CAR$regulates$epithelial$cell$junction$stability$through$control$of$E8cadherin$trafficking$

Penny%E.%Morton,%Alexander%Hicks,%Theodoros%Nastos,%George%Santis,%Maddy%Parsons%

%

Supplemental$figure$legends$

Figure$S1$Car$and$E8cadherin$localisation$in$HBEC$

(A)%FITCBdextran%permeability%assay%of%wildBtype%and%CARGFP%HBEC%cells.%BarBchart%is%

the%mean%of%results%from%three%independent%experiments%normalised%to%wildBtype.%

(B)%Widefield%images%of%WT%and%CARGFP%stained%for%EBcadherin.%(C)%Confocal%

microscopy%of%a%50:50%mix%of%primary%human%bronchial%epithelial%(pBTEC)%cells%

expressing%CARBGFP%and%wildBtype%pBTEC%cells%stained%for%EBcadherin.%Arrows%point%

to%CARBGFP%containing%junctions.%(D)%Confocal%microscopy%of%HECDB1%internalisation%

assay%performed%in%1:1%mix%of%WT%and%CARBGFP%HBEC.%Cells%were%not%acid%stripped%

before%fixation%and%staining%with%antiBmouseB568%to%detect%bounds%HECDB1.%%

Figure$S2$CAR$does$not$reduce$localisation$of$ZO81$or$P8cadherin$to$cell8cell$junctions$$

(A)%Widefield%images%of%WT%and%CARGFP%HBEC%stained%for%ZOB1%(left).%Quantification%

of%ZOB1%intensity%at%cell%junctions%(left).%(B)%Confocal%images%of%1:1%mix%of%WT%and%

CARGFP%HBEC%stained%for%ZOB1.%(C)%Confocal%images%of%WT%and%CARGFP%HBEC%stained%

with%phalloidin%to%detect%FBactin.%(D)%Confocal%images%of%1:1%mix%of%WT%and%CARRFP%

HBEC%stained%for%PBcadherin.%%

Figure$S3$Phosphorylation$of$CAR$does$not$inhibit$Ad5$infection$

(A)%Western%blots%of%CARGFP%immunoprecipitated%from%HBEC%cells%treated%with%6000%

pfu/cell%Ad5GFP%for%30min%or%300nM%CalyculinA%for%15min,%probed%for%phosphoB

serine%or%phosphoBthreonine.%%PBCAR%band%is%the%higher%molecular%weight%species%

arrowed.%(B)%Western%blot%of%untreated%or%Calyculin%A%treated%pHTEC%cells%probed%for%

CAR.%%(C)%FACs%analysis%of%wildBtype,%CARBRFP,%AACARBRFP%and%DDCARBRFP%HBEC%

infected%with%100pfu/cell%Ad5GFP.%(D)%Western%blot%of%lysates%from%WT,%CARGFP,%

AACARGFP%and%DDCARGFP%cells%probed%for%pY418BSrc,%Src%or%HSCB70%as%a%loading%

control.%%

Supplemental$movie$1.$Representative%movie%of$wildBtype%HBEC%grown%in%the%presence%of%calcium.%Calcium%washout%occurred%just%before%frame%51.%Images%were%acquired%at%30%second%intervals.%Playback%rate%of%the%movie%is%20%frames%per%second.%

Supplemental$movie$2.$Representative%movie%of%CARBGFP%HBEC%grown%in%the%presence%of%calcium.%Calcium%washout%occurred%just%before%frame%46.%Images%were%acquired%at%30%second%intervals.%Playback%rate%of%the%movie%is%20%frames%per%second.%

Supplemental$movie$3.$Representative%movie%of%CARBRFP%HBEC%transiently%

expressing%EBcadherinBGFP.%Images%were%acquired%at%10sec%intervals.%Playback%rate%of%

the%movie%is%20%frames%per%second.%%

%