Figure 3 Acridine Orange was added to living untreated ASPC - 1 and MiaPaCa - 2 cells and cells treated with 5 - FU , OMP or the combination of both for 30 minutes or 24 hours . Microscopical life images were taken at 525 nm ( green ) and 650 nm ( red ) to detect changes in the lysosomal pH value ( three images per plate , three plates per group ) . The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann - Whitney - U - test . Significant differences compared to control are marked by * . In ASPC - 1 cells , after 30 min of treatment , intralysosomal acidity increased upon treatment with OMP ( p : 0 . 0051 ) and 5 - FU + OMP ( p < 0 . 0001 ) . After 24 hours , the acidity is increased upon all treatment regimens ( 5 - FU - p : 0 . 0002 ; OMP - p < 0 . 00001 ; OMP + 5 - FU - p : 0 . 037 ) . In MiaPaCa - 2 cells the acidity is elevated after 30 min upon 5 - FU ( p : 0 . 005 ) and decreased after treatment with OMP ( p : 0 . 037 ) , 5 - FU ( p : 0 . 00026 ) and 5 - FU + OMP ( p : 0 . 011 ) after 24 hours .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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  Acridine Orange was added to living untreated ASPC-1 and MiaPaCa-2 cells and cells treated with 5-FU , OMP or the combination of both for 30 minutes or 24 hours. Microscopical life images were taken at 525 nm (green) and 650 nm (red) to detect changes in the lysosomal pH value (three images per plate, three plates per group). The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann-Whitney-U-test. Significant differences compared to control are marked by *. In ASPC-1 cells, after 30 min of treatment, intralysosomal acidity increased upon treatment with OMP (p:0.0051) and 5-FU+OMP (p<0.0001). After 24 hours, the acidity is increased upon all treatment regimens (5-FU - p:0.0002; OMP - p<0.00001; OMP+5-FU - p:0.037). In MiaPaCa-2 cells the acidity is elevated after 30 min upon 5-FU (p:0.005) and decreased after treatment with OMP (p:0.037), 5-FU (p:0.00026) and 5-FU+OMP (p:0.011) after 24 hours.


Figure 4 ( A ) ASPC - 1 cell without treatment ( 800 fold ) . ( B ) ASPC - 1 cell undergoing apoptosis upon 160 µg / ml OMP after 24 hours ( 800 fold ) . Vacuolisation of the cytoplasma and condensation of the nucleus are visible . ( C ) Phagophores and autophagosomes in a segment of an ASPC - 1 cell treated with omeprazole 80 µg / ml for 24 hours ( 2800fold enlargement ) . The phagophores are characterised by a cup - like shape ( white arrows ) . Autophagosomes are closed particles , the number of which is increased in treated cells ( black arrows ) . ( D ) Early phagophores and autophagosomes are also found in MiaPaCa - 2 cells treated with OMP 80 µg / ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex . In contrast to ASPC - 1 cells , early signs of apoptosis such as vacuolization , are also present . ( E ) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa - 2 and ASPC - 1 cells untreated or treated with 5 - FU , OMP or the combination of both for 24 hours with standard errors . Significant differences compared to control are marked by * . In ASPC - 1 cells there were significant differences compared to the control in the OMP group ( p : 0 . 03 ) and the 5 - FU + OMP group ( p : 0 . 03 ) . In MiaPaCa - 2 cells the 5 - FU + OMP group differed significantly from control ( p < 0 . 001 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ASPC-1 cell without treatment (800 fold). (B) ASPC-1 cell undergoing apoptosis upon 160 µg/ml OMP after 24 hours (800 fold). Vacuolisation of the cytoplasma and condensation of the nucleus are visible. (C) Phagophores and autophagosomes in a segment of an ASPC-1 cell treated with omeprazole 80 µg/ml for 24 hours (2800fold enlargement). The phagophores are characterised by a cup-like shape (white arrows). Autophagosomes are closed particles, the number of which is increased in treated cells (black arrows). (D) Early phagophores and autophagosomes are also found in MiaPaCa-2 cells treated with OMP 80 µg/ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex. In contrast to ASPC-1 cells, early signs of apoptosis such as vacuolization, are also present.(E) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa-2 and ASPC-1 cells untreated or treated with 5-FU, OMP or the combination of both for 24 hours with standard errors. Significant differences compared to control are marked by *. In ASPC-1 cells there were significant differences compared to the control in the OMP group (p: 0.03) and the 5-FU+OMP group (p: 0.03). In MiaPaCa-2 cells the 5-FU+OMP group differed significantly from control (p<0.001).


Figure 6 The ASPC - 1 cell line is shown without and upon various treatment regimens ( OMP , 5 - FU or 5 - FU + OMP combination ) . The nodes of this network symbolize metabolite signals , their colours correspond to relative signal intensity ( when compared to an external standard ) as indicated in the heatmap scale below . The signal intensity is linearly related to the intracellular concentration . The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale . The lines between the boxes symbolize strongly simplified metabolic pathways . The colours of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange ( p < 0 . 05 ) , red lines indicate p < 0 . 01 . The most obvious changes is that the PC / Cho ratio is significantly lower in the OMP group compared to the control group . Upon 5 - FU , the Cho / Acetate ratio decrease is the only significant change . In the 5 - FU + OMP group , there are several significant changes , i . e . the FACH2 / CH = CH ratio is significantly higher . Moreover , the Cho / Acetate ratio changed upon 5 - FU + OMP as in the 5 - FU group , but also the citrate / GSH ratio . The cellular biochemical effects involve mainly the fatty acid and phospholipid metabolism pointing to membrane anabolism . Abbreviations : Gln - glutamine , Ala - alanine , PC - phosphatidylcholine , Cho - Choline , Lac1 + FACH2 - methyl group signal of lactate and methylene groups of the fatty acids , Lac2 - methylene group of lactate , CH = CH - protons of methin groups of unsaturated fatty acids .

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

Figure 6  The ASPC-1 cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colours of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. The most obvious changes is that the PC/Cho ratio is significantly lower in the OMP group compared to the control group. Upon 5-FU, the Cho/Acetate ratio decrease is the only significant change. In the 5-FU+OMP group, there are several significant changes, i.e.the FACH2/CH = CH ratio is significantly higher. Moreover, the Cho/Acetate ratio changed upon 5-FU+OMP as in the 5-FU group, but also the citrate/GSH ratio. The cellular biochemical effects involve mainly the fatty acid and phospholipid metabolism pointing to membrane anabolism. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline, Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.


Figure 7 The cell line is shown without and upon various treatment regimens ( OMP , 5 - FU or 5 - FU + OMP combination ) . The nodes of this network symbolize metabolite signals , their colours correspond to relative signal intensity ( when compared to an external standard ) as indicated in the heatmap scale below . The signal intensity is linearly related to the intracellular concentration . The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale . The lines between the boxes symbolize strongly simplified metabolic pathways . The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange ( p < 0 . 05 ) , red lines indicate p < 0 . 01 . Upon OMP , the PC / Cho ratios are significantly lower compared to control . Furthermore the acetate / FACH2 ratio is significantly decreased in the OMP group . The latter also showed a higher CH = CH level , the ratio to FACH2 is , however , decreased . Upon 5 - FU and 5 - FU + OMP , similar changes could be observed . Furthermore , in contrast to ASPC - 1 cells , the Ala / Gln / AMP pathway is also involved . Abbreviations : Gln - glutamine , Ala - alanine , PC - phosphatidylcholine , Cho - Choline . Lac1 + FACH2 - methyl group signal of lactate and methylene groups of the fatty acids , Lac2 - methylene group of lactate , CH = CH - protons of methin groups of unsaturated fatty acids .

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

Figure 7  The cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. Upon OMP, the PC/Cho ratios are significantly lower compared to control. Furthermore the acetate/FACH2 ratio is significantly decreased in the OMP group. The latter also showed a higher CH = CH level, the ratio to FACH2 is, however, decreased. Upon 5-FU and 5-FU+OMP, similar changes could be observed. Furthermore, in contrast to ASPC-1 cells, the Ala/Gln/AMP pathway is also involved. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline. Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.


Figure 8 ( B ) Western blot analysis of LAMP - 1 , a late endosome marker , which was nearly identically distributed over the first two fractions compared to Cathepsin - D , in the controle group , but ocurred only in the first fraction in the OMP treated group . β - COP as an indicator of the Golgi - complex , is found strongly in the lower fraction in the control group , but very weakly in the OMP treated group . Cathepsin - D , an early endosome marker , which can be found in the upper and middle fraction of the control group , but regarding the OMP group , it was only found in the first one .

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

Figure 8(B) Western blot analysis of LAMP-1, a late endosome marker, which was nearly identically distributed over the first two fractions compared to Cathepsin-D, in the controle group, but ocurred only in the first fraction in the OMP treated group. β-COP as an indicator of the Golgi-complex, is found strongly in the lower fraction in the control group, but very weakly in the OMP treated group. Cathepsin - D, an early endosome marker, which can be found in the upper and middle fraction of the control group, but regarding the OMP group, it was only found in the first one.


Figure 9 The experiment was repeated three times with nearly identical results . The LC3 - I fractions can be clearly distinguished above the LC3 - II signals . Bafilomycin A1 elevated slightly the LC3 - II signal intensity compared to control in both cell lines . There was a marked and dose - dependent increase of the signal strength of both fractions when OMP was used . 5 - FU alone did not relevantly influence the LC3 - level . The corresponding β - Actin level is shown below the LC3 - WB to confirm the correctness of this semiquantitative evaluation .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9  The experiment was repeated three times with nearly identical results. The LC3-I fractions can be clearly distinguished above the LC3-II signals. Bafilomycin A1 elevated slightly the LC3-II signal intensity compared to control in both cell lines. There was a marked and dose-dependent increase of the signal strength of both fractions when OMP was used. 5-FU alone did not relevantly influence the LC3-level. The corresponding β-Actin level is shown below the LC3-WB to confirm the correctness of this semiquantitative evaluation.


Figure 10 Quantification of mRNAs in ASPC - 1 cells untreated or treated with OMP 80 µg / ml , 5 - FU 5 µg / ml or the combination of both was performed at different time points throughout 24 hours , and the means of three replicates are shown for every time point . The stars indicate significant differences after 24 hours compared to control . The pro - apoptotic bad - mRNA was upregulated after 5 - FU - and 5 - FU + OMP - treatment . The antiapoptotic bcl - 2 mRNA was downregulated by 5 - FU and bcl - XL by 5 - FU + OMP . OMP led to downregulation of bad and survivin and to upregulation of the mdr - 1mRNA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 10  Quantification of mRNAs in ASPC-1 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. The stars indicate significant differences after 24 hours compared to control. The pro-apoptotic bad-mRNA was upregulated after 5-FU- and 5-FU+OMP-treatment. The antiapoptotic bcl-2 mRNA was downregulated by 5-FU and bcl-XL by 5-FU+OMP. OMP led to downregulation of bad and survivin and to upregulation of the mdr-1mRNA.


Figure 11 mRNA quantification in MiaPaCa - 2 cells untreated or treated with OMP 80 µg / ml , 5 - FU 5 µg / ml or the combination of both was performed at different time points throughout 24 hours , and the means of three replicates are shown for every time point . While the mdr - 1 mRNA is not signicantly changed , the vATPase mRNA is upregulated in the 5 - FU + OMP group after 24 hours compared to control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 11  mRNA quantification in MiaPaCa-2 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. While the mdr-1 mRNA is not signicantly changed, the vATPase mRNA is upregulated in the 5-FU+OMP group after 24 hours compared to control.


Figure 12 The Bad , Puma and Atg12 proteins were detected in MiaPaCa - 2 and ASPC - 1 cells . The Bad expression is slightly increased by 5 - FU and suppressed by OMP and 5 - FU + OMP in ASPC - 1 , but not in MiaPaCa - 2 cells . Puma is induced by OMP in both cell lines indicating involvement of the BH3 - only pathway . Atg12 is strongly enhanced by OMP in both cell lines . Abbreviations : K - control , F - 5 - FU , O - OMP , FO - 5 - FU + OMP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 12  The Bad , Puma and Atg12 proteins were detected in MiaPaCa-2 and ASPC-1 cells. The Bad expression is slightly increased by 5-FU and suppressed by OMP and 5-FU+OMP in ASPC-1, but not in MiaPaCa-2 cells. Puma is induced by OMP in both cell lines indicating involvement of the BH3-only pathway. Atg12 is strongly enhanced by OMP in both cell lines. Abbreviations: K - control, F - 5-FU, O - OMP, FO - 5-FU+OMP.


Figure 1E Representative images of Notch activity indicated by Tg ( Tp1 : mVenus ) ( green ) and matured MG stained with GS ( magenta ) at different stages . Scale Bars : 20 μm . F Quantitative plots showing the numbers of Tp1 + ( left ) , GS + ( right ) cells in the developing retinas at different stages . We collected retinas from 60 hpf ( n = 3 dpf ( n = 12 ) , 5 dpf ( n = 12 ) , and 7 dpf ( n = 12 , Mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1E Representative images of Notch activity indicated by Tg(Tp1:mVenus)( green) and matured MG stained with GS (magenta) at different stages. Scale Bars: 20 μm. F Quantitative plots showing the numbers of Tp1+ (left), GS+ (right) cells in the developing retinas at different stages. We collected retinas from 60 hpf (n = 3 dpf (n = 12), 5 dpf (n = 12), and 7 dpf (n = 12, Mean ± SEM; * p<0.05, ***p<0.001, t-test).


Figure 2A Representative images of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) and mature MGs stained with glutamate synthase ( GS , magenta ) at 4 dpf in Notch ligands ( dla - / - / dlb - / - / dlc - / - / dld - / - / jag1b - / - / jag2b - / - ) mutants . B , C Quantitative plots showing the numbers of Notch activation ( Tp1 + ) cells ( B ) and mature MG ( GS + ) cells ( C ) in A at 4 dpf . In total , we collected 11 fish for wild - type fish ; 8 fish for jag2b - / - mutants ; 15 fish for dla - / - mutants ; 19 fish for dld - / - mutants ; 19 fish for dlc - / - mutants , 11 fish for dlb - / - mutants , 11 fish for jag1b - / - mutants ( mean ± SEM ; * * * p < 0 . 001 , t - test ) .

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

Figure 2A Representative images of Notch activation labeled by Tg(Tp1:mVenus)(green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch ligands (dla-/-/dlb-/-/dlc-/-/dld-/-/jag1b-/-/jag2b-/-) mutants.B, C Quantitative plots showing the numbers of Notch activation (Tp1+) cells (B) and mature MG (GS+) cells (C) in A at 4 dpf. In total, we collected 11 fish for wild-type fish; 8 fish for jag2b-/- mutants; 15 fish for dla-/- mutants; 19 fish for dld-/- mutants; 19 fish for dlc-/- mutants, 11 fish for dlb-/- mutants, 11 fish for jag1b-/- mutants (mean ± SEM; ***p<0.001, t-test).


Figure 4A Upper : representative images showing that GS + MGs ( magenta ) were rarely observed in 5 - dpf jag2b - / - mutants expressing H2B - CFP ( green ) driven by the crx promoter . Bottom : representative images showing that GS + MGs ( magenta ) were frequently rescued underneath PRs when jag2b expression ( green ) was driven by the crx promoter in 5 - dpf jag2b - / - mutants . The area of retina is labeled with solid line , and each layer structure is labeled with dashed lines and marked with outer nuclear layer ( ONL ) , inner nuclear layer ( INL ) , inner plexiform layer ( IPL ) , and ganglion cell layer ( GCL ) . Scale bars : 20μm . The remaining MGs are indicated by white arrowheads , and the rescued MGs are indicated by yellow arrows . B Quantification of GS + MGs ( magenta in A ) with ( green ) or without ( gray ) CFP expression driven by the crx promoter . Ctrl indicates H2B - CFP expression driven by the crx promoter ( n = 16 larvae ) ; rescue indicates jag2b - H2B - CFP expression driven by the crx promoter ( n = 21 larvae , mean ± SEM ; * p < 0 . 05 , ns , no significant difference , pair - wised t - test ) . C The proportions of jag2b - misexpressing PRs ( Green ) with underneath MGs ( GS + , magenta in A ) in 5 - dpf jag2b - / - mutants and all MGs in retina . In total , we analyzed retinae of 16 control ( Ctrl , jag2b - / - , crx : : h2b - ecfp expression ) and 21 rescue ( jag2b - / - , crx : : jag2b expression ) larvae ( Mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Upper: representative images showing that GS+ MGs (magenta) were rarely observed in 5-dpf jag2b-/- mutants expressing H2B-CFP (green) driven by the crx promoter. Bottom: representative images showing that GS+ MGs (magenta) were frequently rescued underneath PRs when jag2b expression (green) was driven by the crx promoter in 5-dpf jag2b-/- mutants. The area of retina is labeled with solid line, and each layer structure is labeled with dashed lines and marked with outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Scale bars: 20μm. The remaining MGs are indicated by white arrowheads, and the rescued MGs are indicated by yellow arrows. B Quantification of GS+ MGs (magenta in A) with (green) or without (gray) CFP expression driven by the crx promoter. Ctrl indicates H2B-CFP expression driven by the crx promoter (n = 16 larvae); rescue indicates jag2b-H2B-CFP expression driven by the crx promoter (n = 21 larvae, mean ± SEM; * p<0.05, ns, no significant difference, pair-wised t-test). C The proportions of jag2b-misexpressing PRs (Green) with underneath MGs (GS+, magenta in A) in 5-dpf jag2b-/- mutants and all MGs in retina. In total, we analyzed retinae of 16 control (Ctrl, jag2b-/- , crx::h2b-ecfp expression) and 21 rescue (jag2b-/-, crx::jag2b expression) larvae (Mean ± SEM; ***p<0.001, t-test).


Figure 5D Representative image showing the GS + , but vsx1 - expression in the progeny of Tp1 + Müller - like differentiating MGs at wild - type ( jag2b + / + ) . Layer structure indicated in ( A ) . E The percentage of the progeny of Müller - like Tp1 + differentiating MG precursors ( red + ) expression GS ( gray ) , vsx1 ( green ) , or both negative ( magenta ) in wild - type ( jag2b + / + ) fish ( n = 14 , Mean ± SEM ; * * * p < 0 . 001 , t - test ) . F Representative image showing vsx1 + , but GS - expression in the progeny of Tp1 + Müller - like differentiating MGs in jag2b - / - mutants . Layer structure indicated in ( A ) . G The percentage of the progeny of Müller - like Tp1 + differentiating MG precursors ( red + ) expressing GS ( gray ) , vsx1 ( green ) , or both negative ( magenta ) in jag2b - / - mutants ( n = 29 , mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5D Representative image showing the GS+, but vsx1- expression in the progeny of Tp1+ Müller-like differentiating MGs at wild-type (jag2b+/+). Layer structure indicated in (A). E The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expression GS (gray), vsx1 (green), or both negative (magenta) in wild-type (jag2b+/+) fish (n = 14, Mean ± SEM; ***p<0.001, t-test). F Representative image showing vsx1+, but GS- expression in the progeny of Tp1+ Müller-like differentiating MGs in jag2b-/- mutants. Layer structure indicated in (A). G The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expressing GS (gray), vsx1 (green), or both negative (magenta) in jag2b-/- mutants (n = 29, mean ± SEM; ***p<0.001, t-test).


Figure 6A Representative images of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) and mature MGs stained with glutamate synthase ( GS , magenta ) at 4 dpf in Notch receptors ( notch1a - / - / notch1b - / - / notch3 - / - ) mutants . Scale Bars : 20 μm . B Quantitative plots showing the numbers of cells with active Notch ( Tp1 + ) and mature MG ( GS + ) cells at 4 dpf . In total , we collected 4 fish for wild - type fish ; 7 fish for notch1a - / - mutants ; 5 fish for notch1b - / - mutants ; 18 fish for notch3 - / - mutants ( Mean ± SEM ; * * p < 0 . 01 ; * * * p < 0 . 001 , t - test ) . C Representative images showing that GS + MGs ( magenta ) were rarely observed when H2B - CFP expression ( green ) was driven by the gfap promoter in 5 - dpf jag2b - / - mutants . The area between the upper dashed and solid lines is the outer nuclear layer ( ONL ) . The area in the middle two dash lines is the inner plexiform layer ( IPL ) . INL , inner nuclear layer ; GCL , ganglion cell layer . D Representative images showing that GS + MGs ( magenta ) were frequently rescued underneath cells with notch3 expression ( green ) driven by the gfap promoter in 5 - dpf jag2b - / - mutants . Layer structure is similar as ( C ) . E The cell number of GS + MGs ( magenta ) with or without CFP expression driven by gfap promoter . Ctrl : H2B - CFP expression ( green ) driven by the gfap promoter ( n = 5 fish ) ; rescue : notch1b ( n = 13 fish ) and notch3 ( n = 17 fish ) NICD - H2B - CFP expression ( green ) driven by the gfap promoter ( Mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , no significant difference , pair - wised t - test ) . F The proportions of notch1b and notch3 NICD - misexpressing cells ( green ) with underneath MGs ( GS + , magenta ) in 5 - dpf jag2b - / - mutants , indicating that gfap promoter - driven notch1b ( n = 13 fish ) or notch3 ( n = 17 fish ) NICD expression could rescue MG loss in jag2b - / - mutants ( Mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of Notch activation labeled by Tg(Tp1:mVenus)( green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch receptors (notch1a-/-/notch1b-/-/notch3-/-) mutants. Scale Bars: 20 μm. B Quantitative plots showing the numbers of cells with active Notch (Tp1+) and mature MG (GS+) cells at 4 dpf. In total, we collected 4 fish for wild-type fish; 7 fish for notch1a-/- mutants; 5 fish for notch1b-/- mutants; 18 fish for notch3-/- mutants (Mean ± SEM; ** p<0.01; ***p<0.001, t-test).C Representative images showing that GS+ MGs (magenta) were rarely observed when H2B-CFP expression (green) was driven by the gfap promoter in 5-dpf jag2b-/- mutants. The area between the upper dashed and solid lines is the outer nuclear layer (ONL). The area in the middle two dash lines is the inner plexiform layer (IPL). INL, inner nuclear layer; GCL, ganglion cell layer. D Representative images showing that GS+ MGs (magenta) were frequently rescued underneath cells with notch3 expression (green) driven by the gfap promoter in 5-dpf jag2b-/- mutants. Layer structure is similar as (C). E The cell number of GS+ MGs (magenta) with or without CFP expression driven by gfap promoter. Ctrl: H2B-CFP expression (green) driven by the gfap promoter (n = 5 fish); rescue: notch1b (n = 13 fish) and notch3 (n = 17 fish) NICD-H2B-CFP expression (green) driven by the gfap promoter (Mean ± SEM; * p<0.05, ***p<0.001, ns, no significant difference, pair-wised t-test). F The proportions of notch1b and notch3 NICD-misexpressing cells (green) with underneath MGs (GS+, magenta) in 5-dpf jag2b-/- mutants, indicating that gfap promoter-driven notch1b (n = 13 fish) or notch3 (n = 17 fish) NICD expression could rescue MG loss in jag2b-/- mutants (Mean ± SEM; ***p<0.001, t-test).


Figure 1Maximum projection ( 2 z - slices ) of HeLa cells immunostained for F - actin ( Phalloidin ) , Tubulin , Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase . Scale bar - 10µm Quantification of F - actin ( Phalloidin ) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in ( A ) , showing the increase in F - actin around centrosomes at early anaphase . Mean actin metaphase = 1 ± 0 . 03996 , n = 92 , mean actin anaphase = 1 . 875 ± 0 . 08895 , n = 121 , Student t - test , * * * * p < 0 . 0001 ; Time - lapse sequence from a representative Hela cell expressing Lifeact - GFP transiting from metaphase to anaphase , showing accumulation of actin in the presumptive centrosomal region , red arrows . Images represent 4z - projection . Scale bar - 10µm . Time - lapse sequence of a representative HeLa cell expressing GFP - alpha - tubulin and RFP - Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules . Images represent 4 z - projection of movies taken every 1min . Bottom - higher magnification view of the centrosomes C1 and C2 from time - lapse above showing a transient increase in actin around the centrosomes in early anaphase . Scale bar = 10µm , 1µm in zoom . T = 0 is one frame before anaphase onset . Dotted circle shows centrosome position . Integrated fluorescence intensity of GFP - alpha - tubulin and RFP - Lifeact around the centrosomes for a population of cells exiting mitosis , showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time . N = 30 centrosomes ( 15 cells ) . T = 0 is one frame before anaphase onset . Graph shows mean + / - SD , values were normalized to intensity at T = 0 . Quantification and comparison of actin ( RFP - Lifeact ) and tubulin ( a - tubulin - GFP ) fluorescence intensity around the centrosomes at metaphase ( - 4 to - 1min ) , early anaphase ( 2 - 5min ) and late anaphase ( 7 - 10min ) for a population of cells . Mean actin intensity at Metaphase = 1 . 002 ± 0 . 0239 , n = 97 , Early Anaphase = 1 . 023 ± 0 . 03047 , n = 101 , Late anaphase = 0 . 9703 ± 0 . 05004 , n = 98 . Tubulin intensity at Metaphase = 1 . 008 ± 0 . 03356 , n = 106 , Early anaphase = 0 . 9748 ± 0 . 04124 , n = 116 , Late anaphase = 0 . 9276 ± 0 . 05382 , n = 101 , One - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Maximum projection (2 z-slices) of HeLa cells immunostained for F-actin (Phalloidin), Tubulin, Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase. Scale bar- 10µm Quantification of F-actin (Phalloidin) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in (A), showing the increase in F-actin around centrosomes at early anaphase. Mean actin metaphase = 1 ± 0.03996, n=92, mean actin anaphase = 1.875 ± 0.08895, n=121, Student t-test, ****p<0.0001;Time-lapse sequence from a representative Hela cell expressing Lifeact-GFP transiting from metaphase to anaphase, showing accumulation of actin in the presumptive centrosomal region, red arrows. Images represent 4z-projection. Scale bar-10µm.Time-lapse sequence of a representative HeLa cell expressing GFP-alpha-tubulin and RFP-Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules. Images represent 4 z-projection of movies taken every 1min. Bottom - higher magnification view of the centrosomes C1 and C2 from time-lapse above showing a transient increase in actin around the centrosomes in early anaphase. Scale bar = 10µm, 1µm in zoom. T=0 is one frame before anaphase onset. Dotted circle shows centrosome position.Integrated fluorescence intensity of GFP-alpha-tubulin and RFP-Lifeact around the centrosomes for a population of cells exiting mitosis, showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time. N=30 centrosomes (15 cells). T=0 is one frame before anaphase onset. Graph shows mean +/- SD, values were normalized to intensity at T=0. Quantification and comparison of actin (RFP-Lifeact) and tubulin (a-tubulin-GFP) fluorescence intensity around the centrosomes at metaphase (-4 to -1min), early anaphase (2-5min) and late anaphase (7-10min) for a population of cells. Mean actin intensity at Metaphase =1.002 ± 0.0239, n=97, Early Anaphase = 1.023 ± 0.03047, n=101, Late anaphase = 0.9703 ± 0.05004, n=98. Tubulin intensity at Metaphase = 1.008 ± 0.03356, n=106, Early anaphase = 0.9748 ± 0.04124, n=116, Late anaphase = 0.9276 ± 0.05382, n=101, One-way ANOVA.


Figure 2Stills from time - lapse of HeLa cells expressing GFP - alpha - tubulin and RFP - lifeact arrested at prometaphase with STLC ( t = 0 ) and forced to exit mitosis with Cdk1 inhibition ( Ro3306 ) imaged every 90 seconds . Scale bar = 10µm and for zoom = 4µm . n = 21 cells from 4 independent experiments . Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in ( A ) , showing a similar accumulation of actin as observed in bipolar divisions . Graph shows mean actin accumulation normalized to t = 0 , errors bars indicate standard deviation . HeLa cells expressing GFP - centrin1 arrested at prometaphase or forced mitotic exit ( Ro3306 5 ' ) and stained with phalloidin . Scale bar - 5µm and for zoom - 2µm . The level of actin around the centrosome in ( C ) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit . STLC arrest 1 ± 0 . 01317 , n = 210 , STLC + Ro - 5mins = 1 . 251 ± 0 . 02441 , n = 144 , Unpaired t - test with Welch correction , * * * * p < 0 . 0001 . Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor . Tubulin intensity decreses during forced mitotic exit . STLC - DMSO - 1 ± 0 . 02407 , n = 180 , STLC - RO - 3306 - 0 . 6516 ± 0 . 02337 , n = 136 . P < 0 . 0001 , Welch ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Stills from time-lapse of HeLa cells expressing GFP-alpha-tubulin and RFP-lifeact arrested at prometaphase with STLC (t=0) and forced to exit mitosis with Cdk1 inhibition (Ro3306) imaged every 90 seconds. Scale bar = 10µm and for zoom = 4µm. n = 21 cells from 4 independent experiments.Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in (A), showing a similar accumulation of actin as observed in bipolar divisions. Graph shows mean actin accumulation normalized to t=0, errors bars indicate standard deviation.HeLa cells expressing GFP-centrin1 arrested at prometaphase or forced mitotic exit (Ro3306 5') and stained with phalloidin. Scale bar- 5µm and for zoom-2µm.The level of actin around the centrosome in (C) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit. STLC arrest 1 ± 0.01317, n=210, STLC+Ro-5mins=1.251 ± 0.02441, n=144, Unpaired t-test with Welch correction, ****p<0.0001. Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor. Tubulin intensity decreses during forced mitotic exit. STLC-DMSO- 1 ± 0.02407, n=180, STLC-RO-3306- 0.6516 ± 0.02337, n=136. P<0.0001, Welch's t test.


Figure 3Maximum projection ( 2 - z slices ) view of Hela cells pre - treated with DMSO and 0 . 2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase . Scale bar - 10µm . Quantification of actin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , showing the reduction in actin accumulation around the centrosomes following CK666 treatment . DMSO - metaphase - 1 ± 0 . 4325 , n = 54 , CK666 - metaphase - 0 . 936 ± 0 . 4604 , n = 43 , DMSO - anaphase - 1 . 8 ± 0 . 9736 , n = 76 , CK666 - anaphase - 1 . 087 ± 0 . 4597 , n = 73 ; one - way ANOVA , p < 0 . 0001 . Date pooled from 3 independent experiments . Z - projection of HeLa cells expressing GFP - centrin1 pre - treated with DMSO or 0 . 2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F - actin . Quantification of the level of actin around the centrosome from ( C ) , which shows the reduction in actin accumulation around centrosomes following CK666 pre - treatment . DMSO - STLC = 1 ± 0 . 02768 , n = 99 , CK666 STLC = 0 . 776 ± 0 . 02186 , n = 87 , DMSO - RO - 3306 = 1 . 339 ± 0 . 03048 , n = 127 , CK666 - RO - 3306 = 0 . 7699 ± 0 . 02246 , n = 70 , one - way ANOVA , p < 0 . 0001 . Quantification of tubulin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit . DMSO - metaphase - 1 ± 0 . 1968 , n = 54 , DMSO - anaphase - 0 . 867 ± 0 . 2345 , n = 78 , CK666 - metaphase - 0 . 7995 ± 0 . 1275 , n = 43 , CK666 - anaphase - 0 . 7407 ± 0 . 1859 , n = 74 ; one - way ANOVA , p < 0 . 0001 . Data pooled from 3 independent experiments . Error bars indicated standard deviation . Quantification of tubulin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , during monopolar exit , showing the failure to reduce tubulin density around centrosomes following CK666 treatment . Data from three independent experiments . Prometaphase arrest ( p . a . ) 1 ± 0 . 34 , n = 27 ; forced mitotic exit ( f . m . e ) 0 . 78 ± 0 . 30 , n = 26 ; p . a . plus CK666 0 . 49 ± 0 . 34 , n = 30 ; f . m . e plus CK666 0 . 92 ± 0 . 37 , n = 30 . One - way ANOVA with Sidak ' s multiple comparisons test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Maximum projection (2-z slices) view of Hela cells pre-treated with DMSO and 0.2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase. Scale bar-10µm. Quantification of actin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the reduction in actin accumulation around the centrosomes following CK666 treatment. DMSO-metaphase-1±0.4325, n=54, CK666-metaphase-0.936±0.4604, n=43, DMSO-anaphase-1.8±0.9736, n=76, CK666-anaphase-1.087±0.4597, n=73; one-way ANOVA, p<0.0001. Date pooled from 3 independent experiments.Z-projection of HeLa cells expressing GFP-centrin1 pre-treated with DMSO or 0.2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F-actin.Quantification of the level of actin around the centrosome from (C), which shows the reduction in actin accumulation around centrosomes following CK666 pre-treatment. DMSO-STLC= 1 ± 0.02768, n=99, CK666 STLC= 0.776 ± 0.02186, n=87, DMSO-RO-3306= 1.339 ± 0.03048, n=127, CK666-RO-3306= 0.7699 ± 0.02246, n=70, one-way ANOVA, p<0.0001.Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit. DMSO-metaphase-1±0.1968, n=54, DMSO-anaphase-0.867±0.2345, n=78, CK666-metaphase-0.7995±0.1275, n=43, CK666-anaphase-0.7407±0.1859, n=74; one-way ANOVA, p<0.0001. Data pooled from 3 independent experiments. Error bars indicated standard deviation.Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, during monopolar exit, showing the failure to reduce tubulin density around centrosomes following CK666 treatment. Data from three independent experiments. Prometaphase arrest (p.a.) 1±0.34 , n=27; forced mitotic exit (f.m.e) 0.78±0.30, n=26; p.a. plus CK666 0.49±0.34, n=30 ; f.m.e plus CK666 0.92±0.37, n=30. One-way ANOVA with Sidak's multiple comparisons test.


Figure 4Z - projection of HeLa cells expressing GFP - centrin1 immunostained for p34 - Arc ( sub - domain Arp2 / 3 ) following prometaphase arrest and forced mitotic exit for 5min . N = 3 independent experiments . Scale bar = 10µm and for zoom 2µm . Levels of p34 - Arc around the centrosome ( 3um area ) were quantified relative to metaphase and increase during forced mitotic exit . STLC arrested = 1 ± 0 . 01576 , n = 153 , STLC + RO - 3306 = 1 . 268 ± 0 . 01748 , n = 191 , * * * * p < 0 . 0001 , Unpaired t - test with Welch correction . Error bars represent standard deviation . Z - projection of centrosomes isolated from cells expressing GFP - centrin1 during prometaphase arrest and forced exit , which were immunostained with p34 - Arc . N = 2 independent experiments . Scale bar = 2µm . Quantification of p34 - Arc levels around the centrosome in ( C ) , relative to metaphase , showing its increase during forced exit . STLC arrested = 1 ± 0 . 00779 , n = 93 , STLC + RO - 3306 = 1 . 249 ± 0 . 02446 , n = 134 , * * * * p < 0 . 0001 , Unpaired t - test with Welch correction . Error bars represent standard deviation . Time - lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time . Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in ( E ) over an area of 2 μm , ( white circle ) , showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit . N = 2 isolations , 5 independent experiments , n = 16 STLC and n = 19 STLC RO . Color switch experiment using green - and red - labelled actin . Red actin was used first , followed by Capping protein , then green - labelled actin . The accumulation of green in the centre indicates that actin is nucleated at the centrosome . Scale bar = 10µm . Quantification of ( G ) over the time . N = 2 isolations , 2 independent experiments , n = 19 STLC and n = 14 STLC RO . Error bars represent standard deviation . Time - lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre - treated with DMSO or CK666 . Scale bar = 10µm . Quantification of ( I ) , showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre - treated with CK666 . N = 2 isolations , 2 independent experiments , n = 17 DMSO , 26 Ro CK666 24 STLC DMSO , 23 STLC CK666 . Error bars represent standard deviation .

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Figure 4Z-projection of HeLa cells expressing GFP-centrin1 immunostained for p34-Arc (sub-domain Arp2/3) following prometaphase arrest and forced mitotic exit for 5min. N=3 independent experiments. Scale bar = 10µm and for zoom 2µm.Levels of p34-Arc around the centrosome (3um area) were quantified relative to metaphase and increase during forced mitotic exit. STLC arrested = 1 ± 0.01576, n=153, STLC+RO-3306= 1.268 ± 0.01748, n=191, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.Z-projection of centrosomes isolated from cells expressing GFP-centrin1 during prometaphase arrest and forced exit, which were immunostained with p34-Arc. N=2 independent experiments. Scale bar = 2µm.Quantification of p34-Arc levels around the centrosome in (C), relative to metaphase, showing its increase during forced exit. STLC arrested = 1 ± 0.00779, n=93, STLC+RO-3306= 1.249 ± 0.02446, n=134, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.Time-lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time. Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in (E) over an area of 2 μm, (white circle), showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit. N=2 isolations, 5 independent experiments, n=16 STLC and n=19 STLC RO.Color switch experiment using green- and red-labelled actin. Red actin was used first, followed by Capping protein, then green-labelled actin. The accumulation of green in the centre indicates that actin is nucleated at the centrosome. Scale bar = 10µm. Quantification of (G) over the time. N=2 isolations, 2 independent experiments, n=19 STLC and n=14 STLC RO. Error bars represent standard deviation.Time-lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre-treated with DMSO or CK666. Scale bar = 10µm. Quantification of (I), showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre-treated with CK666. N=2 isolations, 2 independent experiments, n = 17 DMSO, 26 Ro CK666 24 STLC DMSO, 23 STLC CK666. Error bars represent standard deviation.


Figure 5Phos - tag Western blot of MCF7 - Her2 - 18 cells synchronised into distinct phases of the cell cycle showing the phospho - shift in WASH1 during mitosis . Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment .

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Figure 5Phos-tag Western blot of MCF7-Her2-18 cells synchronised into distinct phases of the cell cycle showing the phospho-shift in WASH1 during mitosis.Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment.


Figure 6Time - lapse of HeLa cells expressing GFP - alpha tubulin and RFP - lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition ( t = 0 ) . Scale bar = 5µm . Quantification of actin around the centrosome in ( A ) , showing the failure to accumulate actin around the centrosome in siWASH . N = 4 experiments . Error bars represent standard deviation . Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody , showing the reduction in WASH levels following treatment of cells with siRNA against WASH . Scale bar = 5µm and 2µm in zoom . Z - projection of HeLa cells expressing GFP - centrin1 treated with control siRNA or siRNA against WASH , arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin . Scale bar - 5µm Quantification of actin around the centrosome in ( F ) , showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH . siControl - STLC = 1 ± 0 . 0189 , n = 165 , siWASH - STLC = 0 . 9138 ± 0 . 0298 , n = 118 , siControl - STLC + RO - 3306 = 1 . 911 ± 0 . 06221 , n = 147 , siWASH - STLC + RO - 3306 = 1 . 103 ± 0 . 0301 , n = 140 . Error bars represent standard deviation . P < 0 . 0001 , one - way ANOVA . Time - lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre - treated with either no antibody or anti - WASH1 antibody for 1 hour . Scale bar - 10µm Quantification of in vitro assay from ( F ) , showing the reduction in actin nucleation around centrosomes when they are pre - treated with anti - WASH1 . Error bars represent standard deviation . 2 independent experiments .

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Figure 6Time-lapse of HeLa cells expressing GFP-alpha tubulin and RFP-lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition (t=0). Scale bar = 5µm.Quantification of actin around the centrosome in (A), showing the failure to accumulate actin around the centrosome in siWASH. N = 4 experiments. Error bars represent standard deviation.Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody, showing the reduction in WASH levels following treatment of cells with siRNA against WASH. Scale bar = 5µm and 2µm in zoom.Z-projection of HeLa cells expressing GFP-centrin1 treated with control siRNA or siRNA against WASH, arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin. Scale bar-5µm Quantification of actin around the centrosome in (F), showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH. siControl-STLC= 1 ± 0.0189, n=165, siWASH-STLC=0.9138 ± 0.0298, n=118, siControl-STLC+RO-3306=1.911 ± 0.06221, n=147, siWASH-STLC+RO-3306=1.103 ± 0.0301, n=140. Error bars represent standard deviation. P<0.0001, one-way ANOVA.Time-lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre-treated with either no antibody or anti-WASH1 antibody for 1 hour. Scale bar- 10µm Quantification of in vitro assay from (F), showing the reduction in actin nucleation around centrosomes when they are pre-treated with anti-WASH1. Error bars represent standard deviation. 2 independent experiments.


Figure 1A TRAF6 expression in differentiating CD4 + T cells . Naïve CD4 + T cells were obtained from wild type C57BL / 6 mice by FACS and activated with anti - CD3 / CD28 ( 1µg , and 2µg / ml ) for the indicated times in the presence of distinct Thelper lineage - directing cytokines or under neutral activation conditions ( Th0 ) . After total RNA extraction and cDNA conversion , RT - PCR determined of Traf6 mRNA in differentiating Th0 , Th1 , Th17 and iTregs was determined . B TRAF6 mRNA expression by human Tregs and non - Treg CD4 + T cell . Human Tregs ( CD3 + / CD4 + / CD8 - / CD25HIGH / CD127low / CD39 + ) and non - Treg CD4 + T cells ( CD3 + / CD4 + / CD8 - / CD25 - ) were obtained from the peripheral blood of healthy donors by FACS after Ficoll - Paque PLUS gradient centrifugation and magnetic bead enrichment of CD4 + T cells . TRAF6 mRNA was measured by qRT - PCR . C , D Evidence of lymphoproliferative disease in Traf6fl / flFoxp3Cre + mice . ( C ) Spleens and lymph nodes were recovered from Traf6fl / flFoxp3Cre + mice and Traf6fl / fl littermates at 8 weeks of age ( Scale bars : 5 mm ) . ( D ) The cellularity of the lymphoid tissues of Traf6fl / flFoxp3Cre + mice and their Traf6fl / fl Foxp3Cre - littermates was determined ( 8 mice / group ) . E , H Effect of Treg - specific TRAF6 - deficiency on baseline T cell activation . The frequencies of effector cells ( CD44high / CD62Llow ) , memory cells ( CD44high / CD62Lhigh ) and naïve cells ( CD44low / CD62Lhigh ) in the CD4 + T cell compartments of Traf6fl / fl and Traf6fl / flFoxp3Cre + mice were determined by flow cytometry ( 5 mice / group ) . F , I Effect of Treg - specific TRAF6 - deficiency on baseline T cell activation . The frequencies of effector cells ( CD44high / CD62Llow ) , memory cells ( CD44high / CD62Lhigh ) and naïve cells ( CD44low / CD62Lhigh ) in the CD8 + T cell compartments of Traf6fl / fl Foxp3Cre - ( wild type ) and Traf6fl / flFoxp3Cre + mice were determined by flow cytometry ( 5 mice / group ) . G , J Impact of TRAF6 - expression on in vitro Treg differentiation . naïve CD4 + T cells were isolated from Traf6fl / flFoxp3Cre + and Traf6fl / fl mice and differentiated into iTregs . Conditions of sub - optimal TGFβ concentrations ( 0 . 5ng / ml , 0 . 05ng / ml ) were tested as well and intracellular FOXP3 was measured after 4 days .

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

Figure 1A TRAF6 expression in differentiating CD4+ T cells. Naïve CD4+ T cells were obtained from wild type C57BL/6 mice by FACS and activated with anti-CD3/CD28 (1µg, and 2µg/ml) for the indicated times in the presence of distinct Thelper lineage-directing cytokines or under neutral activation conditions (Th0). After total RNA extraction and cDNA conversion, RT-PCR determined of Traf6 mRNA in differentiating Th0, Th1, Th17 and iTregs was determined.B TRAF6 mRNA expression by human Tregs and non-Treg CD4+ T cell. Human Tregs (CD3+/CD4+/CD8-/CD25HIGH/CD127low/CD39+) and non-Treg CD4+ T cells (CD3+/CD4+/CD8-/CD25-) were obtained from the peripheral blood of healthy donors by FACS after Ficoll-Paque PLUS gradient centrifugation and magnetic bead enrichment of CD4+ T cells. TRAF6 mRNA was measured by qRT-PCR.C, D Evidence of lymphoproliferative disease in Traf6fl/flFoxp3Cre+ mice. (C) Spleens and lymph nodes were recovered from Traf6fl/flFoxp3Cre+ mice and Traf6fl/fl littermates at 8 weeks of age (Scale bars: 5 mm). (D) The cellularity of the lymphoid tissues of Traf6fl/flFoxp3Cre+ mice and their Traf6fl/fl Foxp3Cre- littermates was determined (8 mice/group).E, H Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD4+ T cell compartments of Traf6fl/fl and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).F, I Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD8+ T cell compartments of Traf6fl/fl Foxp3Cre- (wild type) and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).G, J Impact of TRAF6-expression on in vitro Treg differentiation. naïve CD4+ T cells were isolated from Traf6fl/flFoxp3Cre+ and Traf6fl/fl mice and differentiated into iTregs. Conditions of sub-optimal TGFβ concentrations (0.5ng/ml, 0.05ng/ml) were tested as well and intracellular FOXP3 was measured after 4 days.


Figure 2A In vivo suppressive function of WT and Traf6fl / flFoxp3Cre + Tregs . Tregs from the indicated mice ( CD45 . 2 + ) were isolated by FACS , as were naïve ( CD62Lhigh / CD25 - ) CD4 + responder T cells from congenically distinct ( CD45 . 1 + ) donor mice . Tregs and Tresponders were mixed at a 1 : 5 ( 2x105 : 10x105 ) ratio before injection into Rag2 - / - mice . 7 days later , spleens were harvested , and the relative frequencies and absolute numbers of each transferred cell populations were determined by flow cytometry . B Implanted B16 melanoma growth in Traf6fl / flFoxp3Cre + and wild type ( Traf6fl / flFoxp3Cre - ) mice . 1x105 B16F10 cells were injected subcutaneously ( s . c . ) into the shaved flanks of the indicated mice ( n = 5 / group ) . Tumor volumes were monitored every 3 days . C , D Proinflammatory cytokine production in tumor - bearing mice with and without Treg - specific TRAF6 expression . Cell suspensions of the tumor - draining lymph node and tumor - infiltrating leukocytes ( TILs ) were recovered after 21 days of tumor growth . Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL - 17 and flow cytometry analysis . E , F FOXP3 expression by CD4 + T cells in tumor bearing Traf6fl / flFoxp3Cre + and wild type ( Traf6fl / flFoxp3Cre - ) mice . The frequency of FOXP3 + cells in the CD4 + cells of tumor - draining lymph nodes ( dLN ) , peripheral , non - tumor draining lymph nodes ( pLN ) , spleens , and TILs were determined by flow cytometry .

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Figure 2A In vivo suppressive function of WT and Traf6fl/flFoxp3Cre+ Tregs. Tregs from the indicated mice (CD45.2+) were isolated by FACS, as were naïve (CD62Lhigh/CD25-) CD4+ responder T cells from congenically distinct (CD45.1+) donor mice. Tregs and Tresponders were mixed at a 1:5 (2x105:10x105) ratio before injection into Rag2-/- mice. 7 days later, spleens were harvested, and the relative frequencies and absolute numbers of each transferred cell populations were determined by flow cytometry.B Implanted B16 melanoma growth in Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. 1x105 B16F10 cells were injected subcutaneously (s.c.) into the shaved flanks of the indicated mice (n= 5/group). Tumor volumes were monitored every 3 days.C, D Proinflammatory cytokine production in tumor-bearing mice with and without Treg-specific TRAF6 expression. Cell suspensions of the tumor-draining lymph node and tumor-infiltrating leukocytes (TILs) were recovered after 21 days of tumor growth. Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL-17 and flow cytometry analysis.E, F FOXP3 expression by CD4+ T cells in tumor bearing Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. The frequency of FOXP3+ cells in the CD4+ cells of tumor-draining lymph nodes (dLN), peripheral, non-tumor draining lymph nodes (pLN), spleens, and TILs were determined by flow cytometry.


Figure 3A Assessment of K63 - type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants . 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6 , the enzymatically deficient L74H or C70A TRAF6 mutants , HA - FOXP3 , and FLAG - labeled ubiquitin molecules possessing a single lysine residue ( K63 ) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63 - type only . Cells were lysed and proteins modified by K63 - ubiquitin chains were recovered from lysates by pulling down using a bead - immobilized K63 - specific TUBE reagent . The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre - IP whole cell lysate ( WCL ) . B Co - immunoprecipitation ( co - IP ) of FOXP3 with TRAF6 and its facilitation of K63 - linked ubiquitination . 293T cells expressing the indicated constructs encoding HA - FOXP3 and FLAG - TRAF6 were lysed and incubated with bead immobilized anti - FLAG antibodies . FOXP3 molecules co - IPed in this manner were resolved by SDS PAGE and detected by immunoblotting with anti - HA antibodies . C 293T cells were transfected with the indicated combinations of expression constructs encoding HA - FOXP3 , Myc - TRAF6 , and FLAG - tagged ubiquitin molecules - either wild type ( FLAG - Ub ) , or a variant with a K - to - R mutation at either lysine residue 48 ( K48R ) or 63 ( K63R ) responsible for preventing K48 - and K63 - type polyubiquitination , respectively . . D Endogenous , reciprocal co - IP of FOXP3 and TRAF6 in murine iTreg lysate . Naïve CD62Lhigh / CD25 - / CD4 + T cells were obtained by FACS and activated with anti - CD3 / CD28 antibodies ( 1ug and 2ug / ml , respectively ) in the presence of IL - 2 and TGFβ ( 100U / ml and 5ng / ml , respectively ) for four days before lysis . Antibodies against TRAF6 ( left ) or FOXP3 ( right ) were used to pull down target proteins and their interaction partners , which were resolved under denaturing conditions and visualized by immunoblotting . E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl / flFoxp3Cre + and Traf6wt / wtFoxp3Cre + mice . CD4 + / YFP + cells from Traf6fl / flFoxp3Cre + ( " Traf6 - / - " ) or wild type Traf6wt / wtFoxp3Cre + mice ( " Traf6 + / + " ) were isolated by FACS , lysed and either total FOXP3 ( right ) or K63 - ubiquitin - modified proteins ( left ) were immunoprecipitated . Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti - K63 - ubiquitin and FOXP3 , respectively , and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Assessment of K63-type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants. 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6, the enzymatically deficient L74H or C70A TRAF6 mutants, HA-FOXP3, and FLAG-labeled ubiquitin molecules possessing a single lysine residue (K63) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63-type only. Cells were lysed and proteins modified by K63-ubiquitin chains were recovered from lysates by pulling down using a bead-immobilized K63-specific TUBE reagent. The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre-IP whole cell lysate (WCL).B Co-immunoprecipitation (co-IP) of FOXP3 with TRAF6 and its facilitation of K63-linked ubiquitination. 293T cells expressing the indicated constructs encoding HA-FOXP3 and FLAG-TRAF6 were lysed and incubated with bead immobilized anti-FLAG antibodies. FOXP3 molecules co-IPed in this manner were resolved by SDS PAGE and detected by immunoblotting with anti-HA antibodies.C　293T cells were transfected with the indicated combinations of expression constructs encoding HA-FOXP3, Myc-TRAF6, and FLAG-tagged ubiquitin molecules - either wild type (FLAG-Ub), or a variant with a K-to-R mutation at either lysine residue 48 (K48R) or 63 (K63R) responsible for preventing K48- and K63-type polyubiquitination, respectively. .D Endogenous, reciprocal co-IP of FOXP3 and TRAF6 in murine iTreg lysate. Naïve CD62Lhigh/CD25-/CD4+ T cells were obtained by FACS and activated with anti-CD3/CD28 antibodies (1ug and 2ug/ml, respectively) in the presence of IL-2 and TGFβ (100U/ml and 5ng/ml, respectively) for four days before lysis. Antibodies against TRAF6 (left) or FOXP3 (right) were used to pull down target proteins and their interaction partners, which were resolved under denaturing conditions and visualized by immunoblotting.E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl/flFoxp3Cre+ and Traf6wt/wtFoxp3Cre+mice. CD4+/YFP+ cells from Traf6fl/flFoxp3Cre+ ("Traf6-/-") or wild type Traf6wt/wtFoxp3Cre+mice ("Traf6+/+") were isolated by FACS, lysed and either total FOXP3 (right) or K63-ubiquitin-modified proteins (left) were immunoprecipitated. Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti-K63-ubiquitin and FOXP3, respectively, and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate.


Figure 4A Immunoblot analysis of TRAF6 - mediated ubiquitination of wild type , and mutant FOXP3 molecules . 293T cell lines were transfected with a normal HA - tagged Foxp3 construct , another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines ( 20R ) , or one of 20 single lysine - containing constructs with only the indicated lysine residue available for modification . These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags , respectively . Negative controls did not receive TRAF6 or expressed FOXP3 alone . Labeled FOXP3 proteins were pulled down from cell lysates ( anti - HA ) and ubiquitinated species were visualized by immunoblotting for FLAG . B Assessing the impact of K262 mutation on FOXP3 ubiquitination by TRAF6 . 293T cells were transfected with combinations of expression constructs encoding Myc - TRAF6 , FLAG - ubiquitin , and an HA - tagged FOXP3 molecule that was either normal ( HA - FOXP3 ) or possessed a K - to - R mutation at residue 262 ( K262R ) . FOXP3 proteins were pulled down with anti - HA beads and either ubiquitinated proteins or total FOXP3 proteins were detected by probing for FLAG and HA , respectively .

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Figure 4A Immunoblot analysis of TRAF6-mediated ubiquitination of wild type, and mutant FOXP3 molecules. 293T cell lines were transfected with a normal HA-tagged Foxp3 construct, another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines (20R), or one of 20 single lysine-containing constructs with only the indicated lysine residue available for modification. These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags, respectively. Negative controls did not receive TRAF6 or expressed FOXP3 alone. Labeled FOXP3 proteins were pulled down from cell lysates (anti-HA) and ubiquitinated species were visualized by immunoblotting for FLAG.B Assessing the impact of K262 mutation on FOXP3 ubiquitination by TRAF6. 293T cells were transfected with combinations of expression constructs encoding Myc-TRAF6, FLAG-ubiquitin, and an HA-tagged FOXP3 molecule that was either normal (HA-FOXP3) or possessed a K-to-R mutation at residue 262 (K262R). FOXP3 proteins were pulled down with anti-HA beads and either ubiquitinated proteins or total FOXP3 proteins were detected by probing for FLAG and HA, respectively.


Figure 5A Impact of K63 - ubiquitination loss on FOXP3 ' s gene silencing capacity . Jurkat T cells transfected with a wild type Foxp3 expression vector , one encoding the K262R mutant , or an empty ( control ) vector also received a duel luciferase reporter construct where luciferase expression was under the control of the Il2 promoter . After activation with PMA and ionomycin for 8 hours , luciferase activity was assayed . B IL - 2 production in the presence of wild type and K63 - ubiquitination resistant FOXP3 . Naïve CD4 + T cells isolated from Traf6fl / flCD4Cre + or WT mice were activated overnight by anti - CD3 / CD28 antibodies , then transduced by retroviruses carrying normal Foxp3 , the K262R mutant , or an empty vector . Ires - GFP in the retroviral vector serves as an internal control . GFP + cells were sorted out 48 hours post - transduction . The sorted cells were cultured for one additional day . Culture supernatants were collected for measuring the levels of IL - 2 by ELISA . C Cellular distribution of wild type and K262R FOXP3 . Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant . The relative overlap of FOXP3 protein signal and DAPI - stained nuclei was observed by fluorescence microscopy . ( blue : DAPI , green : FOXP3 , Scale bars : 50 μm ) . D Immunostaining of FOXP3 in Traf6fl / flCD4Cre + and wild type ( Traf6fl / flCD4Cre - ) derived suspensions of lymph node and spleen cells . ( blue : DAPI , green : FOXP3 , Scale bars : 50 μm ) . E Quantification of FOXP3 distribution in murine Tregs . The degree of FOXP3 - nuclear coIocalization in CD4 + / FOXP3 + cells isolated from the lymph nodes and spleens of Traf6fl / flCD4Cre + and wild type ( Traf6fl / flCD4Cre - ) mice was determined by Imagestream analysis , and the frequencies of cells with either a low or high probability of nuclear FOXP3 distribution are shown ( red : nuclear stain , green : FOXP3 , Scale bars : 10 um ) .

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Figure 5A Impact of K63-ubiquitination loss on FOXP3's gene silencing capacity. Jurkat T cells transfected with a wild type Foxp3 expression vector, one encoding the K262R mutant, or an empty (control) vector also received a duel luciferase reporter construct where luciferase expression was under the control of the Il2 promoter. After activation with PMA and ionomycin for 8 hours, luciferase activity was assayed.B IL-2 production in the presence of wild type and K63-ubiquitination resistant FOXP3. Naïve CD4+ T cells isolated from Traf6fl/flCD4Cre+ or WT mice were activated overnight by anti-CD3/CD28 antibodies, then transduced by retroviruses carrying normal Foxp3, the K262R mutant, or an empty vector. Ires-GFP in the retroviral vector serves as an internal control. GFP+ cells were sorted out 48 hours post-transduction. The sorted cells were cultured for one additional day. Culture supernatants were collected for measuring the levels of IL-2 by ELISA.C Cellular distribution of wild type and K262R FOXP3. Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant. The relative overlap of FOXP3 protein signal and DAPI-stained nuclei was observed by fluorescence microscopy. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).D Immunostaining of FOXP3 in Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) derived suspensions of lymph node and spleen cells. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).E Quantification of FOXP3 distribution in murine Tregs. The degree of FOXP3-nuclear coIocalization in CD4+/FOXP3+ cells isolated from the lymph nodes and spleens of Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) mice was determined by Imagestream analysis, and the frequencies of cells with either a low or high probability of nuclear FOXP3 distribution are shown (red: nuclear stain, green:FOXP3, Scale bars: 10 um).


Figure 6The in vitro suppressive potency of wild type - and K262R Foxp3 - expressing T cells . Naïve CD4 + T cells were purified from Thy1 . 1 + BALB / c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262 . These engineered " Tregs " were then co - cultured with naïve responder CD4 + T cells stained with CFSE at the indicated ratios and activated with anti - CD3 / CD28 antibodies . Dilution of CFSE signal by the responder populations ( i . e . proliferation ) was assessed by flow cytometry . The in vitro suppressive potency of wild type - and K262R Foxp3 - expressing T cells . Naïve CD4 + T cells were purified from Thy1 . 1 + BALB / c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262 . These engineered " Tregs " were then co - cultured with naïve responder CD4 + T cells stained with CFSE at the indicated ratios and activated with anti - CD3 / CD28 antibodies . Dilution of CFSE signal by the responder populations ( i . e . proliferation ) was assessed by flow cytometry . C In vivo suppression of colitis by wild type - and K262R mutant - Foxp3 expressing T cells . 2x105 naïve CD4 + transductants from A were mixed with 1x106 colitogenic naïve CD4 + T cells from wild type mice and transferred i . v . into Rag2 - / - mice . Rag2 - / - mice receiving no cell transfers , naïve CD4 + T cells alone , or purified wild type Tregs served as controls ( n = 6 / group ) . Recipient mouse body weights were monitored weekly for each group . D , E After 8 weeks , colons were excised , fixed in buffered formalin , and processed for H and E staining ( D ) and histopathology scoring ( E ) . Scale bars shown in 1 mm ) .

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Figure 6The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.C In vivo suppression of colitis by wild type- and K262R mutant-Foxp3 expressing T cells. 2x105 naïve CD4+ transductants from A were mixed with 1x106 colitogenic naïve CD4+ T cells from wild type mice and transferred i.v. into Rag2-/- mice. Rag2-/- mice receiving no cell transfers, naïve CD4+ T cells alone, or purified wild type Tregs served as controls (n= 6/group). Recipient mouse body weights were monitored weekly for each group.D, E After 8 weeks, colons were excised, fixed in buffered formalin, and processed for H and E staining (D) and histopathology scoring (E). Scale bars shown in 1 mm).


Figure 1B Western blot analysis of liver lysates ( 100 µg ) shows that intein - mediated protein trans - splicing efficiently reconstitutes full - length eGFP . Single AAV : n = 5 ; AAV - intein : n = 5 . Arrows indicate both full - length eGFP and excised inteins . C Quantification of eGFP protein bands . Values are reported as mean ± SEM . Each dot represents the eGFP protein band quantification from animals injected with either single AAV n = 5 or AAV intein n = 5 .

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Figure 1B Western blot analysis of liver lysates (100 µg) shows that intein-mediated protein trans-splicing efficiently reconstitutes full-length eGFP. Single AAV: n=5; AAV-intein: n=5. Arrows indicate both full-length eGFP and excised inteins. C Quantification of eGFP protein bands. Values are reported as mean±SEM. Each dot represents the eGFP protein band quantification from animals injected with either single AAV n=5 or AAV intein n=5.


Figure 2B Western blot analysis of lysates of HEK293 cells 72 hours post - transfection with the various F8 variants . Neg , non - transfected cells . C Chromogenic assay of F8 activity in the medium of transfected cells are reported as International Units / deciliter ( IU / dl ) . Data are presented as mean ± SEM . Each dot within the same group corresponds to a biological replicate : Neg n = 5 ; Wild - type n = 8 ; N6 n = 8 ; SQ n = 7 ; V3 n = 6 . Significant differences between groups were assessed using The Kruskal - Wallis test followed by the posthoc analysis : Nemenyi ' s All - Pairs Rank Comparison Test . The Kruskal - Wallis test p value = 2 . 88e - 05 . * * indicates the significant difference between the N6 and the Wild - type groups : p value ≤ 0 . 01 . * indicates the significant difference between the V3 and the Wild - type groups : p value ≤ 0 . 05 .

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Figure 2B Western blot analysis of lysates of HEK293 cells 72 hours post-transfection with the various F8 variants. Neg, non-transfected cells.C Chromogenic assay of F8 activity in the medium of transfected cells are reported as International Units/deciliter (IU/dl). Data are presented as mean±SEM. Each dot within the same group corresponds to a biological replicate: Neg n=5; Wild-type n=8; N6 n=8; SQ n=7; V3 n=6. Significant differences between groups were assessed using The Kruskal-Wallis test followed by the posthoc analysis: Nemenyi's All-Pairs Rank Comparison Test. The Kruskal-Wallis test p value = 2.88e-05. **indicates the significant difference between the N6 and the Wild-type groups: p value ≤ 0.01.*indicates the significant difference between the V3 and the Wild-type groups: p value ≤ 0.05.


Figure 3A Western blot ( WB ) of protein lysates of HEK293 cells 72 hours post - transfection ( n = 3 ; biological replicates ) with either Npu inteins or Heterologous ( N - intein DnaB + C - intein DnaE ) split - inteins . I + II , N6 split - intein proteins ; I + II ( Het . ) , heterologous split - intein proteins ; I , 5 ' N6 coding sequence ( CDS ) - N - DnaE protein ; I ( N - int B ) , 5 ' N6 CDS - N - DnaB . II , C - DnaE - 3 ' N6 CDS protein . Excised inteins ( ∼ 12kDa ) are present only in the down part of the blot when I + II ( Npu inteins ) are provided . Arrows indicate the full - length N6 protein , single halves and excised inteins . B WB of medium of the transfected cells showing the secreted proteins ( n = 3 ; biological replicates ) . Arrows indicate the full - length N6 protein as well as single halves and excised inteins . C Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units / deciliter ( IU / dl ) . Data are presented as mean ± SEM . Each dot within each group represent a different biological replicate ( n ) : I + II n = 3 ; I + II ( Het . ) n = 3 ; I ( N - int E ) n = 3 ; I ( N - int B ) n = 3 ; II ( C - int E ) n = 3 ; Neg n = 4 . Significant differences between groups were assessed using Kruskal - Wallis rank sum test Kruskal − Wallis , p value = 0 . 013 . * indicates the significant difference between the I + II and the I ( N - int E ) groups : p value ≤ 0 . 05 . * * * indicates the significant difference between the I + II and the Neg groups : p value ≤ 0 . 001 .

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Figure 3A Western blot (WB) of protein lysates of HEK293 cells 72 hours post-transfection (n=3; biological replicates) with either Npu inteins or Heterologous (N-intein DnaB + C-intein DnaE) split-inteins. I + II, N6 split-intein proteins; I+II (Het.), heterologous split-intein proteins; I, 5'N6 coding sequence (CDS)-N-DnaE protein; I (N-int B), 5'N6 CDS-N-DnaB. II, C-DnaE-3'N6 CDS protein. Excised inteins (∼12kDa) are present only in the down part of the blot when I + II (Npu inteins) are provided. Arrows indicate the full-length N6 protein, single halves and excised inteins.B WB of medium of the transfected cells showing the secreted proteins (n=3; biological replicates). Arrows indicate the full-length N6 protein as well as single halves and excised inteins.C Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units/deciliter (IU/dl). Data are presented as mean ±SEM. Each dot within each group represent a different biological replicate (n): I + II n=3; I+II (Het.) n=3; I (N-int E) n=3; I (N-int B) n=3; II (C-int E) n=3; Neg n=4. Significant differences between groups were assessed using Kruskal-Wallis rank sum test Kruskal−Wallis, p value = 0.013. *indicates the significant difference between the I+II and the I (N-int E) groups: p value ≤ 0.05. ***indicates the significant difference between the I+II and the Neg groups: p value ≤0.001.


Figure 4A Western blot ( WB ) of protein lysates of HEK293 cells 72 hpt with the AAV - N6 split - intein plasmids and with the codon - optimised set . I + II , N6 split - intein proteins ; I , 5 ' N6 CDS - N - DnaE protein ; II , C - DnaE - 3 ' N6 CDS protein . ( n = 3 ; biological replicates ) . Arrows indicate the full - length N6 protein , excised inteins and both single halves . Codop : codon - optimised . B WB of medium from the transfected cells showing increased secretion of the Codop - N6 full - length protein compared to the non - codon - optimised . ( n = 3 ; biological replicates ) . Arrows indicate the full - length N6 protein , excised inteins and both single halves . C Chromogenic assay performed on the medium from transfected cells to measure F8 activity levels reported as International Units / deciliter ( IU / dl ) ( n = 3 ; biological replicates ) . Data are presented as mean ± SEM . Significant differences between groups were assessed using Kruskal - Wallis test p value = 0 . 027 . * indicates the significant difference between the I + II ( N6 split - intein ) and the II ( C - DnaE - 3 ' N6 CDS ) groups : p value ≤ 0 . 05 . * * indicates the significant difference between the Codop I + II and the I ( 5 ' N6 CDS - N - DnaE ) codop groups : p value ≤ 0 . 01 . * * indicates the significant difference between the Codop I + II and the II ( C - DnaE - 3 ' N6 CDS ) codop groups : p value ≤ 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot (WB) of protein lysates of HEK293 cells 72 hpt with the AAV-N6 split- intein plasmids and with the codon-optimised set. I + II, N6 split-intein proteins; I, 5' N6 CDS-N-DnaE protein; II, C-DnaE-3'N6 CDS protein. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves. Codop: codon-optimised.B WB of medium from the transfected cells showing increased secretion of the Codop-N6 full-length protein compared to the non-codon-optimised. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves.C Chromogenic assay performed on the medium from transfected cells to measure F8 activity levels reported as International Units/deciliter (IU/dl) (n=3; biological replicates).Data are presented as mean ±SEM. Significant differences between groups were assessed using Kruskal-Wallis test p value = 0.027. *indicates the significant difference between the I+II (N6 split-intein) and the II (C-DnaE-3'N6 CDS) groups: p value ≤ 0.05. **indicates the significant difference between the Codop I+II and the I (5' N6 CDS-N-DnaE) codop groups: p value ≤ 0.01. **indicates the significant difference between the Codop I+II and the II (C-DnaE-3'N6 CDS) codop groups: p value ≤ 0.01.