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 1Tfeb expression in the vasculature ( mice n = 10 ) . ( A ) Representative images of retinal vessels ( p5 ) ( A ) ( scale bar : 50 µm ) of Tfeb - EGFP mice stained with anti - iB4 ( A ) and anti - GFPTfeb expression in the vasculature ( mice n = 10 ) . Representative images of glomerular , arterial ( scale bars : 10 µm ) and interstitial vessels ( scale bar : 50 µm ) of kidney ( p17 ) of Tfeb - EGFP mice stained with anti - CD31 ( B ) and anti - GFPAlterations in the embryonic vasculature in TfebEC - / - mice at E10 . 5 ( mice n = 25 ) . Representative images of whole - mount embryos and yolk sacs ( i , ii ) ( scale bars : 0 . 5 mm ) . Vessels of the head ( iii ) , ocular ( iv ) and intrasomitic regions ( v ) were stained with anti - endomucin Ab ( scale bars : 100 µm ) . Alteration of retinal vascular maturation in TfebiEC - / - at p5 . Representative images of whole mounts of retina and immunostaining of vascular front and vascular plexus with an anti - iB4 Ab ( scale bars : 50 µm ) . Bar graphs indicate the percent of vascular area versus total area of the retina , the percent of vascular density , the number of branches per field , vessel diameter , the number of filopodia per field and the length of filopodia ( mice n = 10 , mean ± SEM ; * * * p < 0 . 0001 versus control mice by Student ' s t - test ) . Alteration of the glomerular ultrastructure in TfebiEC - / - mice at p17 ( mice n = 5 ) . Representative transmission electron micrographs of renal tissue . Magnification : i ) x6000 , ii ) x20000 ) , iii - iv ) x10000 , v - vi ) x25000 ; scale bars : 1µm ) . Asterisks indicate the accumulation of extracellular matrix in mesangium ; white and black triangles indicate the fusion of podocyte foot processes and the lack of endothelial fenestrae , respectively .

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 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 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 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 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

Figure 1Tfeb expression in the vasculature (mice n=10). (A) Representative images of retinal vessels (p5) (A) (scale bar: 50 µm) of Tfeb-EGFP mice stained with anti-iB4 (A) and anti-GFPTfeb expression in the vasculature (mice n=10). Representative images of glomerular, arterial (scale bars: 10 µm) and interstitial vessels (scale bar: 50 µm) of kidney (p17) of Tfeb-EGFP mice stained with anti-CD31 (B) and anti-GFPAlterations in the embryonic vasculature in TfebEC-/- mice at E10.5 (mice n=25). Representative images of whole-mount embryos and yolk sacs (i, ii) (scale bars: 0.5 mm). Vessels of the head (iii), ocular (iv) and intrasomitic regions (v) were stained with anti-endomucin Ab (scale bars: 100 µm).Alteration of retinal vascular maturation in TfebiEC-/- at p5. Representative images of whole mounts of retina and immunostaining of vascular front and vascular plexus with an anti-iB4 Ab (scale bars: 50 µm). Bar graphs indicate the percent of vascular area versus total area of the retina, the percent of vascular density, the number of branches per field, vessel diameter, the number of filopodia per field and the length of filopodia (mice n=10, mean±SEM; ***p< 0.0001 versus control mice by Student's t-test).Alteration of the glomerular ultrastructure in TfebiEC-/- mice at p17 (mice n=5). Representative transmission electron micrographs of renal tissue. Magnification: i) x6000, ii) x20000), iii-iv) x10000, v-vi) x25000; scale bars: 1µm). Asterisks indicate the accumulation of extracellular matrix in mesangium; white and black triangles indicate the fusion of podocyte foot processes and the lack of endothelial fenestrae, respectively.


Figure 2Reduced EC proliferation in the retina ( p5 ) of in TfebiEC - / - mice . Representative images of vessels of the vascular front and vascular plexus in the retina of control and TfebiEC - / - mice stained with anti - iB4 and Ki - 67 antibodies ( scale bars : 50 µm ) . Bar graph indicates the percentage of EC Ki - 67 + nuclei versus total nuclei co - localized with iB4 + vesselsReduced EC proliferation in the kidney ( p17 ) of in TfebiEC - / - mice . ( B ) Representative images of vessels of the kidney in control and TfebiEC - / - mice stained with anti - CD31 and Ki - 67 antibodies ( scale bars : 50 µm ) . Bar graph indicates the percent of EC Ki - 67 + nuclei versus total nuclei co - localized with CD31 + vessels ( mice n = 6 , mean ± SEM ; * * p < 0 . 001 versus control mice by Student ' s t - test ) . Podocin staining is shown to glomerular localization . TFEB silencing reduced EC proliferation . Representative graph of scr - shRNA and sh - TFEB ECs treated for 24 hours with FCS ( 20 % ) or VEGF - A ( 30 ng / ml ) . ( C ) DNA incorporation of EdU was detected by flow cytometry . The percentage of proliferating cells is indicated ( n = 4 , mean ± SEM ; * * * p < 0 . 0001 versus scr - shRNA ECs by Student ' s t - test ) . TFEB silencing reduced EC proliferation . Representative graph of scr - shRNA and sh - TFEB ECs treated for 24 hours with FCS ( 20 % ) or VEGF - A ( 30 ng / ml ) . DNA content was determined by propidium iodide staining and assessed by fluorescences - activated cell sorter analysis ( representative experiment out of 4 with similar results ) . TFEB silencing reduced human EC morphogenesis . Representative images of tube - like - structure of scr - shRNA and sh - TFEB human ECs stained with phalloidin - 555 . Bar graph indicates the percentage of phalloidin + area in sh - TFEB and scr - shRNA ECs ( scale bars : 0 . 25 mm ; n = 6 , mean ± SEM ; * * * p < 0 . 0001 versus scr - shRNA by Student ' s t - test ) .

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 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 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 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

Figure 2Reduced EC proliferation in the retina (p5) of in TfebiEC-/- mice. Representative images of vessels of the vascular front and vascular plexus in the retina of control and TfebiEC-/- mice stained with anti-iB4 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percentage of EC Ki-67+ nuclei versus total nuclei co-localized with iB4+ vesselsReduced EC proliferation in the kidney (p17) of in TfebiEC-/- mice. (B) Representative images of vessels of the kidney in control and TfebiEC-/- mice stained with anti-CD31 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percent of EC Ki-67+ nuclei versus total nuclei co-localized with CD31+ vessels (mice n=6, mean±SEM; **p<0.001 versus control mice by Student's t-test). Podocin staining is shown to glomerular localization.TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). (C) DNA incorporation of EdU was detected by flow cytometry. The percentage of proliferating cells is indicated (n=4, mean±SEM; ***p<0.0001 versus scr-shRNA ECs by Student's t-test).TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). DNA content was determined by propidium iodide staining and assessed by fluorescences-activated cell sorter analysis (representative experiment out of 4 with similar results).TFEB silencing reduced human EC morphogenesis. Representative images of tube-like-structure of scr-shRNA and sh-TFEB human ECs stained with phalloidin-555. Bar graph indicates the percentage of phalloidin+ area in sh-TFEB and scr-shRNA ECs (scale bars: 0.25 mm; n=6, mean±SEM; ***p<0.0001 versus scr-shRNA by Student's t-test).


Figure 3Heatmap showing unsupervised hierarchical clustering of human differentially expressed genes between human scr - shRNA and sh - TFEB ECs . Red : up - regulated genes ; green : down - regulated genes .

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 3Heatmap showing unsupervised hierarchical clustering of human differentially expressed genes between human scr-shRNA and sh-TFEB ECs. Red: up-regulated genes; green: down-regulated genes.


Figure 4Heatmap , Red : up - regulated genes ; green : down - regulated genes . qPCR Data are expressed as relative fold - change compared with the expression in scr - shRNA cells after normalization to the housekeeping gene TBPimmunoblots showing the differentially expressed cell cycle related genes between scr - shRNA and sh - TFEB ECs . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with specific Abs . The bar graph shows the densitometric analysis expressed as the ratio between the cell cycle genes and α - tubulinModulation of cell cycle genes expression in the lung ECs derived from control and TfebiEC - / - mice . Data are expressed as relative fold - change compared with the expression in ECs of control mice after normalization to the housekeeping gene TBPAnalysis of TFEB binding and modulation of the CDK4 promoter in human ECs . ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for CDK4 . Bar graph shows the percent enrichment ( n = 3 , mean ± SD ) . Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs . Analysis of TFEB modulation of CDK4 promoter in human ECs . Bar graph shows the relative luciferase activity % evaluated in control and TFEBS142A human ECs after transfection of CDK4 full promoter and CDK4 promoter deleted of TFEB binding siteModulation of Rb protein in sh - TFEB ECs . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with phospho - Rb and total Rb Abs . The bar graph shows the densitometric analysis of the immunoblotting expressed as % of sh - TFEB vs scr - shRNA and the ratio between phospho - Rb and total Rb in the different

O B-PANEL_START 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 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 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 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 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 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 4Heatmap, Red: up-regulated genes; green: down-regulated genes.qPCR Data are expressed as relative fold-change compared with the expression in scr-shRNA cells after normalization to the housekeeping gene TBPimmunoblots showing the differentially expressed cell cycle related genes between scr-shRNA and sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with specific Abs. The bar graph shows the densitometric analysis expressed as the ratio between the cell cycle genes and α-tubulinModulation of cell cycle genes expression in the lung ECs derived from control and TfebiEC-/- mice. Data are expressed as relative fold-change compared with the expression in ECs of control mice after normalization to the housekeeping gene TBPAnalysis of TFEB binding and modulation of the CDK4 promoter in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for CDK4. Bar graph shows the percent enrichment (n=3, mean±SD).Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. Analysis of TFEB modulation of CDK4 promoter in human ECs. Bar graph shows the relative luciferase activity % evaluated in control and TFEBS142A human ECs after transfection of CDK4 full promoter and CDK4 promoter deleted of TFEB binding siteModulation of Rb protein in sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with phospho-Rb and total Rb Abs. The bar graph shows the densitometric analysis of the immunoblotting expressed as % of sh-TFEB vs scr-shRNA and the ratio between phospho-Rb and total Rb in the different conditions


Figure 5Increase of Vegfr2 expression in the vasculature of TfebEC - / - mice . Representative immunostaining images of TfebEC - / - embryonic vessels ( E10 . 5 ) stained with anti - endomucin and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graph indicates the Vegfr2 mean intensity only in endomucin + vessel areas ( embryos n = 6 , mean ± SEM ; * * * p < 0 . 0001 versus control embryos by Student ' s t - test ) . Increase of Vegfr2 expression in the vasculature of TfebiEC - / - mice . Representative immunostaining images ( i ) and detail ( ii ) of the vascular front and vascular plexus of the retina ( p5 ) of control and TfebiEC - / - mice with anti - iB4 and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graph indicates the Vegfr2 mean intensity only in iB4 + vessel areasIncrease of Vegfr2 expression in the vasculature of TfebiEC - / - mice . Representative immunostaining images of the glomerulus ( p17 ) of control and TfebiEC - / - mice with anti - podocin , anti - CD31 and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graphs indicate the Vegfr2 mean intensity in CD31 + vessel areasVEGFR2 expression is regulated by TFEB . qPCR of VEGFR2 in lung ECs obtained from control and TfebiEC - / - mice ( D ) Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB . qPCR of VEGFR2 in lung ECs obtained from control and in human sh - TFEB Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with anti - VEGFR2 and α - tubulin Abs . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α - tubulinAnalysis of TFEB binding to the VEGFR2 promoter in human ECs . ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for VEGFR2 .

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 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 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 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 O O O O 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 5Increase of Vegfr2 expression in the vasculature of TfebEC-/- mice. Representative immunostaining images of TfebEC-/- embryonic vessels (E10.5) stained with anti-endomucin and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in endomucin+ vessel areas (embryos n=6, mean±SEM; ***p< 0.0001 versus control embryos by Student's t-test).Increase of Vegfr2 expression in the vasculature of TfebiEC-/- mice. Representative immunostaining images (i) and detail (ii) of the vascular front and vascular plexus of the retina (p5) of control and TfebiEC-/- mice with anti-iB4 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in iB4+ vessel areasIncrease of Vegfr2 expression in the vasculature of TfebiEC-/- mice. Representative immunostaining images of the glomerulus (p17) of control and TfebiEC-/- mice with anti-podocin, anti-CD31 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graphs indicate the Vegfr2 mean intensity in CD31+ vessel areasVEGFR2 expression is regulated by TFEB. qPCR of VEGFR2 in lung ECs obtained from control and TfebiEC-/- mice (D) Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB. qPCR of VEGFR2 in lung ECs obtained from control and in human sh-TFEB Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with anti-VEGFR2 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulinAnalysis of TFEB binding to the VEGFR2 promoter in human ECs. ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for VEGFR2.


Figure 6Silenced TFEB alters the localization of VEGFR2 . FACS analysis of surface VEGFR2 expression on human scr - shRNA and sh - TFEB ECs . Bar graph shows the ratio between total and PM VEGFR2Silenced TFEB alters the localization of VEGFR2 . Representative TIRF and epifluorescence images of human scr - shRNA and sh - TFEB ECs after staining with anti - VEGFR2 Ab ( scale bars : 10 µm ) . Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by TIRFSilenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction . Representative immunoblot of PM biotinylated portion and total cell lysates of scr - shRNA and sh - TFEB ECs after VEGF - A stimulation ( 30 ng / ml ) . Blots of total or PM cell lysates were probed with anti - VEGFR2 . Blots of total cell lysates were probed with anti - p - Y1175 - VEGFR2 , anti - PLCγ , p - PLCγ , anti - ERK - 1 / 2 , anti - pERK1 / 2 , anti - p - Src , anti - Src , anti CD31 and α - tubulin Abs . The bar graphs ( i , ii ) show densitometric analysis of stimulated versus unstimulated scr - shRNA and sh - TFEB ECs expressed as : ( i ) % of VEGFR2 on PM fraction ( n = 3 , mean ± SEM ; ANOVA p < 0 . 02 ; * * p < 0 . 001 versus scr - shRNA by Bonferroni post - test ) , ( ii ) % of VEGFR2 totalRegulaton of autophagy and lysosome pathway by TFEB silencing . qPCR showing the differentially expressed autophagy and lysosome related genes between human scr - shRNA and sh - TFEB ECs . Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene TBPRegulaton of autophagy and lysosome pathway by TFEB silencing . immunoblots showing the differentially expressed autophagy and lysosome related genes between human scr - shRNA and sh - TFEB ECs . Immunoblots of total lysates from human scr - shRNA and sh - TFEB ECs probed with anti - ULK - 1 , anti - ATG9 , anti - LC3 - I / II , anti - LAMP - 1 and α - tubulin Abs . The bar graph shows the densitometric analysis expressed as the ratio between scr - shRNA and sh - TFEB ECsTFEB silencing inhibits VEGFR2 internalization . Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF - A

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 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 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 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 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 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

Figure 6Silenced TFEB alters the localization of VEGFR2. FACS analysis of surface VEGFR2 expression on human scr-shRNA and sh-TFEB ECs. Bar graph shows the ratio between total and PM VEGFR2Silenced TFEB alters the localization of VEGFR2. Representative TIRF and epifluorescence images of human scr-shRNA and sh-TFEB ECs after staining with anti-VEGFR2 Ab (scale bars: 10 µm). Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by TIRFSilenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction. Representative immunoblot of PM biotinylated portion and total cell lysates of scr-shRNA and sh-TFEB ECs after VEGF-A stimulation (30 ng/ml). Blots of total or PM cell lysates were probed with anti-VEGFR2. Blots of total cell lysates were probed with anti-p-Y1175-VEGFR2, anti-PLCγ, p-PLCγ, anti-ERK-1/2, anti-pERK1/2, anti-p-Src, anti-Src, anti CD31 and α-tubulin Abs. The bar graphs (i,ii) show densitometric analysis of stimulated versus unstimulated scr-shRNA and sh-TFEB ECs expressed as: (i) % of VEGFR2 on PM fraction (n=3, mean±SEM; ANOVA p<0.02; **p<0.001 versus scr-shRNA by Bonferroni post-test), (ii) % of VEGFR2 totalRegulaton of autophagy and lysosome pathway by TFEB silencing. qPCR showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBPRegulaton of autophagy and lysosome pathway by TFEB silencing. immunoblots showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. Immunoblots of total lysates from human scr-shRNA and sh-TFEB ECs probed with anti-ULK-1, anti-ATG9, anti-LC3-I/II, anti-LAMP-1 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between scr-shRNA and sh-TFEB ECsTFEB silencing inhibits VEGFR2 internalization. Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF-A stimulation


Figure 7Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs . ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for MYO1C . Bar graph shows the percent enrichment ( n = 3 , mean ± SD ) . Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs . qPCR of MYO1C expression in scr - shRNA , sh - TFEB , sh - MYO1C and sh - TFEB + sh - MYO1C ECs . Data are expressed as relative fold change compared with the expression in scr - shRNA ECs after normalization to the housekeeping gene TBPMYO1C silencing reverses the effect of TFEB silencing on the up - regulation of PM VEGFR2 . Analyses were performed on human ECs carrying appropriate scr - shRNA or sh - TFEB in the presence or absence of sh - MYO1C . Representative western blot of MYOC1 , total and PM biotinylated VEGFR2 ( representative experiment out of 4 with similar results ) . Representative immunostaining images of the vascular plexus of the retina ( p5 ) ( E ) and glomerulus ( p17 ) ( F ) of control and TfebiEC - / - mice with anti - CD31 and anti - MYO1C Abs ( scale bars : 50 µm ) . Bar graphs indicate the Myo1C mean intensity only in vessel areas CD31 + Representative TIRF and epifluorescence images of VEGFR2 in scr - shRNA , sh - TFEB , sh - MYO1C and sh - TFEB + sh - MYO1C human ECs ( scale bars : 10 µm ) . Bar graph shows the ratio between PM and total

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 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 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

Figure 7Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for MYO1C. Bar graph shows the percent enrichment (n=3, mean±SD).Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs. qPCR of MYO1C expression in scr-shRNA, sh-TFEB, sh-MYO1C and sh-TFEB+sh-MYO1C ECs. Data are expressed as relative fold change compared with the expression in scr-shRNA ECs after normalization to the housekeeping gene TBPMYO1C silencing reverses the effect of TFEB silencing on the up-regulation of PM VEGFR2. Analyses were performed on human ECs carrying appropriate scr-shRNA or sh-TFEB in the presence or absence of sh-MYO1C. Representative western blot of MYOC1, total and PM biotinylated VEGFR2 (representative experiment out of 4 with similar results).Representative immunostaining images of the vascular plexus of the retina (p5) (E) and glomerulus (p17)(F) of control and TfebiEC-/- mice with anti-CD31 and anti-MYO1C Abs (scale bars: 50 µm). Bar graphs indicate the Myo1C mean intensity only in vessel areas CD31+Representative TIRF and epifluorescence images of VEGFR2 in scr-shRNA, sh-TFEB, sh-MYO1C and sh-TFEB+sh-MYO1C human ECs (scale bars: 10 µm). Bar graph shows the ratio between PM and total VEGFR2


Figure 8Analysis of TFEB binding to DLEU2 and SMC4 promoters in human ECs . ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for DLEU2 and SMC4 . DLEU2 expression is regulated by TFEB . qPCR of DLEU2 in human scr - shRNA , sh - TFEB or control and TFEBS142A ECs ( left panel ) and lung ECs obtained from control and TfebiEC - / - mice ( right panel ) . Data are expressed as relative fold - change compared with the expression in scr - shRNA and control cells after normalization to the housekeeping gene TBPHuman miR - 15a - 5p and miR - 16 - 5p are regulated by TFEB . qPCR of miR - 15a - 5p ( left panel ) and miR - 16 - 5p ( right panel ) in sh - TFEB or TFEBS142A ECs . Data are expressed as relative fold - change compared with the expression in scr - shRNA and control cells after normalization to the housekeeping gene RNU44VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . qPCR of VEGFR2 in human scr - shRNA and sh - TFEB ECs and in control treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p mimics Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . VEGFR2 in human scr - shRNA and sh - TFEB ECs and in control treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p mimics Representative western blot of VEGFR2 expression under the same experimental conditions previously reported . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α - tubulinVEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . qPCR of VEGFR2 in control and TFEBS142A ECs treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p inhibitors . Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . VEGFR2 in control and TFEBS142A ECs treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p inhibitors . Representative western blot of VEGFR2 expression under the same experimental conditions previously reported . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α -

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 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 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 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 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 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

Figure 8Analysis of TFEB binding to DLEU2 and SMC4 promoters in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for DLEU2 and SMC4.DLEU2 expression is regulated by TFEB. qPCR of DLEU2 in human scr-shRNA, sh-TFEB or control and TFEBS142A ECs (left panel) and lung ECs obtained from control and TfebiEC-/- mice (right panel). Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene TBPHuman miR-15a-5p and miR-16-5p are regulated by TFEB. qPCR of miR-15a-5p (left panel) and miR-16-5p (right panel) in sh-TFEB or TFEBS142A ECs. Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene RNU44VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulinVEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in control and TFEBS142A ECs treated with a specific miR-control, miR-15a-5p and miR-16-5p inhibitors. Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. VEGFR2 in control and TFEBS142A ECs treated with a specific miR-control, miR-15a-5p and miR-16-5p inhibitors. Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin


Figure 1A - D Heat maps of the occurrence frequency of effective variants within each genotype in DENV1 ( A ) , DENV2 ( B ) , DENV3 ( C ) , and DENV4 ( D ) . Residues with the occurrence frequency < 20 % before 1995 were shown in the heat maps . Red square frames represent ten stable substitutions , including 171T in DENV1 - I ( A ) , 131Q , 170T , 340T , and 380V in DENV2 - Asian American ( B ) , 226K and 228E in DENV2 - Asian I ( B ) , 301S and 377I in DENV3 - I ( C ) , and 265A in DENV4 - I ( D ) . Gray boxes represent the data that were not available . E - H Growth kinetics and plaque morphology DENV2 - Asian I 16681 strain ( 16681 , U87411 ) ( E ) , DENV2 - Asian American DENV2 / US / BID - V1164 / 1986 strain ( BID , EU482568 ) ( F ) , DENV3 - I PF89 / 27643 strain ( PF89 , AY744677 ) ( G ) , and DENV4 - I H241 strain ( H241 , AY947539 ) ( H ) . Vero cells were infected with each mutant at a multiplicity of infection ( MOI ) of 0 . 01 , and the cell supernatant was collected at Days 1 , 3 , 5 , 7 , and 9 for the plaque assay . Data information : In ( E - H ) , data are presented as mean ± SEM of n = 4 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 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

Figure 1A-D Heat maps of the occurrence frequency of effective variants within each genotype in DENV1 (A), DENV2 (B), DENV3 (C), and DENV4 (D). Residues with the occurrence frequency <20% before 1995 were shown in the heat maps. Red square frames represent ten stable substitutions, including 171T in DENV1-I (A), 131Q, 170T, 340T, and 380V in DENV2-Asian American (B), 226K and 228E in DENV2-Asian I (B), 301S and 377I in DENV3-I (C), and 265A in DENV4-I (D). Gray boxes represent the data that were not available.E-H Growth kinetics and plaque morphology DENV2-Asian I 16681 strain (16681, U87411) (E), DENV2-Asian American DENV2/US/BID-V1164/1986 strain (BID, EU482568) (F), DENV3-I PF89/27643 strain (PF89, AY744677) (G), and DENV4-I H241 strain (H241, AY947539) (H). Vero cells were infected with each mutant at a multiplicity of infection (MOI) of 0.01, and the cell supernatant was collected at Days 1, 3, 5, 7, and 9 for the plaque assay. Data information: In (E-H), data are presented as mean ± SEM of n = 4 biological replicates.


Figure 2One - week - old mosquitoes were thoracically infected with DENV2 - Asian I ( A ) , DENV2 - Asian American ( B ) , DENV3 - I mutants , respectively . A . aegypti mosquitoes were infected intrathoracically with 10 p . f . u . of DENV . The DENV viral loads were assessed at 3 days postinfection via qRT - PCR and were normalized against A . aegypti actin . Data information : one dot represents one mosquito , and the horizontal lines represent the median of the results . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) . One - week - old mosquitoes were thoracically infected with DENV4 - I ( D ) mutants , respectively . A . aegypti mosquitoes were infected intrathoracically with 10 p . f . u . of DENV . The DENV viral loads were assessed at 3 days postinfection via qRT - PCR and were normalized against A . aegypti actin . Data information : one dot represents one mosquito , and the horizontal lines represent the median of the results . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) . Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV2 - Asian I ( E ) , were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) . Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV2 - Asian American ( F ) , DENV3 - I ( G ) were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : , n = 6 biological replicates . In ( F ) , n = 5 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) . Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV4 - I ( H ) were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) . I , J Infection of DENV2 - Asian I mutants on hu - moDC ( I ) and hu - moMØ ( J ) . The cells were infected with 0 . 01 MOI of the indicated viruses . The infected cells were detected at 48 h postinfection by qRT - PCR , and DENV viral loads were normalized against human GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the unpaired t - test ( I , J ) . The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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 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 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 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 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 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

Figure 2One-week-old mosquitoes were thoracically infected with DENV2-Asian I (A), DENV2-Asian American (B), DENV3-I mutants, respectively. A. aegypti mosquitoes were infected intrathoracically with 10 p.f.u. of DENV. The DENV viral loads were assessed at 3 days postinfection via qRT-PCR and were normalized against A. aegypti actin. Data information: one dot represents one mosquito, and the horizontal lines represent the median of the results. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).One-week-old mosquitoes were thoracically infected with DENV4-I (D) mutants, respectively. A. aegypti mosquitoes were infected intrathoracically with 10 p.f.u. of DENV. The DENV viral loads were assessed at 3 days postinfection via qRT-PCR and were normalized against A. aegypti actin. Data information: one dot represents one mosquito, and the horizontal lines represent the median of the results. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV2-Asian I (E), were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV2-Asian American (F), DENV3-I (G) were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: , n = 6 biological replicates. In (F), n = 5 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV4-I (H) were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).I, J Infection of DENV2-Asian I mutants on hu-moDC (I) and hu-moMØ (J). The cells were infected with 0.01 MOI of the indicated viruses. The infected cells were detected at 48 h postinfection by qRT-PCR, and DENV viral loads were normalized against human GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the unpaired t-test (I, J). The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


Figure 3A The five - year occurrence frequency of residues in 226th and 228th sites from < 1995 to 2016 - 2020 . Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . The viremia of infected AG6 mice from Day 1 to Day 7 was measured using qRT - PCR . Data information : , n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) . Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . Two days postinfection , infected mice were euthanized , and the viral loads in the bone marrow ( C ) were measured by qRT - PCR . Data information : n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) . Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . Two days postinfection , infected mice were euthanized , and the viral loads in the spleen ( D ) , and footpad ( E ) were measured by qRT - PCR . Data information : n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) . Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . The survival rates of mice ( F ) were recorded daily . Data information : In ( F ) , n = 10 biological replicates . In ( F ) , the data were analyzed statistically using the log - rank ( Mantel - Cox ) test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) . Mosquito infectivity was determined by qRT - PCR at 8 days after the blood meal . The number at the top of each column represents the infected number / total number ( H ) . Data information : In ( H one dot represents one mosquito , and the horizontal lines represent the median of the results . In ( H , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and blood samples were collected for DENV2 viremia detection by qRT - PCR ( J ) . Data information : In ( J ) , n = 4 biological replicates . In J ) , the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test J ) . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) . Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and Mosquitoes were collected at 8 days after the blood meal for qRT - PCR to compare the infection rate of DENV2 ( K Data information : In K , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) . Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and Mosquitoes were collected at 8 days after the blood meal for qRT - PCR to compare the infection rate of DENV2 L ) . The number at the top of each column represents the infected number / total number ( L ) . Data information : In L ) , one dot represents one mosquito , and the horizontal lines represent the median of the results . In , L ) , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Figure 3A The five-year occurrence frequency of residues in 226th and 228th sites from <1995 to 2016-2020.Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. The viremia of infected AG6 mice from Day 1 to Day 7 was measured using qRT-PCR. Data information: , n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. Two days postinfection, infected mice were euthanized, and the viral loads in the bone marrow (C) were measured by qRT-PCR. Data information: n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. Two days postinfection, infected mice were euthanized, and the viral loads in the spleen (D), and footpad (E) were measured by qRT-PCR. Data information: n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. The survival rates of mice (F) were recorded daily. Data information: In (F), n = 10 biological replicates. In (F), the data were analyzed statistically using the log-rank (Mantel-Cox) test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Mosquito infectivity was determined by qRT-PCR at 8 days after the blood meal. The number at the top of each column represents the infected number/total number (H). Data information: In (H one dot represents one mosquito, and the horizontal lines represent the median of the results. In (H, the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and blood samples were collected for DENV2 viremia detection by qRT-PCR (J). Data information: In (J), n = 4 biological replicates. In J), the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test J). The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and Mosquitoes were collected at 8 days after the blood meal for qRT-PCR to compare the infection rate of DENV2 (K Data information: In K, the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and Mosquitoes were collected at 8 days after the blood meal for qRT-PCR to compare the infection rate of DENV2 L). The number at the top of each column represents the infected number/total number (L). Data information: In L), one dot represents one mosquito, and the horizontal lines represent the median of the results. In , L), the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).