time point (nControl=233; nβH-spectrinKD=368; of 5 different embryos each). The color-coded scale bar is depicted on the left. Dark blue indicates a cell area bigger than the initial value (>1) and yellow indicates an area constricted to less than 1/5 of the initial area (<0.2). Three different cell behaviors in βH-spectrin knock-down embryos were identified: cells that constricted, cells that showed impaired constriction and cells that expanded.(G) Quantification of the percentage of cells that constricted (to an area < 50%, yellow), cells that showed impaired constriction (to an area >50 and <100%, green) and cells that expanded (to an area >100%, blue) after 7 min in control embryos and in βH-spectrin knock-down embryos.(H,I) Graph showing the average area over time of control cells (H, n=235 cells) and cells in βH-spectrin knock-down embryos (I) that constricted (yelow, n=274), of cells that showed impaired constriction (green, n=232) and of cells that expanded (blue, n=32). The lines indicate the mean value and the semi-transparent region the standard deviation.(J) Boxplots showing the distribution of apical cell area values 7.5 min after the onset of ventral furrow formation of ventral cells in control embryos, of ventral cells in βH-spectrin knock-down embryos that constricted, and ventral cells in βH-spectrin knock-down embryos that displayed impaired constriction and expanded. Sample numbers are equal to I. The values are presented as percentage of initial cell area. ANOVA result (all data points): F(3,717)=763.9, p=9.6e-223; Cohen's d compared to the control: dconstricted=0.1, dimpaired=1.5, dexpanded=3.7. Due to a big effect size (high sample number), statistical significance was assessed based on Cohen's d. A Cohen's d < 0.5 was considered not significant (n. s.).(K) Quantification of the peak constriction rate and expansion rate relative to control cells. Control cells and cells in βH-spectrin knock-down embryos that showed impaired constriction or expanded were compared. The mean value and standard deviation are shown. Please note the logarithmic scale. While in βH-spectrin knock-down cells the constriction rate was ~24% lower (mean value of 0.8) compared to control cells, the expansion rate increased to ~60% (mean value of 1.6). The absolute change of the expansion rate was 2.5 times higher than the change of the constriction rate. ANOVA result (all data points): F(3,717)=349.2, p=9.7e-140; pairwise comparison of βH-spectrin knock-down cells to control using Cohen's d>1.4.


Figure 1D Quantification of the frequency of cell clusters with a strong polarised PAR - 3 centre . Data information : Data are presented as means ± SEM in ( D ) n = 3 experiments in ( D ) Two - way ANOVA analysis in ( D ) P values were listed in the graphs . F Line - scan profiles of PAR - 3 at the cell - cell interface of 2 - cell mESC doublets . Line scans were sectioned and fitted to each 5 % along the cell - cell interface length . Data information : ; individual line - scans and mean valued line - scans in ( F ) 15 - 32 cell clusters were analysed for each column in every experiment ; 15 cell doublets for each condition from one experiment in ( F ) H , I Representative images of centralised ZO - 1 puncta and polarised Golgi apparatus , labelled by GM130 ( H ) and quantification of the frequency of cell doublets with a polarised ZO - 1 or Golgi apparatus centre ( I ) . Data information : Data are presented as means ± SEM in ; ( I ) ; n = 3 experiments in ( I ) student ' s t - test analysis in ( I ) P values were listed in the graphs . scale bars : 10 µm . J , K Representative images of polarised centrosomes ( J ) , labelled with γ - tubulin and distance between centrosomes ( K ) normalised to the length of the long axis of the doublets in 2 - cell clusters . Data information : individual values in dots and median values in bars in ( K ) . student ' s t - test analysis in ( K ) . P values were listed in the graphs . scale bars : 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of the frequency of cell clusters with a strong polarised PAR-3 centre. Data information: Data are presented as means ± SEM in (D) n = 3 experiments in (D) Two-way ANOVA analysis in (D) P values were listed in the graphs.F Line-scan profiles of PAR-3 at the cell-cell interface of 2-cell mESC doublets. Line scans were sectioned and fitted to each 5% along the cell-cell interface length. Data information: ; individual line-scans and mean valued line-scans in (F) 15-32 cell clusters were analysed for each column in every experiment; 15 cell doublets for each condition from one experiment in (F)H, I Representative images of centralised ZO-1 puncta and polarised Golgi apparatus, labelled by GM130 (H) and quantification of the frequency of cell doublets with a polarised ZO-1 or Golgi apparatus centre (I). Data information: Data are presented as means ± SEM in ; (I); n = 3 experiments in (I) student's t-test analysis in (I) P values were listed in the graphs. scale bars: 10 µm.J, K Representative images of polarised centrosomes (J), labelled with γ-tubulin and distance between centrosomes (K) normalised to the length of the long axis of the doublets in 2-cell clusters. Data information: individual values in dots and median values in bars in (K). student's t-test analysis in (K). P values were listed in the graphs. scale bars: 10 µm.


Figure 2quantification of the frequency of cell clusters with polarised mCherry - PAR - 6B ( C ) in control and mitomycin division - blocked mESC live cells after 24 hours in Matrigel . Data information : Data in ( C ) are presented as means ± SEM . n = 3 experiments . At least 20 clusters were analysed for each column in every experiment . Two - way ANOVA analysis ; P values were listed in the graphs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2quantification of the frequency of cell clusters with polarised mCherry-PAR-6B (C) in control and mitomycin division-blocked mESC live cells after 24 hours in Matrigel. Data information: Data in (C) are presented as means ± SEM. n = 3 experiments. At least 20 clusters were analysed for each column in every experiment. Two-way ANOVA analysis; P values were listed in the graphs.


Figure 3A , B Immunofluorescence of PAR - 3 ( A ) and proportions of mESC clusters with a strong PAR - 3 centre ( B ) in wild - type ( W4 ) and E - cadherin knock - out ( Cdh1 KO ) mESCs at 24 hrs in Matrigel . Data information : Data are presented as means ± SEM in ( B ) n = 3 experiments in ( B ) Two - way ANOVA analysis in ( B ) , P values were listed in the graphs . scale bars : 10 µm . C Line - scan profiles of PAR - 3 at the cell - cell interface in wild - type control , mitomycin C treated and Cdh1 KO control , mitomycin C treated 2 - cell doublets . D Ratio of PAR - 3 pixel intensity values at central and surrounding regions in > 2 - cell mESC clusters . Data information : individual and mean - valued line - scans in ( C ) individual cell cluster values ( small dots ) , mean experimental values ( large dots ) and means of 3 experiments ( bars ) ± SEM in ( D ) 17 - 50 cell clusters were analysed for each column in every experiment ; 15 doublets for each condition in ( C ) ; 17 - 30 cell clusters were analysed for each condition in every experiment in ( D ) ; Two - way ANOVA analysis in ( D ) , P values were listed in the graphs . scale bars : 10 µm . E , F Representative images of PAR - 3 immunofluorescence ( E ) and line - scan profiles of PAR - 3 at the cell - cell interface ( F ) in WT homogeneous or WT ( ES - E14 ) / Cdh1 KO chimeric mESC 2 - cell doublets . * , WT mESCs . Data information : individual and mean - valued line - scans in ( F ) ; 15 doublets for each condition in ( F ) G , H Representative images of ZO - 1 puncta and Golgi apparatus ( G ) and proportions of mESC doublets with central ZO - 1 puncta or polarised Golgi apparatus ( H ) in WT and Cdh1 KO mESC doublets . Data information : Data are presented as means ± SEM in ( H ) ; student ' s t - test analysis in ( H ) ; P values were listed in the graphs . scale bars : 10 µm . I Distance between centrosomes in cell doublets . The distance was normalised to the length of the long axis of the doubles . Data information : individual values in dots and median values in bars in ( I ) ; 35 - 40 doublets in each condition in ( I ) Two - way ANOVA analysis in ( I ) P values were listed in the graphs . J Fluorescence recovery after photobleaching ( FRAP ) of E - cadherin - eGFP at the centre - most or side 2 µm regions of division - blocked mESC doublets ' cell - cell interfaces . K Mobile fraction of E - cadherin - eGFP calculated from the plotting of ( J ) . Data information : exponential association fitting curves ± SD in ( J ) ; means ± SD in ( K ) . 35 - 40 doublets in each condition in ( I ) ; 15 - 18 doublets for each condition in ( J ) & ( K ) . Two - way ANOVA analysis in ( K ) ; P values were listed in the graphs .

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

Figure 3A, B Immunofluorescence of PAR-3 (A) and proportions of mESC clusters with a strong PAR-3 centre (B) in wild-type (W4) and E-cadherin knock-out (Cdh1 KO) mESCs at 24 hrs in Matrigel. Data information: Data are presented as means ± SEM in (B) n = 3 experiments in (B) Two-way ANOVA analysis in (B), P values were listed in the graphs. scale bars: 10 µm.C Line-scan profiles of PAR-3 at the cell-cell interface in wild-type control, mitomycin C treated and Cdh1 KO control, mitomycin C treated 2-cell doublets. D Ratio of PAR-3 pixel intensity values at central and surrounding regions in >2-cell mESC clusters. Data information: individual and mean-valued line-scans in (C) individual cell cluster values (small dots), mean experimental values (large dots) and means of 3 experiments (bars) ± SEM in (D) 17-50 cell clusters were analysed for each column in every experiment; 15 doublets for each condition in (C); 17-30 cell clusters were analysed for each condition in every experiment in (D); Two-way ANOVA analysis in (D), P values were listed in the graphs. scale bars: 10 µm.E, F Representative images of PAR-3 immunofluorescence (E) and line-scan profiles of PAR-3 at the cell-cell interface (F) in WT homogeneous or WT (ES-E14) /Cdh1 KO chimeric mESC 2-cell doublets. *, WT mESCs. Data information: individual and mean-valued line-scans in (F); 15 doublets for each condition in (F)G, H Representative images of ZO-1 puncta and Golgi apparatus (G) and proportions of mESC doublets with central ZO-1 puncta or polarised Golgi apparatus (H) in WT and Cdh1 KO mESC doublets. Data information: Data are presented as means ± SEM in (H); student's t-test analysis in (H); P values were listed in the graphs. scale bars: 10 µm.I Distance between centrosomes in cell doublets. The distance was normalised to the length of the long axis of the doubles. Data information: individual values in dots and median values in bars in (I); 35-40 doublets in each condition in (I) Two-way ANOVA analysis in (I) P values were listed in the graphs.J Fluorescence recovery after photobleaching (FRAP) of E-cadherin-eGFP at the centre-most or side 2 µm regions of division-blocked mESC doublets' cell-cell interfaces. K Mobile fraction of E-cadherin-eGFP calculated from the plotting of (J). Data information: exponential association fitting curves ± SD in (J); means ± SD in (K). 35-40 doublets in each condition in (I); 15-18 doublets for each condition in (J) & (K). Two-way ANOVA analysis in (K); P values were listed in the graphs.


Figure 4A - C Immunofluorescence of PAR - 3 and P - cadherin ( A ) , proportions of mESC clusters with a positive PAR - 3 centre ( B ) and line - scans of PAR - 3 at the cell - cell interface ( C ) in control , P - cadherin knock - down by RNAi , mitomycin treated and P - cadherin knock - down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel . Data information : Data are presented as means ± SEM in ( B ) ; individual and mean - valued line - scans in ( C ) n = 3 experiments in ( B ) , 15 - 21 clusters were analysed for each column in every experiment ; 15 - 21 line - scans in ( C ) Two - way ANOVA analysis in ( B ) P values were listed in the graphs . scale bars : 5 µm . D - F Immunofluorescence of PAR - 3 and JAM - A ( D ) , proportions of mESC clusters with a positive PAR - 3 centre ( E ) and line - scans of PAR - 3 at the cell - cell interface ( F ) in control , JAM - A knock - down by RNAi , mitomycin treated and JAM - A knock - down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel . Data information : Data are presented as means ± SEM in ( E ) , individual and mean - valued line - scans in ( F ) n = 3 experiments in ( E ) , 15 - 21 clusters were analysed for each column in every experiment ; 15 - 21 line - scans in ( F ) , Two - way ANOVA analysis in ( E ) , P values were listed in the graphs . scale bars : 5 µm . G - I Immunofluorescence of PAR - 3 and Nectin - 2 ( G ) , proportions of mESC clusters with a positive PAR - 3 centre ( H ) and line - scans of PAR - 3 at the cell - cell interface ( I ) in control , Nectin - 2 knock - down by RNAi , mitomycin treated and Nectin - 2 knock - down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel . Data information : Data are presented as means ± SEM in ( H ) ; individual and mean - valued line - scans in ( I ) . n = 3 experiments in ( H ) , 15 - 21 clusters were analysed for each column in every experiment ; 15 - 21 line - scans in ( I ) . Two - way ANOVA analysis in ( H ) ; P values were listed in the graphs . scale bars : 5 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Immunofluorescence of PAR-3 and P-cadherin (A), proportions of mESC clusters with a positive PAR-3 centre (B) and line-scans of PAR-3 at the cell-cell interface (C) in control, P-cadherin knock-down by RNAi, mitomycin treated and P-cadherin knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (B) ; individual and mean-valued line-scans in (C) n = 3 experiments in (B), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (C) Two-way ANOVA analysis in (B) P values were listed in the graphs. scale bars: 5 µm.D-F Immunofluorescence of PAR-3 and JAM-A (D), proportions of mESC clusters with a positive PAR-3 centre (E) and line-scans of PAR-3 at the cell-cell interface (F) in control, JAM-A knock-down by RNAi, mitomycin treated and JAM-A knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (E), individual and mean-valued line-scans in (F) n = 3 experiments in (E), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (F), Two-way ANOVA analysis in (E), P values were listed in the graphs. scale bars: 5 µm.G-I Immunofluorescence of PAR-3 and Nectin-2 (G), proportions of mESC clusters with a positive PAR-3 centre (H) and line-scans of PAR-3 at the cell-cell interface (I) in control, Nectin-2 knock-down by RNAi, mitomycin treated and Nectin-2 knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (H); individual and mean-valued line-scans in (I). n = 3 experiments in (H), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (I). Two-way ANOVA analysis in (H); P values were listed in the graphs. scale bars: 5 µm.


Figure 5A , B Immunofluorescence of PAR - 3 ( A ) and proportions of mESC doublets with polarised PAR - 3 ( B ) in wild - type ( ES - E14 ) and E - cadherin knock - out ( Cdh1 KO ) cells at 30 hours in 0 . 5 % agarose . Data information : Data are presented as means ± SEM in ( B ) ; n = 3 experiments in ( B ) Two - way ANOVA analysis in ( B ) P values were listed in the graphs . scale bars : 5 µm . C Immunofluorescence of PAR - 3 in cell division blocked mESCs cultured on E - cadherin or Fibronectin coated glass topped with or without Matrigel for 24 hours . Data information : scale bars : 5 µm . D Heatmap of PAR - 3 in cells from one experiment of ( C ) . Squared frames were fitted to the main bodies of the cells . A pixel was the average value in each 10 % along the width of the frames . The heatmaps were stacks of 15 cells in one experiment . E PAR - 3 levels at the central 20 % of the heatmaps from three experiments of ( C ) . F Ratios between PAR - 3 in the 2 . 5 µm diameter central core and whole cell surface from three experiments of ( C ) . Data information : ; individual cell values ( small dots ) , mean experimental values ( large dots ) and means of experiments ± SEM ( bars ) in ( E ) & ( F ) . n = 3 experiments in ( E ) , ( F ) ; at least 15 clusters were analysed for each column in every experiment ; 15 - 20 cells for each column in every experiment of ( E ) & ( F ) . Two - way ANOVA analysis in ( E ) , ( F ) ; P values were listed in the graphs . scale bars : 5 µm .

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

Figure 5A, B Immunofluorescence of PAR-3 (A) and proportions of mESC doublets with polarised PAR-3 (B) in wild-type (ES-E14) and E-cadherin knock-out (Cdh1 KO) cells at 30 hours in 0.5% agarose. Data information: Data are presented as means ± SEM in (B); n = 3 experiments in (B) Two-way ANOVA analysis in (B) P values were listed in the graphs. scale bars: 5 µm.C Immunofluorescence of PAR-3 in cell division blocked mESCs cultured on E-cadherin or Fibronectin coated glass topped with or without Matrigel for 24 hours. Data information: scale bars: 5 µm.D Heatmap of PAR-3 in cells from one experiment of (C). Squared frames were fitted to the main bodies of the cells. A pixel was the average value in each 10% along the width of the frames. The heatmaps were stacks of 15 cells in one experiment. E PAR-3 levels at the central 20% of the heatmaps from three experiments of (C). F Ratios between PAR-3 in the 2.5 µm diameter central core and whole cell surface from three experiments of (C). Data information: ; individual cell values (small dots), mean experimental values (large dots) and means of experiments ± SEM (bars) in (E) & (F). n = 3 experiments in (E), (F); at least 15 clusters were analysed for each column in every experiment; 15 - 20 cells for each column in every experiment of (E) & (F). Two-way ANOVA analysis in (E), (F); P values were listed in the graphs. scale bars: 5 µm.


Figure 6D Percentage of cell clusters with different cavities relative to total cell clusters at different time points . The analysis was compared between the closed / rosette category among the conditions . E Percentage of cell clusters with closed cavities relative to total cell clusters with cavities calculated from ( D ) . Data information : Data are presented as means ± SEM in ( D ) & ( E ) . n = 20 ( 48 hours WT & Cdh1 KO ) , 30 ( 72 hours WT ) and 32 ( 72 hours Cdh1 KO ) images in ( A ) ; 3 experiments in ( D ) & ( E ) , at least 25 clusters were analysed for each column in every experiment . Two - way ANOVA analysis in ( D ) & ( E ) ; P values were listed in the graphs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6D Percentage of cell clusters with different cavities relative to total cell clusters at different time points. The analysis was compared between the closed/rosette category among the conditions. E Percentage of cell clusters with closed cavities relative to total cell clusters with cavities calculated from (D). Data information: Data are presented as means ± SEM in (D) & (E). n = 20 (48 hours WT & Cdh1 KO), 30 (72 hours WT) and 32 (72 hours Cdh1 KO) images in (A); 3 experiments in (D) & (E), at least 25 clusters were analysed for each column in every experiment. Two-way ANOVA analysis in (D) & (E); P values were listed in the graphs.


Figure 1Immunoblot of whole cell lysates from multiple human lineage - committed cell - lines using a C - terminal anti - JARID2 antibody . The size of canonical form is 140kDa but an additional strong band was detected at around ~ 80kDa . The blot is divided according to cell - types , which are indicated in the figure . qPCR measurements of JARID2 mRNA levels in different cell lines . The RNA levels were measured relative to 18S rRNA . Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates . The data is represented as mean ± SE . Immunoblot showing that the ~ 80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA . Non - silencing control ( NSC ) was used as a transfection control .

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

Figure 1Immunoblot of whole cell lysates from multiple human lineage-committed cell-lines using a C-terminal anti-JARID2 antibody. The size of canonical form is 140kDa but an additional strong band was detected at around ~80kDa. The blot is divided according to cell-types, which are indicated in the figure.qPCR measurements of JARID2 mRNA levels in different cell lines. The RNA levels were measured relative to 18S rRNA. Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates. The data is represented as mean ± SE.Immunoblot showing that the ~80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA. Non-silencing control (NSC) was used as a transfection control.


Figure 2qPCR measurements of RNA levels ( n = 3 ) for the three isoforms of JARID2 in HaCaT cells . The RNA levels are plotted relative to 18S rRNA . Level of JARID2 isoform - 1 is significantly higher than that of isoform - 2 ( * * * P < 0 . 001 ) and isoform - 3 ( * * * * P < 0 . 0001 ) . Agarose gel showing RT - PCR products amplified using primers in exon3 and exon15 ( 2 . 9kb ) as well as RT - PCR product corresponding to JARID2 isoform - 1 ( 3 . 7kb ) amplified using the first ( exon 1 ) and the last exon ( exon18 ) . Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2 . CRISPR - Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in ( E ) . Immunoblot revealed that ~ 80kDa form was removed from JARID2 KO lines . Immunoblot after a full length JARID2 isoform - 1 was expressed from an exogenous vector in HaCaT cells showed an increase ( 2 - 3 fold ) in ~ 80kDa level . Immunoprecipitation using anti - JARID2 antibody and followed by mass spectrometry identification of the ~ 80kDa band detected JARID2 peptides spanning only C - terminus of JARID2 . The identified peptides are shown as red arrows on schematic of JARID2 sequence .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O 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 2qPCR measurements of RNA levels (n=3) for the three isoforms of JARID2 in HaCaT cells. The RNA levels are plotted relative to 18S rRNA. Level of JARID2 isoform-1 is significantly higher than that of isoform-2 (***P<0.001) and isoform-3 (****P<0.0001).Agarose gel showing RT-PCR products amplified using primers in exon3 and exon15 (2.9kb) as well as RT-PCR product corresponding to JARID2 isoform-1 (3.7kb) amplified using the first (exon 1) and the last exon (exon18). Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2.CRISPR-Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in (E). Immunoblot revealed that ~80kDa form was removed from JARID2 KO lines.Immunoblot after a full length JARID2 isoform-1 was expressed from an exogenous vector in HaCaT cells showed an increase (2-3 fold) in ~80kDa level.Immunoprecipitation using anti-JARID2 antibody and followed by mass spectrometry identification of the ~80kDa band detected JARID2 peptides spanning only C-terminus of JARID2. The identified peptides are shown as red arrows on schematic of JARID2 sequence.


Figure 3Immunoblot showing increase in expression levels of differentiation markers Involucrin ( IVL ) and Transglutaminase - 1 ( TGase - 1 ) as differentiation progressed indicating that keratinocyte differentiation protocol was successful . Protein levels on day 0 ( D0 ) , day 1 ( D1 ) , day 3 ( D3 ) and day 6 ( D6 ) of differentiation are shown . Immunoblot for JARID2 during D0 , D1 , D3 and D6 of differentiation ( as in B ) are shown . Effect of JARID2 removal on levels of differentiation markers Involucrin ( IVL ) , Keratin - 1 ( KRT1 ) and Keratin - 10 ( KRT10 ) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN - JARID2 . The effect of exogenous expression of full - length JARID2 ( FL - JARID2 ) in JARID2 knockout is also shown . Data from wildtype HaCaTs , two independent JARID2 knockout ( KO1 and KO2 ) HaCaT lines as well as KO cells exogenously expressing empty vector control , ΔN - JARID2 and FL - JARID2 are shown . Levels of Involucrin ( IVL ) , Keratin - 1 ( KRT1 ) and Keratin - 10 ( KRT10 ) mRNAs as above but measured on day 3 of differentiation .

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

Figure 3Immunoblot showing increase in expression levels of differentiation markers Involucrin (IVL) and Transglutaminase-1 (TGase-1) as differentiation progressed indicating that keratinocyte differentiation protocol was successful. Protein levels on day 0 (D0), day 1 (D1), day 3 (D3) and day 6 (D6) of differentiation are shown. Immunoblot for JARID2 during D0, D1, D3 and D6 of differentiation (as in B) are shown.Effect of JARID2 removal on levels of differentiation markers Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN-JARID2. The effect of exogenous expression of full-length JARID2 (FL-JARID2) in JARID2 knockout is also shown. Data from wildtype HaCaTs, two independent JARID2 knockout (KO1 and KO2) HaCaT lines as well as KO cells exogenously expressing empty vector control, ΔN-JARID2 and FL-JARID2 are shown. Levels of Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as above but measured on day 3 of differentiation.


Figure 4A metagene plot showing enrichment of full - length JARID2 in human induced pluripotent cells at down - regulated genes ( blue ) as compared to up - regulated genes ( grey ) in JARID2 KO cells . The plot is centered on Transcription Start Sites ( TSS ) of genes and distance from TSS is indicated on the x - axis . Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down - regulated genes ( blue ) as compared to up - regulated genes ( grey ) in JARID2 KO cells . The plots are centered on Transcription Start Sites ( TSS ) of genes and distance from TSS is indicated on the x - axes . Levels of H3K27me3 in WT , JARID2 KO lines and JARID2 KO lines expressing ΔN - JARID2 . Histone H3 and Ponceau staining is used as a loading control . A bar - plot showing percentage of down - and up - regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O 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 metagene plot showing enrichment of full-length JARID2 in human induced pluripotent cells at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plot is centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axis.Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plots are centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axes.Levels of H3K27me3 in WT, JARID2 KO lines and JARID2 KO lines expressing ΔN-JARID2. Histone H3 and Ponceau staining is used as a loading control.A bar-plot showing percentage of down- and up-regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation.


Figure 1B . Dot blot of tau levels in media from HEK293T cells overexpressing tau and FLAG - DnaJC5 compared to empty vector and tau . Intracellular protein levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels shown below ; data are mean ± SEM , n = 8 , * * * p < 0 . 001 . C . Light / heavy ratio of tau peptides in label - free mass spectroscopy of media from HEK293T cells overexpressing tau or tau and DnaJC5 compared to 100 ng 15N labelled recombinant tau . Light peptides represent endogenous tau peptides . Data are mean ± SEM , n = 3 biological repeats , * * * 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B. Dot blot of tau levels in media from HEK293T cells overexpressing tau and FLAG-DnaJC5 compared to empty vector and tau. Intracellular protein levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=8, ***p<0.001.C. Light/heavy ratio of tau peptides in label-free mass spectroscopy of media from HEK293T cells overexpressing tau or tau and DnaJC5 compared to 100 ng 15N labelled recombinant tau. Light peptides represent endogenous tau peptides. Data are mean ± SEM, n=3 biological repeats, ***p<0.001.


Figure 2A . Western blot of endogenous tau levels from M17 neuroblastoma cells overexpressing FLAG - DnaJC5 or empty vector . Media was concentrated prior to analysis . Intracellular DnaJC5 was detected with FLAG antibody . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . B . Western blottau levels from concentrated media from ex - vivo organotypic slice cultures of wildtype mice overexpressing GFP - AAV9 or FLAG - DnaJC5 - AAV9 . DnaJC5 is detected by FLAG antibody . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . C . Western blottau levels from concentrated media from ex - vivo organotypic slice cultures of Cspa + / + or Cspa - / - mice . DnaJC5 was detected by DnaJC5 antibody ( Synaptic Systems ) . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

Figure 2A. Western blot of endogenous tau levels from M17 neuroblastoma cells overexpressing FLAG-DnaJC5 or empty vector. Media was concentrated prior to analysis. Intracellular DnaJC5 was detected with FLAG antibody. Quantification of extracellular tau levels are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.B. Western blottau levels from concentrated media from ex-vivo organotypic slice cultures of wildtype mice overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. DnaJC5 is detected by FLAG antibody. Quantification of extracellular tau levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.C. Western blottau levels from concentrated media from ex-vivo organotypic slice cultures of Cspa+/+ or Cspa-/- mice. DnaJC5 was detected by DnaJC5 antibody (Synaptic Systems). Quantification of extracellular tau levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


Figure 3A . FLAG - DnaJC5 overexpression facilitates extracellular release of wildtype ( WT ) , P301L and R406W tau by dot blot in HEK293T cells . Intracellular protein levels are shown by Western blot ; DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels shown below ; data are mean ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . B . Overexpression of FLAG - DnaJC5 induces extracellular release of wildtype ( WT ) and A53T α - synuclein in HEK293T cells . Extracellular protein levels shown by dot blot ( quantification below , mean ± SEM , n = 3 * * * p < 0 . 001 , * * p < 0 . 01 by one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis ) and intracellular proteins levels were analyzed by Western blot , DnaJC5 was detected by FLAG antibody . C . DnaJC5 overexpression induces extracellular release of GFP - tagged wildtype ( WT ) , A315T and Q343R TDP - 43 in HEK293T cells . Intracellular DnaJC5 was detected by FLAG antibody . Extracellular protein levels are analyzed by dot blot ( quantification below , mean ± SEM , n = 4 ) * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

Figure 3A. FLAG-DnaJC5 overexpression facilitates extracellular release of wildtype (WT), P301L and R406W tau by dot blot in HEK293T cells. Intracellular protein levels are shown by Western blot; DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.B. Overexpression of FLAG-DnaJC5 induces extracellular release of wildtype (WT) and A53T α-synuclein in HEK293T cells. Extracellular protein levels shown by dot blot (quantification below, mean ±SEM, n=3 ***p<0.001, ** p<0.01 by one-way analysis of variance with Tukey's multiple comparison post-hoc analysis) and intracellular proteins levels were analyzed by Western blot, DnaJC5 was detected by FLAG antibody.C. DnaJC5 overexpression induces extracellular release of GFP-tagged wildtype (WT), A315T and Q343R TDP-43 in HEK293T cells. Intracellular DnaJC5 was detected by FLAG antibody. Extracellular protein levels are analyzed by dot blot (quantification below, mean ±SEM, n=4) ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


Figure 4A . Dot blot of tau levels in media of cells overexpressing the phospho - deficient FLAG - DnaJC5 S10A compared to FLAG - DnaJC5 WT . Intracellular levels shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of tau levels are mean ± SEM , n = 6 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . B . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated tau release in HEK293T cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected by FLAG antibody . C . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated α - synuclein release in M17 cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 by one - way ANOVA with a Tukey ' s post - hoc analysis . DnaJC5 is detected by FLAG antibody . D . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated TDP - 43 release in M17 cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected by FLAG antibody .

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

Figure 4A. Dot blot of tau levels in media of cells overexpressing the phospho-deficient FLAG-DnaJC5 S10A compared to FLAG-DnaJC5 WT. Intracellular levels shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.B. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated tau release in HEK293T cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody.C. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated α-synuclein release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 by one-way ANOVA with a Tukey's post-hoc analysis. DnaJC5 is detected by FLAG antibody.D. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated TDP-43 release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody.


Figure 5A . Confocal microscopy of tau ( red pseudocolor ) and Hsp70 ( green pseudocolor ) colocalization ( merged image ) levels in wild type primary neurons overexpressing GFP - AAV9 or FLAG - DnaJC5 - AAV9 . Boxed area are 5X digital zoom on a 60X lens shown below . Scale bars are 20 µm and 5 µm as indicated . Only neurons overexpressing virus were imaged . B . Quantification of tau - Hsp70 co - localization ( mean Pearson ' s coefficient ± SEM , n = 18 , * p < 0 . 05 , one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis ) . C . Western blot of immunoprecipitatedtau from HEK293T reveals tau exists in an intracellular complex with DnaJC5 and Hsc70 . DnaJC5 was detected with FLAG antibody . D . Western blot of HEK293T intracellular lysates show the DnaJC5 / Hsc70 / tau complex is disrupted by the addition of increasing amounts of YM - 01 ( 0 , 10 , 30 , 100 µM ) in a dose - dependent fashion . DnaJC5 was detected by FLAG antibody . E . The DnaJC5 / Hsc70 complex is disrupted when Hsc70 activity is inhibited by a dominant - negative point mutation , E175S . Western blot of immunoprecipitated DnaJC5 from HEK293T cells overexpressing WT or DN E175S Hsc70 . DnaJC5 is immunopreicipitated by DnaJC5 antibody ( Synaptic Systems ) . DnaJC5 was detected on Western blot by FLAG antibody . F . Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM - 01 . Quantification of extracellular tau levels are mean ± SEM , n = 6 , * p < 0 . 05 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . Intracellular levels shown by Western blot . DnaJC5 was detected on Western blot by FLAG antibody . G . Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM - 01 ( 10µM ) . FLAG - DnaJC5 was transduced into neurons using AAV9 . Quantification of extracellular tau levels are mean ± SEM , n = 6 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected on Western blot by FLAG antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal microscopy of tau (red pseudocolor) and Hsp70 (green pseudocolor) colocalization (merged image) levels in wild type primary neurons overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. Boxed area are 5X digital zoom on a 60X lens shown below. Scale bars are 20 µm and 5 µm as indicated. Only neurons overexpressing virus were imaged.B. Quantification of tau-Hsp70 co-localization (mean Pearson's coefficient ± SEM, n=18, *p <0.05, one-way analysis of variance with Tukey's multiple comparison post-hoc analysis).C. Western blot of immunoprecipitatedtau from HEK293T reveals tau exists in an intracellular complex with DnaJC5 and Hsc70. DnaJC5 was detected with FLAG antibody.D. Western blot of HEK293T intracellular lysates show the DnaJC5/Hsc70/tau complex is disrupted by the addition of increasing amounts of YM-01 (0, 10, 30, 100 µM) in a dose-dependent fashion. DnaJC5 was detected by FLAG antibody.E. The DnaJC5/Hsc70 complex is disrupted when Hsc70 activity is inhibited by a dominant-negative point mutation, E175S. Western blot of immunoprecipitated DnaJC5 from HEK293T cells overexpressing WT or DN E175S Hsc70. DnaJC5 is immunopreicipitated by DnaJC5 antibody (Synaptic Systems). DnaJC5 was detected on Western blot by FLAG antibody.F. Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM-01. Quantification of extracellular tau levels are mean ± SEM, n=6, *p<0.05 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. Intracellular levels shown by Western blot. DnaJC5 was detected on Western blot by FLAG antibody.G. Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM-01 (10µM). FLAG-DnaJC5 was transduced into neurons using AAV9. Quantification of extracellular tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected on Western blot by FLAG antibody.


Figure 6A . Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG - DnaJC5 reduces extracellular tau levels as shown by dot blot . Intracellular levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . B . Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG - DnaJC5 reduces extracellular α - synuclein levels as shown by dot blot . Intracellular levels are shown by Western blotDnaJC5 was detected by FLAG antibody . Quantification of extracellular synuclein levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . C . Dominant - negative Hsc70 E175S blocks tau release when overexpressed in HEK293T cells as shown by dot blot . Intracellular levels are shown by Western blot . Quantification of extracellular tau levels is shown as average ± SEM , n = 4 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . D . Knockdown of Hsc70 reduces extracellular tau release ( dot blot ) in HEK293T cells overexpressing FLAG - DnaJC5 . Intracellular levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . E . A mutant tau that does not bind Hsc70 is not released when FLAG - DnaJC5 is overexpressed in HEK293T cells . Dot blot and quantification of extracellular tau levels are shown . Intracellular protein levels are shown with representative Western blot , DnaJC5 was detected by FLAG antibody . Data are mean ± SEM , n = 3 p < 0 . 001 . one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . F . Dot blot of Hsp70 isoform overexpression effect on extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot . HSPA variants were detected with V5 antibody . Data are mean ± SEM , n = 3 p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . G . Dot blot of different FLAG - DnaJ overexpression effect on extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot . DnaJ levels were detected by FLAG antibody . Data are mean ± SEM , n = 3 ; * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . H . Dot blot of increasing FLAG - DnaJC7 levels on FLAG - DnaJC5 overexpression - mediated extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot , DnaJ proteins were detected by FLAG antibody . Quantification of extracellular levels are mean ± SEM , n = 4 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular tau levels as shown by dot blot. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.B. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular α-synuclein levels as shown by dot blot. Intracellular levels are shown by Western blotDnaJC5 was detected by FLAG antibody. Quantification of extracellular synuclein levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.C. Dominant-negative Hsc70 E175S blocks tau release when overexpressed in HEK293T cells as shown by dot blot. Intracellular levels are shown by Western blot. Quantification of extracellular tau levels is shown as average ± SEM, n=4, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.D. Knockdown of Hsc70 reduces extracellular tau release (dot blot) in HEK293T cells overexpressing FLAG-DnaJC5. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.E. A mutant tau that does not bind Hsc70 is not released when FLAG-DnaJC5 is overexpressed in HEK293T cells. Dot blot and quantification of extracellular tau levels are shown. Intracellular protein levels are shown with representative Western blot, DnaJC5 was detected by FLAG antibody. Data are mean ± SEM, n=3 p<0.001. one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.F. Dot blot of Hsp70 isoform overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. HSPA variants were detected with V5 antibody. Data are mean ± SEM, n=3 p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.G. Dot blot of different FLAG-DnaJ overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. DnaJ levels were detected by FLAG antibody. Data are mean ± SEM, n=3; *** p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.H. Dot blot of increasing FLAG-DnaJC7 levels on FLAG-DnaJC5 overexpression-mediated extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot, DnaJ proteins were detected by FLAG antibody. Quantification of extracellular levels are mean ± SEM, n=4.


Figure 1 ( B ) The Western blot analysis of GFP , Spd2 and αTub ( loading control ) in the brain extracts from wild type ( Oregon R , OR ) , Spd2WT - OE and Spd2DK - OE third instar larva . Representative immune blot images are shown on the left . The signal intensities of Spd2 bands were quantified and normalised against αTub values . The values relative to control of each biological replicate ( n = 2 ) and their means are shown as dots and bars , respectively , in a scatted dot plot on the right . The numbers above the bars indicate means ± Range . GFP - Spd2DK proteins in Spd2DK - OE larval brain extracts showed approximately 2 . 04 - fold higher accumulation than GFP - Spd2WT proteins in Spd2WT - OE brain extracts . ( C ) The Western blot analysis of Spd2 , CycB and αTub in OR , fzrRNAi , Spd2WT - RES and Spd2DK - RES brain extracts . The signal intensities of Spd2 bands were quantified and normalised against αTub values . The values relative to control and their means are presented in a scatted dot plot on the right ( biological replicate n = 2 ) . Means ± Range are shown above the bars . RFP - Spd2DK proteins in Spd2DK - RES larval brain extracts showed approximately 2 . 08 - fold higher accumulation than RFP - Spd2WT proteins in Spd2WT - RES brain extracts . ( D ) Larval brains of OR , Spd2WT - RES , fzrRNAi and Spd2WT - OE flies were stained by an anti - Spd2 antibody . Representative images of NBs are shown on the top . Scale bar : 10 µm . The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured , and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots ( see Materials and Methods for detail ) . The lines indicate means ± SD . The numbers of NBs ( n ) and the numbers of brains analysed ( N ) are control : n = 29 ( N = 6 ) , Spd2WT - RES : n = 30 ( N = 4 ) , fzrRNA : n = 30 ( N = 6 ) , and Spd2WT - OE : n = 27 ( N = 10 ) . P values were calculated using non - parametric two - tailed Mann - Whitney tests . Based on these results , the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype ( or control ) : Spd2WT - RES : fzrRNAi : Spd2DK - RES : Spd2WT - OE : Spd2DK - OE = 1 : 1 . 00 ~ 1 . 18 : 1 . 06 ~ 1 . 29 : 2 . 08 ~ 2 . 45 ( = 1 . 00 ~ 1 . 18 x 2 . 08 ) : 1 . 93 ~ 3 . 62 : 3 . 94 ~ 7 . 38 ( = 1 . 93 ~ 3 . 62 x 2 .

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

Figure 1(B) The Western blot analysis of GFP, Spd2 and αTub (loading control) in the brain extracts from wild type (Oregon R, OR), Spd2WT-OE and Spd2DK-OE third instar larva. Representative immune blot images are shown on the left. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control of each biological replicate (n = 2) and their means are shown as dots and bars, respectively, in a scatted dot plot on the right. The numbers above the bars indicate means ± Range. GFP-Spd2DK proteins in Spd2DK-OE larval brain extracts showed approximately 2.04-fold higher accumulation than GFP-Spd2WT proteins in Spd2WT-OE brain extracts. (C) The Western blot analysis of Spd2, CycB and αTub in OR, fzrRNAi, Spd2WT-RES and Spd2DK-RES brain extracts. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control and their means are presented in a scatted dot plot on the right (biological replicate n = 2). Means ± Range are shown above the bars. RFP-Spd2DK proteins in Spd2DK-RES larval brain extracts showed approximately 2.08-fold higher accumulation than RFP-Spd2WT proteins in Spd2WT-RES brain extracts.(D) Larval brains of OR, Spd2WT-RES, fzrRNAi and Spd2WT-OE flies were stained by an anti-Spd2 antibody. Representative images of NBs are shown on the top. Scale bar: 10 µm. The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured, and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots (see Materials and Methods for detail). The lines indicate means ± SD. The numbers of NBs (n) and the numbers of brains analysed (N) are control: n= 29 (N = 6), Spd2WT-RES: n = 30 (N = 4), fzrRNA: n = 30 (N = 6), and Spd2WT-OE: n = 27 (N = 10). P values were calculated using non-parametric two-tailed Mann-Whitney tests. Based on these results, the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype (or control) : Spd2WT-RES: fzrRNAi: Spd2DK-RES: Spd2WT-OE: Spd2DK-OE = 1: 1.00~1.18: 1.06~1.29: 2.08~2.45 (= 1.00~1.18 x 2.08): 1.93~3.62: 3.94 ~ 7.38 (= 1.93~3.62 x 2.04


Figure 2 ( C ) In time lapse movies , NBs that showed an active centrosome that was attached to the cortex ( " Attached " ) and NBs with both two centrosomes being inactivated and detached from the cortex ( " Detached " ) in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs . The numbers of NBs analysed from at least four brains ( n ) are Control : 29 , fzrRNAi : 80 , Spd2WT - RES : 45 , Spd2DK - RES : 52 , Spd2WT - OE : 43 , Spd2DK - OE : 40 , Spd2WT - OE + fzrRNAi : 72 . p - values are fzrRNAi : 0 . 0069 , Spd2WT - RES : 0 . 2479 , Spd2DK - RES : 0 . 0116 , Spd2WT - OE : < 0 . 0001 , Spd2DK - OE : < 0 . 0001 , Spd2WT - OE + fzrRNAi : 0 . 0008 ( Pearson ' s chi - squared tests were performed between control and each condition ) . ( E ) The signal intensities of the indicated centriolar and PCM proteins at two centrosomes were quantified in individual interphase NBs in Spd2WT - RES and Spd2DK - RES larval brains , and their asymmetric distributions of their centrosomal signals between the two centrosomes are presented as Asymmetric Indexes ( see Materials and Method ) in a scatted dot plot . Red bars represent means ± SD . The numbers of NBs analysed from at least 3 different brains of Spd2WT - RES and Spd2DK - RES ( n ) were 40 and 40 for Asl , 40 and 41 for RFP - Spd2 , 42 and 37 for Cnn , 40 and 41 for γTub , and 40 and 40 for AurA .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(C) In time lapse movies, NBs that showed an active centrosome that was attached to the cortex ("Attached") and NBs with both two centrosomes being inactivated and detached from the cortex ("Detached") in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs. The numbers of NBs analysed from at least four brains (n) are Control: 29, fzrRNAi: 80, Spd2WT-RES: 45, Spd2DK-RES: 52, Spd2WT-OE: 43, Spd2DK-OE: 40, Spd2WT-OE + fzrRNAi: 72. p-values are fzrRNAi: 0.0069, Spd2WT-RES: 0.2479, Spd2DK-RES: 0.0116, Spd2WT-OE: <0.0001, Spd2DK-OE: <0.0001, Spd2WT-OE + fzrRNAi: 0.0008 (Pearson's chi-squared tests were performed between control and each condition).(E) The signal intensities of the indicated centriolar and PCM proteins at two centrosomes were quantified in individual interphase NBs in Spd2WT-RES and Spd2DK-RES larval brains, and their asymmetric distributions of their centrosomal signals between the two centrosomes are presented as Asymmetric Indexes (see Materials and Method) in a scatted dot plot. Red bars represent means ± SD. The numbers of NBs analysed from at least 3 different brains of Spd2WT-RES and Spd2DK-RES (n) were 40 and 40 for Asl, 40 and 41 for RFP-Spd2, 42 and 37 for Cnn, 40 and 41 for γTub, and 40 and 40 for AurA.


Figure 3 ( D ) Division axis deviations were separately analysed in NBs with apically attached interphase centrosomes ( " attached " ) and those with centrosomes being detached from the cortex ( " detached " ) for each line , using the same dataset as in Fig . 3C . The same control data as in Fig . 3C is shown for reference . fzrRNA detached ( mean ± SD : 34 . 62 ° ± 23 . 39 ) , Spd2DK - OE detached ( 64 . 01 ° ± 33 . 93 ) , and Spd2WT - OE + fzrRNAi attached ( 39 . 55 ° ± 27 . 08 ) and detached ( 59 . 08 ° ± 34 . 88 ) brains showed statistically significant differences from control , while NBs with cortically attached centrosomes in any of the lines did not show significant differences . n : the number of NB divisions analysed from at least four brains for each line . p - values were calculated using Mann - Whitney U tests comparing with control and are shown above the dots .

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

Figure 3(D) Division axis deviations were separately analysed in NBs with apically attached interphase centrosomes ("attached") and those with centrosomes being detached from the cortex ("detached") for each line, using the same dataset as in Fig. 3C. The same control data as in Fig. 3C is shown for reference. fzrRNA detached (mean ± SD: 34.62° ± 23.39), Spd2DK-OE detached (64.01° ± 33.93), and Spd2WT-OE+fzrRNAi attached (39.55° ± 27.08) and detached (59.08° ± 34.88) brains showed statistically significant differences from control, while NBs with cortically attached centrosomes in any of the lines did not show significant differences. n: the number of NB divisions analysed from at least four brains for each line. p-values were calculated using Mann-Whitney U tests comparing with control and are shown above the dots.


Figure 4 ( B ) Selected images from time - lapse microscopy of NBs in control ( wor > mCh - Tub , pUbq - GFP - Fzr , top panels ) and Spd2DK - OE ( lower panels ) larval brains ( Movies EV1 , EV10 ) . In the control NB , the daughter centrosomes ( blue arrowheads ) first matured ( - 5 . 0 min ) and was segregated into the daughter NB ( 12 . 0 min ) , while the mother centrosome ( yellow arrowheads ) matured a little later ( 0 . 0 min ) and segregated into the GMC . In contrast , in the Spd2DK - OE NB , both centrosomes lost microtubule nucleation activity in interphase ( red arrowheads ) . The centrosome that matured earlier ( blue arrowheads , - 6 . 0 min ) was incorrectly segregated into the GMC ( 12 . 0 min ) while the centrosome that matured later ( yellow arrowheads , 0 . 0 min ) was segregated into the NB ( 12 . 0 min ) . Dotted white lines outline the dividing NBs and the newly formed GMC , which are also marked by asterisks .

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

Figure 4(B) Selected images from time-lapse microscopy of NBs in control (wor>mCh-Tub, pUbq-GFP-Fzr, top panels) and Spd2DK-OE (lower panels) larval brains (Movies EV1, EV10). In the control NB, the daughter centrosomes (blue arrowheads) first matured (-5.0 min) and was segregated into the daughter NB (12.0 min), while the mother centrosome (yellow arrowheads) matured a little later (0.0 min) and segregated into the GMC. In contrast, in the Spd2DK-OE NB, both centrosomes lost microtubule nucleation activity in interphase (red arrowheads). The centrosome that matured earlier (blue arrowheads, -6.0 min) was incorrectly segregated into the GMC (12.0 min) while the centrosome that matured later (yellow arrowheads, 0.0 min) was segregated into the NB (12.0 min). Dotted white lines outline the dividing NBs and the newly formed GMC, which are also marked by asterisks.


Figure 5 ( C , D ) Representative images of interphase NBs in fixed wor > lacZ ( control , top panels ) and HA - Spd2WT - OE larval brains expressing Polo - GFP from the native promoter and stained for DAPI and Spd2 ( C ) . Asymmetric indexes of Polo - GFP in interphase NBs wor > lacZ ( control , top panels ) and HA - Spd2WT - OE larval brains ( D ) . 38 and 39 NBs ( n ) from at least three brains were analysed in each line . The asymmetric distribution of Polo - GFP at interphase centrosomes was unaffected by HA - Spd2WT overexpression . ( E ) Representative images of interphase NBs in fixed mCh - Spd2WT - RES ( top panels ) and mCH - Spd2DK - RES larval brains and stained for DAPI , γTub and Cnn . Both multichannel and single - channel images of mCh - Spd2 , γTub and Cnn are shown . Dotted yellow squares highlight centrosomes and their magnified images are shown in insets . Similar to Spd2DK - RES NBs , in mCh - Spd2CONS - RES NBs , Spd2 and γTub were more symmetrically accumulated at the two centrosomes than in mCh - Spd2WT - RES NBs , but Cnn was still asymmetrically distributed . ( G , H ) FRAP analyses of the centrosomal Spd2 fluorescent signals in Spd2WT - RES and Spd2DK - OE NBs . ( G ) Representative images from time - lapse movies ( Movies EV13 , EV14 ) of Spd2 fluorescent signals in Spd2WT - RES NBs ( top panels ) and Spd2DK - OE NBs ( lower panels ) upon photobleaching . ( H ) The recoveries of the fluorescent intensities of centrosomal Spd2 signals after photobleaching were measured in Spd2WT - RES ( n = 7 ) and Spd2DK - OE NBs ( n = 5 ) . Means ± SEM of the normalised fluorescent intensities are shown in a line graph . Centrosomal Spd2 signals recovered more fully after photobleaching in Spd2DK - OE NBs than in Spd2WT - RES NBs .

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

Figure 5(C, D) Representative images of interphase NBs in fixed wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains expressing Polo-GFP from the native promoter and stained for DAPI and Spd2 (C). Asymmetric indexes of Polo-GFP in interphase NBs wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains (D). 38 and 39 NBs (n) from at least three brains were analysed in each line. The asymmetric distribution of Polo-GFP at interphase centrosomes was unaffected by HA-Spd2WT overexpression.(E) Representative images of interphase NBs in fixed mCh-Spd2WT-RES (top panels) and mCH-Spd2DK-RES larval brains and stained for DAPI, γTub and Cnn. Both multichannel and single-channel images of mCh-Spd2, γTub and Cnn are shown. Dotted yellow squares highlight centrosomes and their magnified images are shown in insets. Similar to Spd2DK-RES NBs, in mCh-Spd2CONS-RES NBs, Spd2 and γTub were more symmetrically accumulated at the two centrosomes than in mCh-Spd2WT-RES NBs, but Cnn was still asymmetrically distributed.(G, H) FRAP analyses of the centrosomal Spd2 fluorescent signals in Spd2WT-RES and Spd2DK-OE NBs. (G) Representative images from time-lapse movies (Movies EV13, EV14) of Spd2 fluorescent signals in Spd2WT-RES NBs (top panels) and Spd2DK-OE NBs (lower panels) upon photobleaching. (H) The recoveries of the fluorescent intensities of centrosomal Spd2 signals after photobleaching were measured in Spd2WT-RES (n = 7) and Spd2DK-OE NBs (n = 5). Means ± SEM of the normalised fluorescent intensities are shown in a line graph. Centrosomal Spd2 signals recovered more fully after photobleaching in Spd2DK-OE NBs than in Spd2WT-RES NBs.


Figure 2 ( C ) Temporal analysis of antigen - specific CD8 + T cell responses . Peripheral blood was obtained on days 4 , 7 , 14 , 21 , 42 and 88 post - immunisation with WT ( triangles ; n = 3 - 9 ) , CSPSIINFEKL ( orange squares ; n = 4 - 8 ) or UIS4SIINFEKL ( blue circles ; n = 4 - 10 ) sporozoites , or no parasites ( diamonds ; n = 2 - 5 ) and stained for Kb - SIINFEKL + CD11ahi CD8 + T cells . Line graph shows mean values ( ± SEM ) from representative experiments . ( * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Welch ' s t - test comparing CSPSIINFEKL and UIS4SIINFEKL ) . See Appendix Table S1 for the number of mice used per timepoint and per group . See Appendix Table S4 for exact p - values . ( D - H ) C57BL / 6 mice ( n = 4 per group ) , which received 2x106 CFSE - labelled OT - I splenocytes , were immunised with 10 , 000 γ - radiation attenuated WT , CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously . 5 days later , mice were sacrificed , spleens harvested and splenocytes assessed for ( D ) CFSE dilution of CD8 + T cells , ( E ) CFSE dilution of antigen experienced Kb - SIINFEKL + CD11ahi CD8 + T cells and stained ex vivo ( F - H ) for effector CD8 + T cell surface markers . Shown are flow cytometry plots of Kb - SIINFEKL co - staining with markers of effector phenotypes : ( F ) CD11ahi , ( G ) CD62Llo and ( H ) CD49dhi .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(C) Temporal analysis of antigen-specific CD8+ T cell responses. Peripheral blood was obtained on days 4, 7, 14, 21, 42 and 88 post-immunisation with WT (triangles; n=3-9), CSPSIINFEKL (orange squares; n=4-8) or UIS4SIINFEKL (blue circles; n=4-10) sporozoites, or no parasites (diamonds; n=2-5) and stained for Kb-SIINFEKL+ CD11ahi CD8+ T cells. Line graph shows mean values (±SEM) from representative experiments. (*, p<0.05; **, p<0.01; ***, p<0.001; Welch's t-test comparing CSPSIINFEKL and UIS4SIINFEKL). See Appendix Table S1 for the number of mice used per timepoint and per group. See Appendix Table S4 for exact p-values.(D-H) C57BL/6 mice (n=4 per group), which received 2x106 CFSE-labelled OT-I splenocytes, were immunised with 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously. 5 days later, mice were sacrificed, spleens harvested and splenocytes assessed for (D) CFSE dilution of CD8+ T cells, (E) CFSE dilution of antigen experienced Kb-SIINFEKL+ CD11ahi CD8+ T cells and stained ex vivo (F-H) for effector CD8+ T cell surface markers. Shown are flow cytometry plots of Kb-SIINFEKL co-staining with markers of effector phenotypes: (F) CD11ahi, (G) CD62Llo and (H) CD49dhi.