Fig 1.
Generation of CD46-/- RPTEC/TERT1 mass culture.
a Workflow for generation of RPTEC/TERT1 CD46 knock-out cells. b FACS analysis of CD46 knock-out in mass culture for gRNA3436 and gRNA3437.
Fig 2.
Confirmation of homozygous knock-out of CD46 -/- RPTEC/TERT1 cells.
a Sanger sequencing results of gene knock-out. Manual alignment. b Sanger sequencing results of gene knock-out. Analysis of in-del by TIDE online tool. c Confirmation of CD46 knock-out in clone 1E3 on protein level by FACS analysis.
Fig 3.
Relative loss of factor I cofactor activity in CD46 -/- RPTEC/TERT1 cells.
a+b Overlay histograms show C4d (a) or C4c (b) deposition after classical complement pathway activation on wild-type (WT; control-transfected) and knock-out (KO; 1E3) RPTEC/TERT1 cells. KO: dark-grey tinted area with solid line, WT: light-grey tinted area with dotted line, KO without W6/32 as trigger of classical pathway activation: solid line, WT without W6/32: dotted line. c Detection of anti-HLA class I (W6/32) antibody on CD46 -/-(KO) and wild-type (WT) RPTEC/TERT1 cells. d Detection of C4d deposition. e Detection of C4c deposition. c-e Mean values and standard deviations (T-shaped whiskers) of geometric mean of fluorescence intensity of 4 experiments are shown. No ab = without W6/32 in the incubation mixture (only 50% serum) PE phycoerythrin, FI fluorescence intensity, n.s. not significant.
Fig 4.
Characterisation of RPTEC/TERT1 CD46 knock-out clone 1E3.
a Immunofluorescence of E-cadherin and ZO1. b Gamma-glutamyltransferase activity. c FACS analysis of CD13 (Aminopeptidase N). D: Dome formation.