D . Representative time - lapse spinning disk confocal of n = 3 of COS7 stably expressing GFP - TMD ( ER ) ( green ) SiR - lysosome ( orange ) . Zoom insets highlight regions of interest containing a static ( upper panels ) or mobile endosome ( lower panels ) as denoted by arrows . First row shows ER ( green ) , second row shows MDA generated ER movement ( magenta ) , third row shows a merge of two channels fourth row shows a merge of ER ( green ) LE / Lys ( orange ) . Scale bars : full 10µm , zoom inset 3µm . relate to movies EV2 3 . E . Kymograph of mobile vesicle in panel ( D ) . F . MDA of ( D ) . Mobility of static mobile endosomes was quantified normalized to static endosomes . Graph represents mean ± s . d . of analyses at multiple locations within same ( n = 20 per condition from 3 independent experiments ) . acquired for 90 frames at 4Hz . Significance two - student t - , * * * * P < 0 . 0001 .

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

D. Representative time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (ER) (green) and SiR-lysosome (orange). Zoom insets highlight regions of interest containing a static (upper panels) or mobile endosome (lower panels) as denoted by the white arrows. First row shows ER (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Scale bars: full size 10µm, zoom inset 3µm. Images relate to movies EV2 and 3. E. Kymograph of the mobile vesicle in panel (D). F. MDA quantification of (D). Mobility of static and mobile endosomes was quantified and normalized to the static endosomes. Graph represents mean ± s.d. of analyses at multiple locations within the same cell(n=20 cells per condition from 3 independent experiments). All images acquired for 90 frames at 4Hz. Significance two-tailed student t-test, **** P<0.0001.


C . Representative time - lapse spinning disk confocal of n = 3 of live COS7 stably expressing GFP - TMD ( green ) marked ER SiR - lysosome - stained LE / Lys ( orange ) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by arrow . First row represents ER ( GFP - TMD ) ( green ) , second row shows MDA generated ER movement ( magenta ) , third row shows a merge of two preceding channels fourth row shows a merge of ER ( green ) LE / Lys ( orange ) . Samples were treated with either DMSO or 2µm paclitaxel . Scale bar : 30µm zoom insets 3µm . acquired for 90 frames at 1Hz . related to movie EV7 8 D . Kymograph of panel ( C ) . E . MDA of panel ( C ) normalized to control , showing average ER movement surrounding LE / Lys under control or treated conditions overall average ER mobility under control or treatment conditions . Graph represents mean ± s . d . of analyses at multiple locations within same ( n = 20 per condition from 3 independent experiments ) . Significance two - student t - * * * * P < 0 . 0001 ; ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) marked ER and SiR-lysosome-stained LE/Lys (orange) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either DMSO or 2µm paclitaxel. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz.Images related to movie EV7 and 8 D. Kymograph of panel (C). E. MDA quantification of panel (C) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n = 20 cells per condition from 3 independent experiments). Significance two-tailed student t-test **** P<0.0001; ns = not significant.


F . Representative of n = 3 of COS7 stably expressing GFP - TMD ( green ) to mark ER SiR - lysosome stained LE / Lys ( orange ) merged . Zoom insets highlight region of interest containing a mobile endosome as denoted by arrow . First row represents ER ( GFP - TMD ) ( green ) , second row shows MDA generated ER movement ( magenta ) , third row shows a merge of two preceding channels fourth row shows a merge of ER ( green ) LE / Lys ( orange ) . Samples were treated with either siControl or siKTN1 . Scale bar : 30µm zoom insets 3µm . acquired for 90 frames at 1Hz . related to movie EV9 10 G . Kymograph of panel ( F ) . H . MDA of panel ( F ) normalized to control , showing average ER movement surrounding LE / Lys under control or treated conditions overall average ER mobility under control or treatment conditions , respectively . Graph represents mean ± s . d . of analyses at multiple locations within same ( n = 20 per condition from 3 independent experiments ) . Significance two - student t - . * * * * P < 0 . 0001 ; ns = not significant

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

F. Representative images of n = 3 of COS7 cells stably expressing GFP-TMD (green) to mark the ER and SiR-lysosome stained LE/Lys (orange) merged. Zoom insets highlight region of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either siControl or siKTN1. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz. Images related to movie EV9 and 10 G. Kymograph of panel (F). H. MDA quantification of panel (F) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions, respectively. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=20 cells per condition from 3 independent experiments). Significance two-tailed student t-test. **** P<0.0001; ns = not significant


A . Representative time - lapse spinning disk confocal of n = 3 live COS7 stably expressing mCherry - KDEL ( green ) transiently expressing GFP - RAB7 ( orange ) . Zoom insets shows a region of interest containing a mobile RAB7 - positive endosome as denoted by arrows . First row represents ER ( mCherry - KDEL ) ( green ) , second row shows MDA - generated ER movement ( magenta ) , third row shows a merge of two preceding channels fourth row shows a merge of ER ( green ) GFP - RAB7 ( orange ) . Scale bar : 10µm , zoom insets 3µm . related to movie EV11 . were acquired for 90 frames at 1Hz .

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

A. Representative time-lapse spinning disk confocal images of n = 3 live COS7 cells stably expressing mCherry-KDEL (green) and transiently expressing GFP-RAB7 (orange). Zoom insets shows a region of interest containing a mobile RAB7-positive endosome as denoted by the white arrows. First row represents the ER (mCherry-KDEL) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and GFP-RAB7 (orange). Scale bar: 10µm, zoom insets 3µm. Images related to movie EV11. Images were acquired for 90 frames at 1Hz.


B . Kymograph of panel ( A ) . C . MDA of panel ( A ) figures EV2A - G normalized to 1 showing average ER movement surrounding static endosomes mobile endosomes . Graph represents mean ± s . d . of analyses at multiple locations within same ( n = 15 per condition from 3 independent experiments ) . acquired for 90 frames at 1Hz . Significance two - student t - . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ns = not significant .

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

B. Kymograph of panel (A). C. MDA quantification of panel (A) and figures EV2A-G normalized to 1 showing average ER movement surrounding static endosomes and mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. *P<0.05, ** P<0.01, **** P<0.0001 ns = not significant.


E . Representative time - lapse spinning disk confocal of n = 3 of live COS7 stably expressing GFP - TMD ( green ) in lower panels transiently expressing mScarlet - RILP ( magenta ) SiR - lysosome - stained LE / Lys ( orange ) . Zoom insets shows region of interest containing ER ( GFP - TMD ) . First row represents ER ( GFP - TMD ) ( green ) , second row shows MDA - generated ER movement ( magenta ) , third row shows a merge of two preceding channels . Scale bar : 10µm , zoom insets 3µm . related to movie EV19 movie EV20 . were acquired for 90 frames at 1Hz . F . MDA of panel ( E ) figure EV3A normalized to 1 showing total average ER movement . Graph represents mean ± s . d . of analyses at multiple locations within same ( n = 15 per condition from 3 independent experiments ) . acquired 90 frames at 1Hz . Significance two - student t - . * * * * P < 0 . 0001 ns = not significant .

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

E. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) and in lower panels transiently expressing mScarlet-RILP (magenta) and SiR-lysosome-stained LE/Lys (orange). Zoom insets shows region of interest containing ER (GFP-TMD). First row represents ER (GFP-TMD) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels. Scale bar: 10µm, zoom insets 3µm. Images related to movie EV19 and movie EV20. Images were acquired for 90 frames at 1Hz. F. MDA quantification of panel (E) and figure EV3A normalized to 1 showing total average ER movement. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired 90 frames at 1Hz. Significance two-tailed student t-test. **** P<0.0001 ns = not significant.


B . Representative stills of spinning disk confocal of n = 3 of COS7 stably expressing TMD - GFP ( green ) SiR - lysosome - stained endosomes / lysosomes ( orange ) . COS7 in middle panels overexpress mCherry - RILP ( magenta ) . COS7 in lower panels overexpress mCherry - TMEM55B ( magenta ) . Zoom insets show region of interest containing ER ( TMD - GFP ) ( green ) junction of skeletonized ER where number of ER junctions representing quantified average . Scale bars : 15µm . zoom insets : 3µm . C . of number of ER junctions per µm2 from panel ( B ) as resulted from ER junction . Graph represents mean ± s . d . of analyses at multiple locations within one ( n = 15 from 3 independent experiments ) . Significance two - student t - . * * * P < 0 . 001 .

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

B. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange). COS7 cells in the middle panels overexpress mCherry-RILP (magenta). The COS7 cells in the lower panels overexpress mCherry-TMEM55B (magenta). Zoom insets show region of interest containing the ER (TMD-GFP) (green) and junction analysis of the skeletonized ER where the number of ER junctions representing the quantified average. Scale bars: 15µm. zoom insets: 3µm. C. Quantification of the number of ER junctions per µm2 from panel (B) as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell (n=15 from 3 independent experiments). Significance two-tailed student t-test. ***P<0.001.


A . Representative of time - lapse spinning disk confocal of n = 3 of COS7 stably expressing GFP - TMD ( green ) SiR - lysosome - stained LE / Lys ( orange ) treated with either siControl or co - treated with siVAPA , siVAPB siMOSPD2 . Zoom insets show regions of interest containing mobile LE / Lys as denoted by arrow . First row represents ER ( TMD - GFP ) ( green ) , second row shows MDA generated ER movement ( magenta ) , third row shows a merge of two preceding channels fourth row shows a merge of ER ( green ) SiR - lysosome stained LE / Lys ( orange ) . Scale bar : 5µm , zoom insets 3µm . related to movie EV40 47 . were acquired for 90 frames at 1Hz .

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

A. Representative images of time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (green) and SiR-lysosome-stained LE/Lys (orange) treated with either siControl or co-treated with siVAPA, siVAPB and siMOSPD2. Zoom insets show regions of interest containing mobile LE/Lys as denoted by the white arrow. First row represents ER (TMD-GFP) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and SiR-lysosome stained LE/Lys (orange). Scale bar: 5µm, zoom insets 3µm. Images related to movie EV40 and 47. Images were acquired for 90 frames at 1Hz.


B . Representative stills of spinning disk confocal of n = 3 of COS7 stably expressing TMD - GFP ( green ) SiR - lysosome - stained endosomes / lysosomes ( orange ) treated with siControl or with siVAPA , siVAPB siMOSPD2 . Zoom insets show region of interest containing ER ( TMD - GFP ) ( green ) junction of skeletonized ER where number of ER junctions representing quantified average . Scale bars : 10µm . zoom insets : 3µm .

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

B. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange) treated with siControl or with siVAPA, siVAPB and siMOSPD2. Zoom insets show region of interest containing ER (TMD-GFP) (green) and junction analysis of skeletonized ER where number of ER junctions representing the quantified average. Scale bars: 10µm. zoom insets: 3µm.


C . MDA of ER of panels ( A , B ) fig . EV4B - G normalized to control showing average ER movement surrounding mobile endosomes . Graph represents mean ± s . d . of analyses at multiple locations within same ( n = 30 per condition from 3 independent experiments ) . acquired for 90 frames at 1Hz . Significance two - student t - . * * P < 0 . 01 , * * * * P < 0 . 0001 , ns = not significant D . MDA of SiR - lysosome - stained LE / Lys of ( A , B ) fig . EV4B - G normalized to 1 showing total average endosomal movement . Graph represents mean ± s . d . of n = 15 per condition from 3 independent experiments . Significance two student t - . ns = not significant . E . of number of ER junctions per µm2 from ( B ) fig . H - M as resulted from ER junction . Graph represents mean ± s . d . of analyses at multiple locations within one of n = 15 from 3 independent experiments . Significance two - student t - . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns = not significant .

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

C. MDA quantification of ER of panels (A, B) and fig. EV4B-G normalized to control cells showing average ER movement surrounding mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=30 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. **P<0.01, ****P< 0.0001, ns=not significant D. MDA quantification of SiR-lysosome-stained LE/Lys of (A, B) and fig. EV4B - G normalized to 1 showing total average endosomal movement. Graph represents mean ±s.d. of n = 15 cells per condition from 3 independent experiments. Significance two tailed student t-test. ns= not significant. E. Quantification of number of ER junctions per µm2 from (B) and fig. H - M as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell of n = 15 from 3 independent experiments. Significance two-tailed student t-test. *P<0.05, **P<0.01 ***P<0.001, ****P< 0.0001, ns=not significant.


ASPC - 1 , MiaPaCa - 2 , Colo - 357 , Panc - 1 , Panc - 89 PancTu - 1 were grown in absence or presence of indicated concentrations of omeprazole , 5 - fluorouracile , pantoprazole or gemcitabine , respectively , for 4 days to determine IC50 values . IC50 ies sigmoidicities are gradually differing between lines . In lower concentrations a slight growth - stimulatory effect ( hormesis ) was observed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ASPC-1, MiaPaCa-2, Colo-357, Panc-1, Panc-89 and PancTu-1 cells were grown in the absence or presence of the indicated concentrations of omeprazole, 5-fluorouracile, pantoprazole or gemcitabine, respectively, for 4 days to determine the IC50 values. The IC50 ies and the sigmoidicities are gradually differing between the cell lines. In lower concentrations a slight growth-stimulatory effect (hormesis) was observed.


lines ASPC - 1 , MiaPaCa - 2 , Colo357 , Panc - 1 , Panc89 PancTu1 were untreated or treated with 5 - FU alone or in combination with indicated concentrations of OMP for 4 days . At lower doses of 5 - FU alone ( black lines , 0 µg / ml OMP ) a growth - stimulatory effect ( hormesis ) was observed in lines ASPC - 1 , Panc - 1 PancTu - 1 . data points show means of 8 measurements . curves are fitted in these lines using Brain - Cousens model ( see sub - section 4 . 11 ) . In MiaPaCa - 2 Panc - 89 no hormesis occured , these curves were fitted using a three parameter logistic model . In Colo357 curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations ( data points shown ) . red , green blue lines indicate dose - effect curves of 5 - FU when various concentrations of OMP were added ( 10 , 20 40 µg / ml , respectively ) . In ASPC - 1 Panc - 1 hormesis of 5 - FU was reversed in PancTu - 1 it was mitigated by OMP depending on of 5 - FU . In MiaPaCa - 2 we found an additive interaction of 5 - FU with OMP . In Panc - 89 interaction was antagonistic .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

The cell lines ASPC-1, MiaPaCa-2 , Colo357, Panc-1, Panc89 and PancTu1 were untreated or treated with 5-FU alone or in combination with the indicated concentrations of OMP for 4 days. At lower doses of 5-FU alone (black lines, 0 µg/ml OMP) a growth-stimulatory effect (hormesis) was observed in the cell lines ASPC-1, Panc-1 and PancTu-1. The data points show the means of 8 measurements. The curves are fitted in these cell lines using the Brain-Cousens model (see sub-section 4.11). In MiaPaCa-2 and Panc-89 cells no hormesis occured, these curves were fitted using a three parameter logistic model. In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations (data points shown). The red, green and blue lines indicate the dose-effect curves of 5-FU when various concentrations of OMP were added (10, 20 and 40 µg/ml, respectively). In ASPC-1 and Panc-1 cells the hormesis of 5-FU was reversed and in PancTu-1 cell it was mitigated by OMP depending on the concentration of 5-FU. In MiaPaCa-2 cells we found an additive interaction of 5-FU with OMP. In Panc-89 cells the interaction was antagonistic.


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

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O

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.


( A ) ASPC - 1 without treatment ( 800 fold ) . ( B ) ASPC - 1 undergoing apoptosis upon 160 µg / ml OMP after 24 hours ( 800 fold ) . Vacuolisation of cytoplasma condensation of nucleus are visible . ( C ) Phagophores autophagosomes in a segment of an ASPC - 1 treated with omeprazole 80 µg / ml for 24 hours ( 2800fold enlargement ) . phagophores are characterised by a cup - like shape ( arrows ) . Autophagosomes are closed particles , number of which is increased in treated ( black arrows ) . ( D ) Early phagophores autophagosomes are also found in MiaPaCa - 2 treated with OMP 80 µg / ml after 24 hours in a perinuclear region containing lysosomes Golgi . In contrast to ASPC - 1 , early signs of apoptosis such as vacuolization , are also present .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 numbers of autophagosomes lysosomes per in MiaPaCa - 2 ASPC - 1 untreated or treated with 5 - FU , OMP or combination of both for 24 hours with standard errors . Significant differences compared to control are marked by * . In ASPC - 1 there were significant differences compared to control in OMP group ( p : 0 . 03 ) 5 - FU + OMP group ( p : 0 . 03 ) . In MiaPaCa - 2 5 - FU + OMP group differed significantly from control ( p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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).


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

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(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.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

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.


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

O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O

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.


Bad , Puma Atg12 were detected in MiaPaCa - 2 ASPC - 1 . Bad expression is slightly increased by 5 - FU suppressed by OMP 5 - FU + OMP in ASPC - 1 , but not in MiaPaCa - 2 . Puma is induced by OMP in both lines indicating involvement of BH3 - only pathway . Atg12 is strongly enhanced by OMP in both 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O

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.


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

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

E 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).


A Representative of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) mature MGs stained with glutamate synthase ( GS , magenta ) at 4 dpf in Notch ligands ( dla - / - / dlb - / - / dlc - / - / dld - / - / jag1b - / - / jag2b - / - ) mutants .

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

A 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 numbers of Notch activation ( Tp1 + ) ( B ) mature MG ( GS + ) ( 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 ± ; * * * p < 0 . 001 , t - ) .

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

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).


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

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

A 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).


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

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

D 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).


A Representative of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) 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 numbers of with active Notch ( Tp1 + ) mature MG ( GS + ) 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 ± ; * * p < 0 . 01 ; * * * p < 0 . 001 , t - ) .

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

A 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 showing that GS + MGs ( magenta ) were rarely observed when H2B - CFP expression ( green ) was driven by gfap promoter in 5 - dpf jag2b - / - mutants . between upper dashed solid lines is outer layer ( ONL ) . in middle two dash lines is inner plexiform layer ( IPL ) . INL , inner layer ; GCL , ganglion layer . D Representative showing that GS + MGs ( magenta ) were frequently rescued underneath with notch3 expression ( green ) driven by gfap promoter in 5 - dpf jag2b - / - mutants . Layer structure is similar as ( C ) . E number of GS + MGs ( magenta ) with or without CFP expression driven by gfap promoter . Ctrl : H2B - CFP expression ( green ) driven by gfap promoter ( n = 5 fish ) ; rescue : notch1b ( n = 13 fish ) notch3 ( n = 17 fish ) NICD - H2B - CFP expression ( green ) driven by gfap promoter ( Mean ± ; * p < 0 . 05 , * * * p < 0 . 001 , ns , no significant difference , pair - wised t - ) . F proportions of notch1b notch3 NICD - misexpressing ( 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 ± ; * * * p < 0 . 001 , t - ) .

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

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).


Maximum projection ( 2 z - slices ) of HeLa immunostained for F - actin ( Phalloidin ) , Tubulin , Pericentrin DAPI at metaphase early anaphase showing actin accumulation around centrosomes in early anaphase . Scale bar - 10µm of F - actin ( Phalloidin ) around pericentrin positive centrosomes in HeLa immunostained as in ( A ) , showing 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 - , * * * * p < 0 . 0001 ;

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

Maximum 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 from a representative Hela expressing Lifeact - GFP transiting from metaphase to anaphase , showing accumulation of actin in presumptive centrosomal region , red arrows . represent 4z - projection . Scale bar - 10µm .

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

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 of a representative HeLa expressing GFP - alpha - tubulin RFP - Lifeact transiting from metaphase to anaphase showing changes in actin microtubules . represent 4 z - projection of movies taken every 1min . Bottom - higher magnification view of centrosomes C1 C2 from time - lapse above showing a transient increase in actin around centrosomes in early anaphase . Scale bar = 10µm , 1µm in zoom . T = 0 is one frame before anaphase onset . Dotted circle shows centrosome position .

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

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 of GFP - alpha - tubulin RFP - Lifeact around centrosomes for a population of exiting mitosis , showing actin accumulation around centrosomes a simultaneous decrease in tubulin over time . N = 30 centrosomes ( 15 ) . T = 0 is one frame before anaphase onset . Graph shows mean + / - SD , values were normalized to at T = 0 . comparison of actin ( RFP - Lifeact ) tubulin ( a - tubulin - GFP ) around centrosomes at metaphase ( - 4 to - 1min ) , early anaphase ( 2 - 5min ) late anaphase ( 7 - 10min ) for a population of . Mean actin 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Stills from time - lapse of HeLa expressing GFP - alpha - tubulin RFP - lifeact arrested at prometaphase with STLC ( t = 0 ) forced to exit mitosis with Cdk1 inhibition ( Ro3306 ) imaged every 90 seconds . Scale bar = 10µm for zoom = 4µm . n = 21 from 4 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Stills 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.


of actin around centrosomes when 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 .

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

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 expressing GFP - centrin1 arrested at prometaphase or forced mitotic exit ( Ro3306 5 ' ) stained with phalloidin . Scale bar - 5µm for zoom - 2µm .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

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.


level of actin around centrosome in ( C ) was quantified normalised relative to metaphase 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 - with Welch correction , * * * * p < 0 . 0001 . of tubulin around centrosomes during prometaphase arrested or forced to exit with Cdk1 inhibitor . Tubulin 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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.


Maximum projection ( 2 - z slices ) view of Hela pre - treated with DMSO 0 . 2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around centrosomes during anaphase . Scale bar - 10µm . of actin around centrosomes for treated with DMSO or 0 . 2mM CK666 , showing reduction in actin accumulation around 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 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Maximum 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 expressing GFP - centrin1 pre - treated with DMSO or 0 . 2mM CK666 during prometaphase arrest forced mitotic exit stained with phalloidin for F - actin .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

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.


of level of actin around centrosome from ( C ) , which shows 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 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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.


of tubulin around centrosomes for treated with DMSO or 0 . 2mM CK666 , showing 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 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


of tubulin around centrosomes for treated with DMSO or 0 . 2mM CK666 , during monopolar exit , showing 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 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

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.


Z - projection of HeLa expressing GFP - centrin1 immunostained for p34 - Arc ( sub - domain Arp2 / 3 ) following prometaphase arrest forced mitotic exit for 5min . N = 3 independent experiments . Scale bar = 10µm for zoom 2µm .

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

Z-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.


of p34 - Arc around centrosome ( 3um ) were quantified relative to metaphase 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 - with Welch correction . Error bars represent standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 expressing GFP - centrin1 during prometaphase arrest forced exit , which were immunostained with p34 - Arc . N = 2 independent experiments . Scale bar = 2µm .

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

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.


of p34 - Arc around 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 - with Welch correction . Error bars represent standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 on centrosomes isolated from prometaphase arrested forced to exit mitosis showing centrosomal actin nucleation over time . Scale bar = 10µm of actin nucleation from isolated centrosomes as in ( E ) over an of 2 μm , ( circle ) , showing an increase in actin nucleation around centrosomes isolated from undergoing forced mitotic exit . N = 2 isolations , 5 independent experiments , n = 16 STLC n = 19 STLC RO .

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

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 - red - labelled actin . Red actin was used first , followed by Capping , then green - labelled actin . accumulation of green in centre indicates that actin is nucleated at centrosome . Scale bar = 10µm . of ( G ) over time . N = 2 isolations , 2 independent experiments , n = 19 STLC n = 14 STLC RO . Error bars represent standard deviation .

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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 actin filament formation at centrosomes isolated from undergoing forced exit when pre - treated with DMSO or CK666 . Scale bar = 10µm . of ( I ) , showing that centrosomal actin nucleation fails when centrosomes isolated from 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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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.


Phos - tag Western blot of MCF7 - Her2 - 18 synchronised into distinct phases of cycle showing phospho - shift in WASH1 during mitosis .

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Phos-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 of WASH1 isolated from STLC treated prometaphase arrested when compared to forced to exit mitosis following Ro3306 treatment .

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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.


Time - lapse of HeLa expressing GFP - alpha tubulin RFP - lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase forced to exit mitosis with Ro3306 addition ( t = 0 ) . Scale bar = 5µm .

Time-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.


of actin around centrosome in ( A ) , showing failure to accumulate actin around centrosome in siWASH . N = 4 experiments . Error bars represent standard deviation .

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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 treated with siRNA control siRNA against WASH1 probed with WASH1 antibody , showing reduction in WASH following treatment of with siRNA against WASH . Scale bar = 5µm 2µm in zoom .

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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 expressing GFP - centrin1 treated with control siRNA or siRNA against WASH , arrested at prometaphase or forced to exit mitosis immunostained with phalloidin . Scale bar - 5µm of actin around centrosome in ( F ) , showing failure to accumulated actin around centrosome during forced exit in 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 .

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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 with centrosomes isolated from undergoing forced exit were pre - treated with either no antibody or anti - WASH1 antibody for 1 hour . Scale bar - 10µm of in vitro from ( F ) , showing 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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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.


A TRAF6 expression in differentiating CD4 + T . Naïve CD4 + T were obtained from wild type C57BL / 6 mice by FACS activated with anti - CD3 / CD28 ( 1µg , 2µg / ml ) for indicated times in presence of distinct Thelper lineage - directing cytokines or under neutral activation conditions ( Th0 ) . After total extraction cDNA conversion , RT - PCR determined of Traf6 mRNA in differentiating Th0 , Th1 , Th17 iTregs was determined .

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A 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 non - Treg CD4 + T . Human Tregs ( CD3 + / CD4 + / CD8 - / CD25HIGH / CD127low / CD39 + ) non - Treg CD4 + T ( CD3 + / CD4 + / CD8 - / CD25 - ) were obtained from peripheral blood of healthy donors by FACS after Ficoll - Paque PLUS gradient centrifugation magnetic bead enrichment of CD4 + T . TRAF6 mRNA was measured by qRT - PCR .

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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 lymph nodes were recovered from Traf6fl / flFoxp3Cre + mice Traf6fl / fl at 8 of ( Scale bars : 5 mm ) . ( D ) cellularity of lymphoid tissues of Traf6fl / flFoxp3Cre + mice their Traf6fl / fl Foxp3Cre - was determined ( 8 mice / group ) .

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 activation . frequencies of effector ( CD44high / CD62Llow ) , memory ( CD44high / CD62Lhigh ) naïve ( CD44low / CD62Lhigh ) in CD4 + T compartments of Traf6fl / fl Traf6fl / flFoxp3Cre + mice were determined by flow cytometry ( 5 mice / group ) .

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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 activation . frequencies of effector ( CD44high / CD62Llow ) , memory ( CD44high / CD62Lhigh ) naïve ( CD44low / CD62Lhigh ) in CD8 + T compartments of Traf6fl / fl Foxp3Cre - ( wild type ) Traf6fl / flFoxp3Cre + mice were determined by flow cytometry ( 5 mice / group ) .

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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 were isolated from Traf6fl / flFoxp3Cre + Traf6fl / fl mice differentiated into iTregs . Conditions of sub - optimal TGFβ concentrations ( 0 . 5ng / ml , 0 . 05ng / ml ) were tested as well intracellular FOXP3 was measured after 4 days .

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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.


A In vivo suppressive function of WT Traf6fl / flFoxp3Cre + Tregs . Tregs from indicated mice ( CD45 . 2 + ) were isolated by FACS , as were naïve ( CD62Lhigh / CD25 - ) CD4 + responder T from congenically distinct ( CD45 . 1 + ) donor mice . Tregs Tresponders were mixed at a 1 : 5 ( 2x105 : 10x105 ) before injection into Rag2 - / - mice . 7 days later , spleens were harvested , relative frequencies absolute numbers of each transferred populations were determined by flow cytometry .

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A 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 + wild type ( Traf6fl / flFoxp3Cre - ) mice . 1x105 B16F10 were injected subcutaneously ( s . c . ) into shaved flanks of indicated mice ( n = 5 / group ) . volumes were monitored every 3 days .

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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 - bearing mice with without Treg - specific TRAF6 expression . suspensions of - draining lymph node - infiltrating leukocytes ( TILs ) were recovered after 21 days of growth . Ex vivo stimulation with PMA ionomycin in presence of Golgistop for 5 hour preceded intracellular for IFNγ IL - 17 flow cytometry .

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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 in bearing Traf6fl / flFoxp3Cre + wild type ( Traf6fl / flFoxp3Cre - ) mice . frequency of FOXP3 + in CD4 + of - draining lymph nodes ( dLN ) , peripheral , non - draining lymph nodes ( pLN ) , spleens , TILs were determined by flow cytometry .

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.


A Assessment of K63 - type ubiquitination of FOXP3 upon expression of wild type catalytically deficient TRAF6 mutants . 293T were transfected with given combinations of vectors encoding wild type TRAF6 , enzymatically deficient L74H or C70A TRAF6 mutants , HA - FOXP3 , FLAG - labeled ubiquitin molecules possessing a single lysine residue ( K63 ) that restrain possible ubiquitin monomer linkages on polyubiquitinated to K63 - type only . were lysed modified by K63 - ubiquitin chains were recovered from lysates by pulling down using a bead - immobilized K63 - specific TUBE reagent . presence of FOXP3 among these modified was determined by immunoblotting as were of FOXP3 TRAF6 in pre - IP whole lysate ( WCL ) .

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A 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 its facilitation of K63 - linked ubiquitination . 293T expressing indicated constructs encoding HA - FOXP3 FLAG - TRAF6 were lysed incubated with bead immobilized anti - FLAG antibodies . FOXP3 molecules co - IPed in this manner were resolved by SDS PAGE detected by immunoblotting with anti - HA antibodies .

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 were transfected with indicated combinations of expression constructs encoding HA - FOXP3 , Myc - TRAF6 , 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 - K63 - type polyubiquitination , respectively . .

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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 TRAF6 in murine iTreg lysate . Naïve CD62Lhigh / CD25 - / CD4 + T were obtained by FACS activated with anti - CD3 / CD28 antibodies ( 1ug 2ug / ml , respectively ) in presence of IL - 2 TGFβ ( 100U / ml 5ng / ml , respectively ) for four days before lysis . Antibodies against TRAF6 ( left ) or FOXP3 ( right ) were used to pull down target their interaction partners , which were resolved under denaturing conditions visualized by immunoblotting .

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 FOXP3 pools of Traf6fl / flFoxp3Cre + Traf6wt / wtFoxp3Cre + mice . CD4 + / YFP + from Traf6fl / flFoxp3Cre + ( " Traf6 - / - " ) or wild type Traf6wt / wtFoxp3Cre + mice ( " Traf6 + / + " ) were isolated by FACS , lysed either total FOXP3 ( right ) or K63 - ubiquitin - modified ( left ) were immunoprecipitated . of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti - K63 - ubiquitin FOXP3 , respectively , of total FOXP3 actin were also measured by immunoblot of whole lysate .

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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.


A Immunoblot of TRAF6 - mediated ubiquitination of wild type , mutant FOXP3 molecules . 293T lines were transfected with a normal HA - tagged Foxp3 construct , another encoding a ubiquitination resistant mutant in which lysine residues were replaced by arginines ( 20R ) , or one of 20 single lysine - containing constructs with only indicated lysine residue available for modification . These lines also received expression vectors encoding TRAF6 ubiquitin molecules labeled with Myc FLAG tags , respectively . Negative controls did not receive TRAF6 or expressed FOXP3 alone . Labeled FOXP3 were pulled down from lysates ( anti - HA ) ubiquitinated species were visualized by immunoblotting for FLAG .

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A 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 impact of K262 mutation on FOXP3 ubiquitination by TRAF6 . 293T were transfected with combinations of expression constructs encoding Myc - TRAF6 , FLAG - ubiquitin , an HA - tagged FOXP3 molecule that was either normal ( HA - FOXP3 ) or possessed a K - to - R mutation at residue 262 ( K262R ) . FOXP3 were pulled down with anti - HA beads either ubiquitinated or total FOXP3 were detected by probing for FLAG HA , respectively .

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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.


A Impact of K63 - ubiquitination loss on FOXP3 ' s gene silencing capacity . Jurkat T transfected with a wild type Foxp3 expression vector , one encoding K262R mutant , or an empty ( control ) vector also received a duel luciferase reporter construct where luciferase expression was under control of Il2 promoter . After activation with PMA ionomycin for 8 hours , luciferase activity was assayed .

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A 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 presence of wild type K63 - ubiquitination resistant FOXP3 . Naïve CD4 + T isolated from Traf6fl / flCD4Cre + or WT mice were activated overnight by anti - CD3 / CD28 antibodies , then transduced by retroviruses carrying normal Foxp3 , K262R mutant , or an empty vector . Ires - GFP in retroviral vector serves as an internal control . GFP + were sorted out 48 hours post - transduction . sorted were cultured for one additional day . Culture supernatants were collected for measuring of IL - 2 by ELISA .

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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.