hBD3 enters cells more quickly than polyI:C and does not co-localise with polyI:C or endosome marker EEA1.

Wildtype BMDM were treated with (A)TAMRA-hBD3 or (B) FITC-polyI:C (pI:C) and imaged at 2 (i),10 (ii) and 30 (iii) minutes. BMDM treated for 30 min with (C) FITC-pI:C (green) or (D) TAMRA-hBD3 were stained with anti-EEA1 antibody. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) staining shown in C(i) and D(i); FITC-pI:C (green) and TAMRA-hBD3 (red) are shown in C(ii) and D(ii) respectively; EEA1 staining is shown in C(iii) and D(iii) (pseudocoloured red and green respectively); pI:C and hBD3 colocalisation with EEA1 are demonstrated C(iv) in and D(iv) merged images respectively, with Pearson co-efficients of r = 0.615(C(iv)) and r = 0.205(D(iv)). (E-i-viii) BMDM were treated (30 min) with FITC-pI:C and TAMRA-hBD3 and immunostained with EEA1-1 antibody. One representative cell from each slide is shown with filters allowing visualisation of single entities, E(i) DAPI staining for nucleus; E(ii) TAMRA-hBD3; E(iii) FITC-pI:C; E(iv) EEA1-1-Cy5 (blue), E(v) merge of Eii,iii and iv. E(vi)-(viii) are merged images showing only two components to assess co-localisation, (vi) polyI:C (green), EEA1-1 (pseudocoloured red) and hBD3 invisible; (vii) polyI:C (green) hBD3 (red) and EEA1-1 invisible; (viii) hBD3 (red) and EEA1-1(pseudocoloured green) and polyI:C invisible. Images are representative of all cells treated, and visualised by confocal microscopy. Additional single colour images of EEA1 pseudocolouring to complement 5E are shown in S3 Fig. Scale bars indicate 10 microns.