Figure 6A Quantification of TMRM fluorescence by flow cytometry in U2OS : GFP ‐ parkin cells treated with 25 μM antimycin A ( anti A ) , 25 μM antimycin A and 10 μM oligomycin ( anti A + oligo ) , and 20 μM CCCP ( CCCP ) for the indicated time , represented as a fraction of fluorescence intensity compared to DMSO ‐ treated cells . Error bars represent the mean ± s . e . m . ( n = 3 experiments ) . B U2OS : GFP ‐ parkin cells were treated with DMSO ( DMSO ) , 25 μM antimycin A ( anti A ) , 25 μM antimycin A and 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP ( CCCP ) for two hours , then fixed and immunostained for TOM20 ( red ) . Scale bars , 50 μm . C Quantification ofPDHE2 / E3 bp − positive / TOM20 − negative structures from ( B ) ; both the total number ( white bars ) and the number colocalizing with GFP − parkin ( gray bars ) are indicated . Error bars represent the mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 42 - 60 cells in three experiments ) ; ns , not significant ; * * * P < 0 . 001 ) . D U2OS : GFP − parkin cells were treated with DMSO ( DMSO ) , 25 μM antimycin A ( anti A ) , 25 μM antimycin A and 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP ( CCCP ) for two hours , then fixed and immunostained for PDH E2 / E3 bp ( red ) and TOM20 ( blue ) . A merged image of both mitochondrial markers ( mito ) is shown at the far right . Arrows indicate PDH E2 / E3 bp − positive / TOM20 − negative structures colocalizing with GFP − parkin ( green ) . Scale bars , 20 μm .

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

Figure 6A Quantification of TMRM fluorescence by flow cytometry in U2OS:GFP‐parkin cells treated with 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), and 20 μM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO‐treated cells. Error bars represent the mean ± s.e.m. (n = 3 experiments).B U2OS:GFP‐parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for TOM20 (red). Scale bars, 50 μm.C Quantification ofPDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001).D U2OS:GFP−parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). A merged image of both mitochondrial markers (mito) is shown at the far right. Arrows indicate PDH E2/E3 bp−positive/TOM20−negative structures colocalizing with GFP−parkin (green). Scale bars, 20 μm.


Figure 7A Representative immunoblot of whole ‐ cell lysates from U2OS : GFP and GFP ‐ parkin cells treated with DMSO , 25 μM antimycin A ( anti A ) , 25 μM antimycin A with 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP for the indicated time period . B Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from ( A ) . Error bars represent the mean ± s . e . m . ( n = 3 experiments ) . C Quantification of mitochondrial clearance in U2OS : GFP − parkin cells treated as in ( A ) , fixed and immunostained for TOM20 . Data are shown as percentage of cells containing mitochondria , by TOM20 staining , visualized by fluorescence microscopy . Error bars represent the mean ± s . e . m . ( n = 3 experiments , with at least 85 cells quantified per condition , per experiment ) . D U2OS : GFP ‐ parkin cells were treated as in ( A ) for the indicated time period , then fixed and immunostained for TOM20 ( red ) . Cell boundaries are delineated in single ‐ channel images . Scale bars , 20 μm .

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

Figure 7A Representative immunoblot of whole‐cell lysates from U2OS:GFP and GFP‐parkin cells treated with DMSO, 25 μM antimycin A (anti A), 25 μM antimycin A with 10 μM oligomycin (anti A + oligo), or 20 μM CCCP for the indicated time period.B Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from (A). Error bars represent the mean ± s.e.m. (n = 3 experiments).C Quantification of mitochondrial clearance in U2OS:GFP−parkin cells treated as in (A), fixed and immunostained for TOM20. Data are shown as percentage of cells containing mitochondria, by TOM20 staining, visualized by fluorescence microscopy. Error bars represent the mean ± s.e.m. (n = 3 experiments, with at least 85 cells quantified per condition, per experiment).D U2OS:GFP‐parkin cells were treated as in (A) for the indicated time period, then fixed and immunostained for TOM20 (red). Cell boundaries are delineated in single‐channel images. Scale bars, 20 μm.


Figure 1 ( A ) Representative photomicrographs of MAP2 ( green ) and cleaved caspase - 3 ( red ) immunocytochemistry of embryonic cortical neurons from wild type ( upper panels ) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h . Scale bar 15µm . ( B ) Quantification of active ( cleaved ) caspase - 3 in cultured cortical neurons of the indicated genotypes treated for 14h with vehicle or Aβ oligomers ( 2 . 5 , 5 , or 10 µM ) . Data are expressed as percentage cleaved caspase - 3 and MAP2 double positive cells over the total number of MAP2 positive cells . ( C ) Quantification of neurite length in in cultured cortical neurons of the indicated genotypes treated for 24h with vehicle or Aβ oligomers ( 2 . 5 , or 10 µM ) . Data are expressed as mean neurite length in individual neurons , relative to wild type vehicle ( set to 100 units ) .

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

Figure 1(A) Representative photomicrographs of MAP2 (green) and cleaved caspase-3 (red) immunocytochemistry of embryonic cortical neurons from wild type (upper panels) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h. Scale bar 15µm.(B) Quantification of active (cleaved) caspase-3 in cultured cortical neurons of the indicated genotypes treated for 14h with vehicle or Aβ oligomers (2.5, 5, or 10 µM). Data are expressed as percentage cleaved caspase-3 and MAP2 double positive cells over the total number of MAP2 positive cells.(C) Quantification of neurite length in in cultured cortical neurons of the indicated genotypes treated for 24h with vehicle or Aβ oligomers (2.5, or 10 µM). Data are expressed as mean neurite length in individual neurons, relative to wild type vehicle (set to 100 units).


Figure 2 ( A ) Immunohistochemistry for p75NTR in hippocampal CA1 of 5 month wild type , 5xFAD and p75NTR knock - out ( KO ) mice . Scale bar , 20μm . Western blot analysis of p75NTR expression in total lysates of hippocampus from 6 month old wild type ( WT ) and 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain The lower panel shows reprobing for GAPDH to control for equal gel loading . ( C ) Quantification of p75NTR expression in mouse hippocampus relative to GAPDH . ( D ) Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through the hippocampus of 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice of the indicated ages . Scale bar , 400μm . ( E ) Quantification of Aβ plaque burden in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows the percentage of hippocampal area occupied by Aβ plaques ( mean ± SEM , N = 5 mice per group ) . * , P < 0 . 05 and * * , P < 0 . 01 vs 5XFAD . # , P < 0 . 05 vs 5XFAD / KO ( one - way ANOVA followed by post hoc test ) . ( F ) Quantification of the number of Aβ plaques larger than 30um in diameter per um2 in coronal sections through the hippocampus of 9 month old 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice . Color codes are as in panel ( E ) . Histogram shows mean ± SEM , N = 4 mice per group ; * , P < 0 . 05 KO vs . knock - in genotypes . ELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers ( G ) refers to the soluble fraction after Tris - buffered saline extractionELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers ( H ) to the soluble fraction after RIPA buffer extraction of the Tris - buffered saline pellet , ELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils ( I ) to the soluble fraction after formic acid treatment of the RIPA pellet .

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

Figure 2(A) Immunohistochemistry for p75NTR in hippocampal CA1 of 5 month wild type, 5xFAD and p75NTR knock-out (KO) mice. Scale bar, 20μm.Western blot analysis of p75NTR expression in total lysates of hippocampus from 6 month old wild type (WT) and 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain The lower panel shows reprobing for GAPDH to control for equal gel loading. (C) Quantification of p75NTR expression in mouse hippocampus relative to GAPDH.(D) Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through the hippocampus of 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice of the indicated ages. Scale bar, 400μm. (E) Quantification of Aβ plaque burden in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Aβ plaques (mean ± SEM, N=5 mice per group). *, P<0.05 and **, P<0.01 vs 5XFAD. #, P<0.05 vs 5XFAD/KO (one-way ANOVA followed by post hoc test). (F) Quantification of the number of Aβ plaques larger than 30um in diameter per um2 in coronal sections through the hippocampus of 9 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Color codes are as in panel (E). Histogram shows mean ± SEM, N=4 mice per group; *, P<0.05 KO vs. knock-in genotypes. ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers (G) refers to the soluble fraction after Tris-buffered saline extractionELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers (H) to the soluble fraction after RIPA buffer extraction of the Tris-buffered saline pellet,ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils (I) to the soluble fraction after formic acid treatment of the RIPA pellet.


Figure 3 ( A ) Immunostaining for Glial fibrillary acidic protein ( GFAP ) , a marker of astrocytes , and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice . Scale bar , 300μm . Right hand panels show high magnification of the indicated areas . Scale bar , 50μm . ( B ) Quantification of GFAP area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows the percentage of hippocampal area occupied by GFAP immunostaining ( mean ± SEM , N = 5 mice per group ) . ( C ) Immunostaining for Ionized calcium binding adaptor molecule 1 ( Iba1 ) , a marker of microglial cells , and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A . Scale bar , 300μm . Right hand panels show high magnification of the indicated areas . Scale bar , 10μm . ( D ) Quantification of Iba1 area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows the percentage of hippocampal area occupied by Iba1 immunostaining ( mean ± SEM , N = 5 mice per group ) . ( E ) Immunostaining of reticulon 3 ( RTN3 ) , a marker of dystrophic neurites , and Aβ plaques in coronal sections through the hippocampus of 6 month old 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice . Scale bar , 40μm . ( F ) Quantification of RTN3 - positive dystrophic neurite area in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows the percentage of Aβ plaque area that overlapped with RTN3 immunostaining ( mean ± SEM , N = 5 mice per group ) . ( G ) MitoSox staining , a mitochondrial superoxide indicator , and DAPI in coronal sections through the hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice . Scale bar , 60μm . ( H ) Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows MitoSox mean fluorescence intensity in arbitrary units ( mean ± SEM , N = 5 mice per group ) .

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

Figure 3(A) Immunostaining for Glial fibrillary acidic protein (GFAP), a marker of astrocytes, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 50μm. (B) Quantification of GFAP area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by GFAP immunostaining (mean ± SEM, N=5 mice per group). (C) Immunostaining for Ionized calcium binding adaptor molecule 1 (Iba1), a marker of microglial cells, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 10μm. (D) Quantification of Iba1 area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Iba1 immunostaining (mean ± SEM, N=5 mice per group). (E) Immunostaining of reticulon 3 (RTN3), a marker of dystrophic neurites, and Aβ plaques in coronal sections through the hippocampus of 6 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 40μm. (F) Quantification of RTN3-positive dystrophic neurite area in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of Aβ plaque area that overlapped with RTN3 immunostaining (mean ± SEM, N=5 mice per group).(G) MitoSox staining, a mitochondrial superoxide indicator, and DAPI in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 60μm. (H) Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows MitoSox mean fluorescence intensity in arbitrary units (mean ± SEM, N=5 mice per group).


Figure 4 ( A ) Percentage of change in field excitatory postsynaptic potential ( fEPSP ) recorded after theta - burst stimulation ( TBS ) in Schaffer collaterals of hippocampal slices from 6 month old wild type , 5xFAD and 5xFAD / p75NTR mutant mice , as indicated . Results are presented as mean % change normalized to t = 0 ± SEM . N = 6 ( wild type ) , 5 ( 5xFAD ) , 9 ( 5xFAD / KO ) , 7 ( 5xFAD / ΔDD ) and 8 ( 5xFAD / C259A ) slices from 3 mice per genotype , respectively . ( B ) Quantification of fEPSP ( mean % change ± SEM ) in the indicated genotypes at 3 time points . * , P < 0 . 05 ; * * , P < 0 . 01 vs . 5xFAD ( two - way ANOVA followed by post hoc test ) . N numbers as in ( A ) . ( C ) Behavior in the novel object recognition ( NOR ) test of 6 month old wild type , 5XFAD and 5XFAD / p75NTR mutant mice , as indicated . Histograms show mean recognition index ± SEM during training , and 30min , 24h and 14 days after training , corresponding to measures of short term , long term and remote memory , respectively . Bar color codes are as in panel ( A ) . * , P < 0 . 05 vs . 5xFAD ( two - way ANOVA followed by post hoc test ) . N = 12 ( wild type , 5xFAD / ΔDD and 5xFAD / C259A ) , 10 ( 5xFAD ) and 8 ( 5xFAD / KO ) mice per genotype , respectively . ( D ) Training latency in the Barnes maze test of 6 month old wild type , 5XFAD and 5XFAD / p75NTR mutant mice , as indicated . Histograms show mean latency in seconds to find the platform hole ± SEM in 4 consecutive training sessions . Bar color codes are as in panel ( A ) . * , P < 0 . 05 vs . 5xFAD ; # , P < 0 . 05 vs . wild type , 5xFAD / ΔDD or 5xFAD / C259A ( two - way ANOVA followed by post hoc test ) . N = 14 ( wild type and 5xFAD / ΔDD ) , 10 ( 5xFAD , ( 5xFAD / KO and 5xFAD / C259A ) mice per genotype , respectively . N = 14 ( wild type and 5xFAD / ΔDD ) , 10 ( 5xFAD , ( 5xFAD / KO and 5xFAD / C259A ) mice per genotype , respectively . ( E ) Percentage of time spent ( mean ± SEM ) in the target quadrant of the Barnes maze test 30min , 24h and 14 days after training . * , P < 0 . 05 vs . 5xFAD ; # , P < 0 . 05 vs . wild type , 5xFAD / ΔDD or 5xFAD / C259A ( two way ANOVA followed by post hoc test ) .

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

Figure 4(A) Percentage of change in field excitatory postsynaptic potential (fEPSP) recorded after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from 6 month old wild type, 5xFAD and 5xFAD/p75NTR mutant mice, as indicated. Results are presented as mean % change normalized to t=0 ± SEM. N=6 (wild type), 5 (5xFAD), 9 (5xFAD/KO), 7 (5xFAD/ΔDD) and 8 (5xFAD/C259A) slices from 3 mice per genotype, respectively. (B) Quantification of fEPSP (mean % change ± SEM) in the indicated genotypes at 3 time points. *, P<0.05; **, P<0.01 vs. 5xFAD (two-way ANOVA followed by post hoc test). N numbers as in (A).(C) Behavior in the novel object recognition (NOR) test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean recognition index ± SEM during training, and 30min, 24h and 14 days after training, corresponding to measures of short term, long term and remote memory, respectively. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD (two-way ANOVA followed by post hoc test). N=12 (wild type, 5xFAD/ΔDD and 5xFAD/C259A), 10 (5xFAD) and 8 (5xFAD/KO) mice per genotype, respectively.(D) Training latency in the Barnes maze test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean latency in seconds to find the platform hole ± SEM in 4 consecutive training sessions. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two-way ANOVA followed by post hoc test). N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively.N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively. (E) Percentage of time spent (mean ± SEM) in the target quadrant of the Barnes maze test 30min, 24h and 14 days after training. *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two way ANOVA followed by post hoc test).


Figure 5 ( A ) Western blot analysis of CTF beta ( CTFβ ) in hippocampal lysates of 9 month old 5xFAD mice carrying different p75NTR alleles detected using anti - APP - CTF antibody Arrows point to phospho - C99 and C99 CTFβ species Asterisk denotes different species of native and phosphorylated alpha and beta CTFs of lower molecular weights Lower panel shows reprobing for GAPDH . ( B ) Quantification ( mean ± SEM ) of phospho - C99 / C99 CTFβ species , normalized to GAPDH and expressed relative to levels in 5xFAD mice . * , P < 0 . 05 5xFAD vs , 5xFAD / KO ; * * * , P < 0 . 001 5xFAD vs . 5xFAD / ΔDD and 5xFAD / C259A ; # , P < 0 . 05 5xFAD / C259A vs . 5xFAD / KO ; § , P = 0 . 056 5xFAD / ΔDD vs . 5xFAD / KO . N = 5 mice per group . ( C ) Western blot analysis of soluble APP alpha ( sAPPα ) in the soluble Tris - buffered saline fraction of hippocampal homogenate made from 9 moth old 5xFAD mice carrying different p75NTR alleles detected using 6E10 antibody Lower panel shows reprobing for GAPDH . ( D ) Quantification ( mean ± SEM ) normalized to GAPDH and expressed relative to levels in 5xFAD mice . * , P < 0 . 05 5xFAD vs . 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A ; # , P < 0 . 05 5xFAD / C259A vs . 5xFAD / KO ; § , P < 0 . 05 5xFAD / ΔDD vs . 5xFAD / KO . N = 9 mice per group .

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

Figure 5(A) Western blot analysis of CTF beta (CTFβ) in hippocampal lysates of 9 month old 5xFAD mice carrying different p75NTR alleles detected using anti-APP-CTF antibody Arrows point to phospho-C99 and C99 CTFβ species Asterisk denotes different species of native and phosphorylated alpha and beta CTFs of lower molecular weights Lower panel shows reprobing for GAPDH. (B) Quantification (mean ± SEM) of phospho-C99/C99 CTFβ species, normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs, 5xFAD/KO; ***, P<0.001 5xFAD vs. 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P=0.056 5xFAD/ΔDD vs. 5xFAD/KO. N=5 mice per group.(C) Western blot analysis of soluble APP alpha (sAPPα) in the soluble Tris-buffered saline fraction of hippocampal homogenate made from 9 moth old 5xFAD mice carrying different p75NTR alleles detected using 6E10 antibody Lower panel shows reprobing for GAPDH. (D) Quantification (mean ± SEM) normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs. 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P<0.05 5xFAD/ΔDD vs. 5xFAD/KO. N=9 mice per group.


Figure 6 ( A ) Internalization of triple mutant hAPP in wild type , p75NTR knock - out ( KO ) , ΔDD and C259A hippocampal neurons ( 15min time point ) . Counterstaining for p75NTR and DAPI are also shown . Scale bar , 10μm . ( B ) Kinetics of internalization of triple mutant hAPP in hippocampal neurons from wild type , p75NTR knock - out ( KO ) , ΔDD and C259A mice . Shown is mean ± SEM of percentage internalization of total surface APP ( set to 100 % ) . N = 3 independent experiments each performed in duplicate ; * , P < 0 . 05 knock - in vs . KO mutants ; * * , P < 0 . 01 , WT vs . all other genotypes ( 2 - way ANOVA ) . ( C ) Linear transformation of hAPP internalization kinetics shown in ( B ) . IMAX denotes maximal internalization in % . T1 / 2 denotes time for half maximal internalization in minutes . ( D ) Kinetics of internalization of p75NTR in wild type hippocampal neurons in the presence or absence of NGF . Shown is mean ± SEM of percentage internalization of total surface p75NTR ( set to 100 % ) . N = 3 independent experiments each performed in duplicate ; * , P < 0 . 05 ± NGF ( 2 - way ANOVA ) . ( E ) Linear transformation of p75NTR internalization kinetics IMAX denotes maximal internalization in % . T1 / 2 denotes time for half maximal internalization in minutes . ( F ) Internalization of endogenous p75NTR in wild type hippocampal neurons ( 30min time point ) . Counterstaining for MAP2 and DAPI are also shown . Scale bar , 10μm . ( G ) Kinetics of internalization of p75NTR in hippocampal neurons from wild type , ΔDD and C259A mice . Show are mean ± SEM of percentage internalization of total surface p75NTR ( set to 100 % ) . N = 3 independent experiments each performed in duplicate ; * , P < 0 . 05 knock - in mutants vs . WT ( 2 - way ANOVA ) . ( H ) Linear transformation of p75NTR internalization kinetics shown in ( G ) . IMAX denotes maximal internalization in % . T1 / 2 denotes time for half maximal internalization in minutes . ( I ) ( A ) Co - immunoprecipitation between p75NTR and β - adaptin in hippocampal extracts from 6 month wild type and p75NTR mutant mice as indicated . Results shown are representative from 2 independent experiments . Molecular weights are in kDa . IP : immunoprecipitation ; IB : immunoblotting ; WCL , whole cell lysate .

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

Figure 6(A) Internalization of triple mutant hAPP in wild type, p75NTR knock-out (KO), ΔDD and C259A hippocampal neurons (15min time point). Counterstaining for p75NTR and DAPI are also shown. Scale bar, 10μm.(B) Kinetics of internalization of triple mutant hAPP in hippocampal neurons from wild type, p75NTR knock-out (KO), ΔDD and C259A mice. Shown is mean ± SEM of percentage internalization of total surface APP (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 knock-in vs. KO mutants; **, P<0.01, WT vs. all other genotypes (2-way ANOVA). (C) Linear transformation of hAPP internalization kinetics shown in (B). IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes.(D) Kinetics of internalization of p75NTR in wild type hippocampal neurons in the presence or absence of NGF. Shown is mean ± SEM of percentage internalization of total surface p75NTR (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 ± NGF (2-way ANOVA). (E) Linear transformation of p75NTR internalization kinetics IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes.(F) Internalization of endogenous p75NTR in wild type hippocampal neurons (30min time point). Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.(G) Kinetics of internalization of p75NTR in hippocampal neurons from wild type, ΔDD and C259A mice. Show are mean ± SEM of percentage internalization of total surface p75NTR (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 knock-in mutants vs. WT (2-way ANOVA). (H) Linear transformation of p75NTR internalization kinetics shown in (G). IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes. (I) (A) Co-immunoprecipitation between p75NTR and β-adaptin in hippocampal extracts from 6 month wild type and p75NTR mutant mice as indicated. Results shown are representative from 2 independent experiments. Molecular weights are in kDa. IP: immunoprecipitation; IB: immunoblotting; WCL, whole cell lysate.


Figure 7 ( A ) Co - immunoprecipitation between hAPP and p75NTR in hippocampal extracts from 6 month 5xFAD and p75NTR mutant mice as indicated . Results shown are representative from 3 independent experiments . Molecular weights are indicated in kDa . IP : immunoprecipitation ; IB : immunoblotting ; WCL , whole cell lysate . ( B ) Proximity ligation assay ( PLA ) between triple mutant hAPP and p75NTR in hippocampal neurons from wild type and C259A mice ( first and second rows , respectively ) . The third row ( no APP ) shows control PLA reaction in neurons that did not receive hAPP lentivirus . The fourth row ( KO ) shows control PLA reaction in neurons from p75NTR knock - out mice infected with hAPP lentivirus . Counterstaining for MAP2 is also shown . Scale bar , 10μm . ( C ) Quantification of total PLA signals in wild type and p75NTR mutant neurons in the presence or absence of hAPP lentivirus , as indicated . Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments . ( D ) Quantification of cell surface PLA signals in wild type and p75NTR mutant neurons in the presence or absence of triple mutanthAPP lentivirus , as indicated . Live neuron cultures were fed with anti - mouse p75NTR and anti - hAPP antibodies on ice , washed after staining , fixed , and developed with PLA reaction . Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments . ( E ) Internalization of hAPP / p75NTR PLA signals in hippocampal neurons of wild type and p75NTR mutant mice . Live neuron cultures were fed with anti - mouse p75NTR and anti - hAPP antibodies on ice , washed and plates placed at 37 ° C for different periods of time to allow internalization . Internalization was stopped by acid wash , followed by fixation and PLA reaction . Counterstaining for MAP2 and DAPI are also shown . Scale bar , 10μm . ( F ) Kinetics of co - internalization of hAPP and p75NTR in hippocampal neurons from wild type , ΔDD and C259A mice . Show are mean ± SEM of percentage internalization of total surface PLA signal ( set to 100 % ) . ( G ) Linear transformation of p75NTR internalization

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

Figure 7(A) Co-immunoprecipitation between hAPP and p75NTR in hippocampal extracts from 6 month 5xFAD and p75NTR mutant mice as indicated. Results shown are representative from 3 independent experiments. Molecular weights are indicated in kDa. IP: immunoprecipitation; IB: immunoblotting; WCL, whole cell lysate.(B) Proximity ligation assay (PLA) between triple mutant hAPP and p75NTR in hippocampal neurons from wild type and C259A mice (first and second rows, respectively). The third row (no APP) shows control PLA reaction in neurons that did not receive hAPP lentivirus. The fourth row (KO) shows control PLA reaction in neurons from p75NTR knock-out mice infected with hAPP lentivirus. Counterstaining for MAP2 is also shown. Scale bar, 10μm.(C) Quantification of total PLA signals in wild type and p75NTR mutant neurons in the presence or absence of hAPP lentivirus, as indicated. Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments. (D) Quantification of cell surface PLA signals in wild type and p75NTR mutant neurons in the presence or absence of triple mutanthAPP lentivirus, as indicated. Live neuron cultures were fed with anti-mouse p75NTR and anti-hAPP antibodies on ice, washed after staining, fixed, and developed with PLA reaction. Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments. (E) Internalization of hAPP/p75NTR PLA signals in hippocampal neurons of wild type and p75NTR mutant mice. Live neuron cultures were fed with anti-mouse p75NTR and anti-hAPP antibodies on ice, washed and plates placed at 37°C for different periods of time to allow internalization. Internalization was stopped by acid wash, followed by fixation and PLA reaction. Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.(F) Kinetics of co-internalization of hAPP and p75NTR in hippocampal neurons from wild type, ΔDD and C259A mice. Show are mean ± SEM of percentage internalization of total surface PLA signal (set to 100%). (G) Linear transformation of p75NTR internalization kinetics


Figure 8 ( A ) Micrographs of triple mutant hAPP after 15min internalization ( red ) , BACE1 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main images . Scale bar , 5μm . ( B ) Quantification of the proportion of BACE1 that co - localized with hAPP after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % BACE co - localized with hAPP . N = 3 independent experiments , each performed in duplicate ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) . ( C ) Quantification of the proportion of internalized hAPP that co - localized with BACE1 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized hAPP co - localized with BACE1 . N = 3 independent experiments , each performed in duplicate ; * , P < 0 . 05 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) . ( D ) Micrographs of p75NTR after 15min internalization ( red ) , Rab11 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main merged images . Scale bar , 5μm . ( E ) Quantification of the proportion of Rab11 that co - localized with p75NTR after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % Rab11 co - localized with p75NTR . n = 4 ; * , P < 0 . 05 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) . ( F ) Quantification of the proportion of internalized p75NTR that co - localized with Rab11 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized p75NTR co - localized with Rab11 . n = 4 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) . ( G ) Micrographs of triple mutant hAPP after 15min internalization ( red ) , Rab11 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main merged images . Scale bar , 5μm . ( H ) Quantification of the proportion of Rab11 that co - localized with hAPP after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % Rab11 co - localized with hAPP . n = 4 ; * , P < 0 . 05 vs . WT ( one - way ANOVA followed by post hoc test ) . ( I ) Quantification of the proportion of internalized hAPP that co - localized with Rab11 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized hAPP co - localized with Rab11 . n = 4 ; * , P < 0 . 05 vs . WT ( one - way ANOVA followed by post hoc test ) .

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

Figure 8(A) Micrographs of triple mutant hAPP after 15min internalization (red), BACE1 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main images. Scale bar, 5μm. (B) Quantification of the proportion of BACE1 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % BACE co-localized with hAPP. N=3 independent experiments, each performed in duplicate; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (C) Quantification of the proportion of internalized hAPP that co-localized with BACE1 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with BACE1. N=3 independent experiments, each performed in duplicate; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test).(D) Micrographs of p75NTR after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (E) Quantification of the proportion of Rab11 that co-localized with p75NTR after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with p75NTR. n=4; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (F) Quantification of the proportion of internalized p75NTR that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized p75NTR co-localized with Rab11. n=4; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (G) Micrographs of triple mutant hAPP after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (H) Quantification of the proportion of Rab11 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with hAPP. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test). (I) Quantification of the proportion of internalized hAPP that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with Rab11. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test).


Figure 1F smFISH of Arglu1 pre - mRNA ( magenta ) and Arglu1 sisRNA ( green ) . Merged dots ( white , arrowheads ) represent Arglu1 pre - mRNA transcript . G Quantification of Arglu1 pre - mRNA and Arglu1 sisRNA copy number from ( F ) . ( n = 50 cells ) Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum . I RT - PCR of Arglu1 sisRNA , spliced mRNA levels and actin as control after AMOs treatment .

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

Figure 1F smFISH of Arglu1 pre-mRNA (magenta) and Arglu1 sisRNA (green). Merged dots (white, arrowheads) represent Arglu1 pre-mRNA transcript. G Quantification of Arglu1 pre-mRNA and Arglu1 sisRNA copy number from (F). (n = 50 cells) Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.I RT-PCR of Arglu1 sisRNA, spliced mRNA levels and actin as control after AMOs treatment.


Figure 2G Left : Western blot analysis of ARGLU1 protein and GAPDH as loading control in control cells and Arglu1 sisRNA KD cells . Right : Quantification of relative ARGLU1 protein levels normalized to GADPH in Arglu1 sisRNA KD cells vs control cells from western blot analysis ( left ) . ( n = 3 biological replicates ) . M Left : RT - PCR of AXIN1a and AXIN1b spliced levels and actin as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs . Right : Quantification of relative spliced AXIN1a / AXIN1b expression ratio normalized to actin from RT - PCR analysis ( left ) . ( n = 3 biological replicates ) .

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

Figure 2G Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in control cells and Arglu1 sisRNA KD cells. Right: Quantification of relative ARGLU1 protein levels normalized to GADPH in Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).M Left: RT-PCR of AXIN1a and AXIN1b spliced levels and actin as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs. Right: Quantification of relative spliced AXIN1a/AXIN1b expression ratio normalized to actin from RT-PCR analysis (left). (n = 3 biological replicates).


Figure 3B Left : Western blot analysis of ARGLU1 protein and GAPDH as loading control in 1 day Arglu1 sisRNA KD cells vs control cells . Right : Quantification of relative ARGLU1 protein expression level normalized to GADPH in 1 day Arglu1 sisRNA KD cells vs control cells from western blot analysis ( left ) . ( n = 3 biological replicates ) . D Left : smFISH of Arglu1 sisRNA ( green ) coupled with immunostaining of ARGLU1 protein ( red ) shown in z - stack . Right : Zoom in z - slice on the boxed area showing co - localization ( arrowheads ) of Arglu1 sisRNA with ARGLU1 protein . The intensity plot shows the signal quantification and co - localization of Arglu1 sisRNA and ARGLU1 protein in the direction of the white arrow . E Left : smFISH of Arglu1 pre - mRNA ( green ) coupled with ARGLU1 protein ( red ) immunostaining shown in z - stack . Right : Zoom in z - slice on the boxed area showing co - localization ( arrowhead ) of Arglu1 pre - mRNA with ARGLU1 protein . The intensity plot shows the signal quantification and co - localization of Arglu1 pre - mRNA and ARGLU1 protein in the direction of the white arrow . F smFISH of Arglu1 sisRNA ( green ) and Arglu1 pre - mRNA ( magenta ) in 1 day Arglu1 sisRNA KD cells vs control cells . G , H Quantification of Arglu1 sisRNA and Arglu1 pre - mRNA copy number from ( F ) . ( n = 90 cells ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

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

Figure 3B Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in 1 day Arglu1 sisRNA KD cells vs control cells. Right: Quantification of relative ARGLU1 protein expression level normalized to GADPH in 1 day Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).D Left: smFISH of Arglu1 sisRNA (green) coupled with immunostaining of ARGLU1 protein (red) shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowheads) of Arglu1 sisRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 sisRNA and ARGLU1 protein in the direction of the white arrow. E Left: smFISH of Arglu1 pre-mRNA (green) coupled with ARGLU1 protein (red) immunostaining shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowhead) of Arglu1 pre-mRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 pre-mRNA and ARGLU1 protein in the direction of the white arrow.F smFISH of Arglu1 sisRNA (green) and Arglu1 pre-mRNA (magenta) in 1 day Arglu1 sisRNA KD cells vs control cells. G, H Quantification of Arglu1 sisRNA and Arglu1 pre-mRNA copy number from (F). (n = 90 cells). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.


Figure 4A Immunostaining of MED1 ( magenta ) and SC35 ( red ) with transiently expressed mCherry - ARGLU1 protein ( green ) in MCF - 7 cells . Zoom in of boxed area and the intensity plot shows the co - localization and signal quantification of mCherry - ARGLU1 protein , MED1 and SC35 in the direction of the white arrow . B Confocal images of GFP - SRSF2 ( green , control ) and mCherry - ARGLU1 protein ( red ) at the pre - bleach , bleach , and post - bleach time points in FRAP analysis . The boxed area showing the speckle that was being bleached . C Fluorescence intensities of the nuclear speckle over time in GFP - SRSF2 ( green , control ) and mCherry - ARGLU1 protein ( red ) FRAP analysis . ( n = 3 separate nuclear speckles ) . D Immunostaining of ARGLU1 protein ( green ) and SC35 ( red ) in 1 day Arglu1 sisRNA KD cells vs control cells . Zoom in of boxed area and the intensity plot shows the co - localization and signal quantification of ARGLU1 protein and SC35 in the direction of the white arrow . E Confocal images of 125 nM ARGLU1 - GFP protein ( green ) only and ARGLU1 - GFP protein added with 50 nM IVT RNA ( magenta ) , of either Arglu1 intron 2 with UCE ( Intron 2 WT ) or IVT Arglu1 intron without UCE ( Intron 2 ΔUCE ) . White arrowhead indicates ARGLU1 - GFP droplet incorporated with IVT Intron 2 WT and blue arrowhead indicates free ARGLU1 - GFP droplet not incorporated with IVT Intron 2 WT . F Quantification of total ARGLU1 - GFP droplets from ( E ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum . G Quantification of average ARGLU1 - GFP droplets size with / without incorporation of IVT intron 2 WT from ( E , ARGLU1 - GFP + Intron 2 WT ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

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

Figure 4A Immunostaining of MED1 (magenta) and SC35 (red) with transiently expressed mCherry-ARGLU1 protein (green) in MCF-7 cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of mCherry-ARGLU1 protein, MED1 and SC35 in the direction of the white arrow.B Confocal images of GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) at the pre-bleach, bleach, and post-bleach time points in FRAP analysis. The boxed area showing the speckle that was being bleached. C Fluorescence intensities of the nuclear speckle over time in GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) FRAP analysis. (n = 3 separate nuclear speckles).D Immunostaining of ARGLU1 protein (green) and SC35 (red) in 1 day Arglu1 sisRNA KD cells vs control cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of ARGLU1 protein and SC35 in the direction of the white arrow.E Confocal images of 125 nM ARGLU1-GFP protein (green) only and ARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either Arglu1 intron 2 with UCE (Intron 2 WT) or IVT Arglu1 intron without UCE (Intron 2 ΔUCE). White arrowhead indicates ARGLU1-GFP droplet incorporated with IVT Intron 2 WT and blue arrowhead indicates free ARGLU1-GFP droplet not incorporated with IVT Intron 2 WT. F Quantification of total ARGLU1-GFP droplets from (E). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. G Quantification of average ARGLU1-GFP droplets size with/without incorporation of IVT intron 2 WT from (E, ARGLU1-GFP + Intron 2 WT). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.


Figure 6A Western blot analysis of dARGLU1 protein level from dARGLU1 protein immunoprecipitation in S2 cells . Samples with rabbit - IgG antibodies were used as control . ARGLU1 antibody # 1 , but not # 2 , worked for IP . F Left : Western blot analysis of dARGLU1 protein levels and ACTIN5C as loading control in dArglu1 sisRNA KD ovaries vs control ( sibling control , MTD - GAL4 / CyO ) ovaries . Right : Quantification of relative dARGLU1 protein levels in dArglu1 sisRNA KD ovaries vs control ovaries from the western blot analysis . ( n = 3 biological replicates ) G Left : Immunostaining of dARGLU1 protein ( green ) and VASA ( red ) in dArglu1 sisRNA KD germaria vs control ( sibling control , MTD - GAL4 / TM3 ) germaria . The asterisk ( * ) marks the anterior of the germarium . Right : Zoom in of the boxed area and the intensity plot shows the signal quantification of dARGLU1 protein and VASA in the direction of the white arrow . Confocal images of 125 nM dARGLU1 - GFP protein ( green ) only and dARGLU1 - GFP protein added with 50 nM IVT RNA ( magenta ) , of either dArglu1 intron 2 until the 3 ' end of sisRNA ( dArglu1 intron 2 ) or IVT dArglu1 intron 2 starting after 3 ' end of sisRNA ( dArglu1 pre - mRNA ) . White arrowheads indicate dARGLU1 - GFP droplets incorporated with IVT dArglu1 intron 2 and blue arrowheads indicate free dARGLU1 - GFP droplets not incorporated with IVT dArglu1 intron 2 . J Quantification of total dARGLU1 - GFP droplets from ( I ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum . K Quantification of average dARGLU1 - GFP droplets size with / without incorporation of IVT dArglu1 intron 2 from ( I , dARGLU1 - GFP + dArglu1 intron 2 ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

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

Figure 6A Western blot analysis of dARGLU1 protein level from dARGLU1 protein immunoprecipitation in S2 cells. Samples with rabbit-IgG antibodies were used as control. ARGLU1 antibody #1, but not #2, worked for IP.F Left: Western blot analysis of dARGLU1 protein levels and ACTIN5C as loading control in dArglu1 sisRNA KD ovaries vs control (sibling control, MTD-GAL4/CyO) ovaries. Right: Quantification of relative dARGLU1 protein levels in dArglu1 sisRNA KD ovaries vs control ovaries from the western blot analysis. (n = 3 biological replicates)G Left: Immunostaining of dARGLU1 protein (green) and VASA (red) in dArglu1 sisRNA KD germaria vs control (sibling control, MTD-GAL4/TM3) germaria. The asterisk (*) marks the anterior of the germarium. Right: Zoom in of the boxed area and the intensity plot shows the signal quantification of dARGLU1 protein and VASA in the direction of the white arrow.Confocal images of 125 nM dARGLU1-GFP protein (green) only and dARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either dArglu1 intron 2 until the 3' end of sisRNA (dArglu1 intron 2) or IVT dArglu1 intron 2 starting after 3' end of sisRNA (dArglu1 pre-mRNA). White arrowheads indicate dARGLU1-GFP droplets incorporated with IVT dArglu1 intron 2 and blue arrowheads indicate free dARGLU1-GFP droplets not incorporated with IVT dArglu1 intron 2. J Quantification of total dARGLU1-GFP droplets from (I). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. K Quantification of average dARGLU1-GFP droplets size with/without incorporation of IVT dArglu1 intron 2 from (I, dARGLU1-GFP + dArglu1 intron 2). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. D


Figure 7A Confocal images of 125 nM d / hARGLU1 - GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT ( magenta ) or dArglu1 intron 2 ( red ) . White arrowheads indicate free IVT RNA dots that are not co - localized with the d / hARGLU1 - GFP droplets . B Quantification of non co - localized RNA dots from ( A , white arrowheads ) . ( n = 16 biological replicates ) . C , D Quantification of total hARGLU1 - GFP droplets and the average droplet size with / without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from ( A ) . ( n = 3 biological replicates ) . E , F Quantification of total dARGLU1 - GFP droplets and the average droplet size with / without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from ( A ) . ( n = 3 biological replicates ) .

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

Figure 7A Confocal images of 125 nM d/hARGLU1-GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT (magenta) or dArglu1 intron 2 (red). White arrowheads indicate free IVT RNA dots that are not co-localized with the d/hARGLU1-GFP droplets. B Quantification of non co-localized RNA dots from (A, white arrowheads). (n = 16 biological replicates). C, D Quantification of total hARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates). E, F Quantification of total dARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates).


Figure 1Ability of the SARS - CoV - 2 DNA vaccine OC - 2 . 4 to induce antibodies against S protein of the huCoV - 19 / WH01 ( B ) and Beta variants ( D ) Ability of the SARS - CoV - 2 DNA vaccine OC - 2 . 4 to induce antibodies against S protein the ability to neutralize these viruses in vitro ( C and E ) . Also shown is the ability of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 to prime and boost antibodies to S protein ( huCoV - 19 / WH01 ) following priming with a recombinant S protein in adjuvant ( F ) Also shown is the ability of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 to prime the ability of these antibodies to neutralize the Delta and Omicron variants in vitro ( G ) . Finally , three doses of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 induce antibodies that cross react with S proteins from the huCoV - 19 / WH01 , Beta , and Delta variants in mice ( H ) , four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro ( I ) .

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

Figure 1Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein of the huCoV-19/WH01 (B) and Beta variants (D)Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein the ability to neutralize these viruses in vitro (C and E).Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime and boost antibodies to S protein (huCoV-19/WH01) following priming with a recombinant S protein in adjuvant (F)Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime the ability of these antibodies to neutralize the Delta and Omicron variants in vitro (G).Finally, three doses of the universal SARS-CoV-2 DNA vaccine OC-2.4 induce antibodies that cross react with S proteins from the huCoV-19/WH01, Beta, and Delta variants in mice (H),four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro (I).


Figure 2A - D ) T cell responses as determined by ELISpot in Balb / c mice immunized three times with either spike protein in QS21 adjuvant ( rS / QS21 ; A ) , 50µg universal SARS - CoV - 2 vaccine OC - 2 . 4 ( B ) , primed with rS / QS21 and boosted with two doses of OC - 2 . 4 ( C ) , or empty pVAX plasmid ( D ) , using in vivo electroporation . Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens . Data has been given as the number of IFNγ producing ( spot forming ) cells ( SFCs ) per million splenocytes . Each color indicate an individual mice . E - H ) Also shown is analysis of T cell responses in New Zeeland White rabbits , which were immunized four times with either vehicle only ( E ) , or 84µg ( F ) or 840 µg ( G ) universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 using in vivo electroporation . Peripheral blood mononuclear cells ( PBMCs ) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens . Data has been given as the number of IFNγ SFCs per million PBMCs . The dotted line indicates the 50 SFC cut off . Also shown ( H ) is the cumulated IFNγ SFCs per million PBMCs to the RBD , M and N peptide pools added together for each group of immunized rabbits ( 84µg DNA , 840µg DNA , and vehicle only ) at each time point ( day ) . The numbers on the x - axis of graphs A ) to D ) indicates the number of the respective peptide pool ( WH1 variant ) covering a part of the indicated protein .

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

Figure 2A-D) T cell responses as determined by ELISpot in Balb/c mice immunized three times with either spike protein in QS21 adjuvant (rS/QS21; A), 50µg universal SARS-CoV-2 vaccine OC-2.4 (B), primed with rS/QS21 and boosted with two doses of OC-2.4 (C), or empty pVAX plasmid (D), using in vivo electroporation. Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ producing (spot forming) cells (SFCs) per million splenocytes. Each color indicate an individual mice.E-H) Also shown is analysis of T cell responses in New Zeeland White rabbits, which were immunized four times with either vehicle only (E), or 84µg (F) or 840 µg (G) universal SARS-CoV-2 DNA vaccine OC-2.4 using in vivo electroporation. Peripheral blood mononuclear cells (PBMCs) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ SFCs per million PBMCs. The dotted line indicates the 50 SFC cut off. Also shown (H) is the cumulated IFNγ SFCs per million PBMCs to the RBD, M and N peptide pools added together for each group of immunized rabbits (84µg DNA, 840µg DNA, and vehicle only) at each time point (day). The numbers on the x-axis of graphs A) to D) indicates the number of the respective peptide pool (WH1 variant) covering a part of the indicated protein.


Figure 3Three doses of respective vaccine fully or partially protected the mice against severe disease as determined by histological scoring of brochial and alveolar lung tissues ( b ) Three doses of respective vaccine fully or partially protected the mice against severe disease as percent weight loss ( c ) . D ) Three doses of respective vaccine also protect against viral replication in the nose , lungs and spleen . Values have been given as cycle times ( CT ) , where lower values indicate a higher viral load . Statistical comparisons in the graph are shown with lines with one asterisc indicating p < 0 , 05 and two asterics p < 0 ,

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

Figure 3Three doses of respective vaccine fully or partially protected the mice against severe disease as determined by histological scoring of brochial and alveolar lung tissues (b)Three doses of respective vaccine fully or partially protected the mice against severe disease as percent weight loss (c).D) Three doses of respective vaccine also protect against viral replication in the nose, lungs and spleen. Values have been given as cycle times (CT), where lower values indicate a higher viral load. Statistical comparisons in the graph are shown with lines with one asterisc indicating p<0,05 and two asterics p<0,01


Figure 1B , RMD assays performed after DSBs have been generated by sgRNAs / Cas9 . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is indicated . U2OS cells carrying a single copy of the EGFP - SSA reporter ( 3427 - 17 ) were transfected with sgRNA / Cas9 plasmids , and the percentage of GFP - positive cells was determined ( left ) . U2OS cells transfected with the indicated reporters EGFP - SSA - 17 / 6 bp , EGFP - SSA - 0 . 3 kb , EGFP - SSA - 0 . 6 kb and EGFP - SSA - 1 . 0 kb were pooled as a population after selection of the drug marker on the reporters , followed by sgRNA / Cas9 cleavage ( right ) . C , RMD is strongly induced when a DSB is generated close to one repeat . The U2OS ( EGFP - SSA - 3427 - 17 ) cell line carrying a single copy of the reporter was transfected with sgRNA / Cas9 plasmids as indicated , and the percentage of GFP - positive cells was determined . Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when POLD3 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control . Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when RAD52 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control . G , Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when RAD51 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control .

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

Figure 1B, RMD assays performed after DSBs have been generated by sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. U2OS cells carrying a single copy of the EGFP-SSA reporter (3427-17) were transfected with sgRNA/Cas9 plasmids, and the percentage of GFP-positive cells was determined (left). U2OS cells transfected with the indicated reporters EGFP-SSA-17/6 bp, EGFP-SSA-0.3 kb, EGFP-SSA-0.6 kb and EGFP-SSA-1.0 kb were pooled as a population after selection of the drug marker on the reporters, followed by sgRNA/Cas9 cleavage (right).C, RMD is strongly induced when a DSB is generated close to one repeat. The U2OS (EGFP-SSA-3427-17) cell line carrying a single copy of the reporter was transfected with sgRNA/Cas9 plasmids as indicated, and the percentage of GFP-positive cells was determined.Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when POLD3 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when RAD52 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.G, Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when RAD51 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.


Figure 2B , RMD efficiency is decreased when the distance from a DSB to one repeat is increased . RMD assays were performed in U2OS ( EGFP - SSA - 3427 - 17 ) cells using the indicated sgRNAs / Cas9 . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is shown . C and D , Knockdown of POLD3 impairs RMD when a DSB is close to one repeat . RMD frequency was determined in U2OS ( EGFP - SSA - 3427 - 17 ) cells using the indicated sgRNAs / Cas9 with or without depletion of POLD3 by lentivirus - expressed shRNA . Cells infected with a lentiviral empty vector were used as control . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is indicated . The knockdown level of POLD3 was shown by Western blot , and KU70 was used as the loading control .

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Figure 2B, RMD efficiency is decreased when the distance from a DSB to one repeat is increased. RMD assays were performed in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is shown.C and D, Knockdown of POLD3 impairs RMD when a DSB is close to one repeat. RMD frequency was determined in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9 with or without depletion of POLD3 by lentivirus-expressed shRNA. Cells infected with a lentiviral empty vector were used as control. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.


Figure 3A , Cyclin E overexpression results in increased RMD when the DSB is close to one repeat . The U2OS ( EGFP - SSA - 3427 - 3 ) cell line carrying Tet - on - cyclin E plasmid was used , and RMD frequency was determined with or without induction of cyclin E expression by doxycycline ( DOX , 0 . 5 µg / mL ) , which was added 24 hours before transfection of the indicated sgRNAs / Cas9 . Cyclin E expression levels were validated by Western blot , using KU70 as the loading control . B , ATR is required for BIR / RMD upon cyclin E overexpression . RMD frequency was determined in U2OS ( EGFP - SSA - 3427 - 3 ) cells infected with lentivirus - expressed ATR shRNA or the control vector ( Ctrl ) with and without induction of cyclin E expression by DOX ( 0 . 5 µg / ml ) after R29 sgRNA / Cas9 cleavage . ATR knockdown was shown by Western blot with KU70 as the loading control . D , RMD is stimulated when Cas9 - D10A is used to create a nick near the repeat . The repair frequencies of the U2OS ( MMEJ ) reporter cell line ( left ) and U2OS ( EGFP - SSA - 3427 - 17 ) cell line ( right ) were determined after expression of the indicated sgRNAs / Cas9 and sgRNAs / Cas9 - D10A . The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated ( right bottom ) . E , RMD induced by Cas9 - D10A is dependent on POLD3 . U2OS ( EGFP - SSA - 3427 - 17 ) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control and RMD was determined after cleavage by the indicated sgRNAs / Cas9 - D10A . The distance between sgRNA / Cas9 - D10A cleavage sites and the left and right repeat sequences is indicated at the bottom . The knockdown level of POLD3 was shown by Western blot , and KU70 was used as the loading control . F , p27 suppresses BIR / RMD and SSA / RMD . U2OS ( EGFP - SSA - 3427 - 3 ) cells carrying the Tet - on - cyclin E allele were infected with and without p27 lentiviral viruses . RMD was determined before and after induction of cyclin E overexpression by DOX ( 0 . 5 µg / mL ) added 24 hours before transfection with the indicated sgRNAs / Cas9 . Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6 .

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Figure 3A, Cyclin E overexpression results in increased RMD when the DSB is close to one repeat. The U2OS (EGFP-SSA-3427-3) cell line carrying Tet-on-cyclin E plasmid was used, and RMD frequency was determined with or without induction of cyclin E expression by doxycycline (DOX, 0.5 µg/mL), which was added 24 hours before transfection of the indicated sgRNAs/Cas9. Cyclin E expression levels were validated by Western blot, using KU70 as the loading control.B, ATR is required for BIR/RMD upon cyclin E overexpression. RMD frequency was determined in U2OS (EGFP-SSA-3427-3) cells infected with lentivirus-expressed ATR shRNA or the control vector (Ctrl) with and without induction of cyclin E expression by DOX (0.5 µg/ml) after R29 sgRNA/Cas9 cleavage. ATR knockdown was shown by Western blot with KU70 as the loading control.D, RMD is stimulated when Cas9-D10A is used to create a nick near the repeat. The repair frequencies of the U2OS (MMEJ) reporter cell line (left) and U2OS (EGFP-SSA-3427-17) cell line (right) were determined after expression of the indicated sgRNAs/Cas9 and sgRNAs/Cas9-D10A. The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated (right bottom).E, RMD induced by Cas9-D10A is dependent on POLD3. U2OS (EGFP-SSA-3427-17) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control and RMD was determined after cleavage by the indicated sgRNAs/Cas9-D10A. The distance between sgRNA/Cas9-D10A cleavage sites and the left and right repeat sequences is indicated at the bottom. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.F, p27 suppresses BIR/RMD and SSA/RMD. U2OS (EGFP-SSA-3427-3) cells carrying the Tet-on-cyclin E allele were infected with and without p27 lentiviral viruses. RMD was determined before and after induction of cyclin E overexpression by DOX (0.5 µg/mL) added 24 hours before transfection with the indicated sgRNAs/Cas9. Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6.


Figure 4B , Impact of the 5 ' DSB / repeat distance on RMD frequency when the 3 ' DSB / repeat distance is fixed . RMD assays were performed using mES cells carrying the RMD - GFP reporter , and RMD frequency was determined by the number of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated . C , Long - range RMD is dependent on POLD3 when one DSB is close to a repeat . mES cells carrying the RMD - GFP reporter were transfected with nontargeting siRNA ( Ctrl ) or a pool of POLD3 siRNAs ( siPOLD3 ) 24 hours before for the first time , and with transfection of sgRNAs / Cas9 the second time . The graphs show the frequency of GFP + cells normalized to the transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated . D and E , Impact of the 3 ' DSB / repeat distance on RMD frequency when the 5 ' DSB / repeat distance is fixed . RMD assays were performed using mES cells carrying the RMD - GFP reporter , and RMD frequency was determined by the number of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated . F , RMD frequency of two far - apart repeats that are a short distance from the DSB . RMD was determined in mES cells carrying the RMD - GFP reporter using the indicated sgRNAs / Cas9 , with the frequency of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated . G , The roles of POLD3 , RAD51 and RAD52 in RMD of two far - apart repeats that are a short distance from the DSB . RMD was determined in mES cells carrying the RMD - GFP reporter using the indicated sgRNAs / Cas9 after POLD3 ( left ) or RAD51 ( middle ) was depleted by siRNA or RAD52 was knocked out ( right ) . The distance between the sgRNA targeting sites and the repeats is indicated .

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Figure 4B, Impact of the 5' DSB/repeat distance on RMD frequency when the 3' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.C, Long-range RMD is dependent on POLD3 when one DSB is close to a repeat. mES cells carrying the RMD-GFP reporter were transfected with nontargeting siRNA (Ctrl) or a pool of POLD3 siRNAs (siPOLD3) 24 hours before for the first time, and with transfection of sgRNAs/Cas9 the second time. The graphs show the frequency of GFP+ cells normalized to the transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.D and E, Impact of the 3' DSB/repeat distance on RMD frequency when the 5' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.F, RMD frequency of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9, with the frequency of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.G, The roles of POLD3, RAD51 and RAD52 in RMD of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9 after POLD3 (left) or RAD51 (middle) was depleted by siRNA or RAD52 was knocked out (right). The distance between the sgRNA targeting sites and the repeats is indicated.


Figure 5SSA / RMD and BIR / SSA are both dependent on ATM . SSA / RMD and BIR / SSA frequencies in U2OS ( EGFP - SSA - 3427 - 17 ) cells were determined after the addition of ATM inhibitor ( 20mM ku55933 ) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA / Cas9BIR / SSA are both dependent on ATM . BIR / RMD in mES cells carrying the RMD - GFP reporter were determined after the addition of ATM inhibitor ( 20mM ku55933 ) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA / Cas9 or in ATM KO mES cells carrying the RMD - GFP reporter ( right ) . Dependence of RMD on H2AX . SSA / RMD and BIR / RMD frequencies in U2OS ( EGFP - SSA - 3427 - 17 ) cells were determined using the indicated sgRNA / Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by qPCRD , Dependence of RMD on H2AX . BIR / RMD in mES cells carrying the RMD - GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO

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Figure 5SSA/RMD and BIR/SSA are both dependent on ATM. SSA/RMD and BIR/SSA frequencies in U2OS (EGFP-SSA-3427-17) cells were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9BIR/SSA are both dependent on ATM. BIR/RMD in mES cells carrying the RMD-GFP reporter were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9 or in ATM KO mES cells carrying the RMD-GFP reporter (right).Dependence of RMD on H2AX. SSA/RMD and BIR/RMD frequencies in U2OS (EGFP-SSA-3427-17) cells were determined using the indicated sgRNA/Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by qPCRD, Dependence of RMD on H2AX. BIR/RMD in mES cells carrying the RMD-GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO cells


Figure 6A , Frequency of long - range RMD is increased by an additional DSB downstream of the 3 ' repeat . Illustrations show the GFP - RMD reporter , with sgRNAs / Cas9 cleavage sites indicated as red arrows ( left ) . The frequency of BIR / SSA in mES cells carrying the RMD - GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs / Cas9 ( right ) . B and C , The effects of RAD52 , POLD3 , ATM and H2AX inactivation on BIR / RMD with cleavage at L17 / D10k . The frequency of BIR / RMD in mES cells carrying the RMD - GFP reporter was determined using the sgRNAs / Cas9 at L17 / D10k in RAD52 KO cells or after POLD3 depletion by siRNA ( B ) , or after treatment with ATM inhibitor ( ku55933 , 15mM ) or H2AX depletion by siRNA ( C ) .

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Figure 6A, Frequency of long-range RMD is increased by an additional DSB downstream of the 3' repeat. Illustrations show the GFP-RMD reporter, with sgRNAs/Cas9 cleavage sites indicated as red arrows (left). The frequency of BIR/SSA in mES cells carrying the RMD-GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs/Cas9 (right).B and C, The effects of RAD52, POLD3, ATM and H2AX inactivation on BIR/RMD with cleavage at L17/D10k. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter was determined using the sgRNAs/Cas9 at L17/D10k in RAD52 KO cells or after POLD3 depletion by siRNA (B), or after treatment with ATM inhibitor (ku55933, 15mM) or H2AX depletion by siRNA (C).


Figure 7A , Long - range BIR / RMD with identical repeats or divergent repeats . The frequency of BIR / RMD in mES cells carrying the RMD - GFP reporter with identical repeats ( 0 % ) or repeats with 1 % and 3 % divergence was determined after cleavage at L17 / R3 . 3k or L17 / R28 . 4k . B and C , The effect of MSH2 on BIR / RMD when repeat sequences are divergent . The frequency of BIR / RMD was determined after cleavage at L17 / R3 . 3k or L17 / R28 . 4k in mES cells carrying the RMD - GFP reporter with 1 % and 3 % divergent repeats , and with and without MSH2 KO ( B ) or with MSH2 KO and POLD3 depletion by siRNAs ( C ) . The MSH2 KO was validated by Western blot , using KU70 as the loading control as shown in ( B ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , two - tailed non - paired t - test .

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Figure 7A, Long-range BIR/RMD with identical repeats or divergent repeats. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter with identical repeats (0%) or repeats with 1% and 3% divergence was determined after cleavage at L17/R3.3k or L17/R28.4k.B and C, The effect of MSH2 on BIR/RMD when repeat sequences are divergent. The frequency of BIR/RMD was determined after cleavage at L17/R3.3k or L17/R28.4k in mES cells carrying the RMD-GFP reporter with 1% and 3% divergent repeats, and with and without MSH2 KO (B) or with MSH2 KO and POLD3 depletion by siRNAs (C). The MSH2 KO was validated by Western blot, using KU70 as the loading control as shown in (B). Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, two-tailed non-paired t-test.


Figure 1B . Western blot analysis of RAB6A / A ' and RAB6B protein levels in WT , Emx1 - Cre ; RAB6loxP / loxP ( RAB6A KO ) , RAB6B - / - ( RAB6B KO ) and Emx1 - Cre ; RAB6AloxP / loxP ; RAB6B - / - ( RAB6A / B dKO ) E15 . 5 cortical extracts .

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Figure 1B. Western blot analysis of RAB6A/A' and RAB6B protein levels in WT, Emx1-Cre; RAB6loxP/loxP (RAB6A KO), RAB6B-/- (RAB6B KO) and Emx1-Cre; RAB6AloxP/loxP; RAB6B-/- (RAB6A/B dKO) E15.5 cortical extracts.


Figure 2A . PAX6 staining in WT and RAB6A / B dKO E15 . 5 brains . Scale bar = 50µm . B . Percentage of PAX6 + cells located in the upper half of the cortex of WT and RAB6A / B dKO E15 . 5 brains . WT : 4282 cells from N = 6 brains . RAB6A / B dKO : 1241 cells from N = 3 brains . Mann - Whitney U test . Data information : All error bars indicate SD . C . Phospho - Vimentin ( p - Vim ) staining in WT and RAB6A / B dKO E15 . 5 brains . Scale bar = 50µm . D . Percentage p - VIM + cells dividing ectopically , in the upper half of the cortex of WT and RAB6A / B dKO E15 . 5 brains . WT : 1713 cells from N = 6 brains . RAB6A / B dKO : 506 cells from N = 3 brains . Mann - Whitney U test . Data information : All error bars indicate SD . G . Electroporation of RAB6AloxP / loxP ; RAB6B - / - E14 . 5 embryos with mCherry ( control ) or mCherry + CRE ( RAB6A / B dKO ) and live imaging of delamination events at E17 . 5 . H . Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17 . 5 . mCherry : N = 69 cells from 5 movies . mCherry + CRE : N = 52 cells from 4 movies . Fisher ' s exact test , * p ≤ 0 . 05 . Data information : All error bars indicate SD .

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Figure 2A. PAX6 staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm. B. Percentage of PAX6+ cells located in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 4282 cells from N=6 brains. RAB6A/B dKO: 1241 cells from N=3 brains. Mann-Whitney U test. Data information: All error bars indicate SD.C. Phospho-Vimentin (p-Vim) staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm. D. Percentage p-VIM+ cells dividing ectopically, in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 1713 cells from N=6 brains. RAB6A/B dKO: 506 cells from N=3 brains. Mann-Whitney U test. Data information: All error bars indicate SD.G. Electroporation of RAB6AloxP/loxP; RAB6B-/- E14.5 embryos with mCherry (control) or mCherry + CRE (RAB6A/B dKO) and live imaging of delamination events at E17.5. H. Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17.5. mCherry: N=69 cells from 5 movies. mCherry + CRE: N=52 cells from 4 movies. Fisher's exact test, *p ≤ 0.05. Data information: All error bars indicate SD.