Figure 3A C57BL / 6N mice pups at P3 received 1 μg of mouse Sema3C - Fc ( right eye ) or control IgG - Fc ( left eye ) . The retinal vasculature was examined at P5 by whole - mount CD31immunofluorescence staining . Representative images show the total retinal vasculature and sprouting tips at the vascular front ( iii and iv , arrows ) and the enlarged intercapillary space ( ii and iv , asterisks ) . Scale bar , 100 μm and 20 μm ( iii and iv ) . B , C ( B ) Retinal vessel outgrowth measured as the total area from the central optic disk to the vascular front normalized to control IgG - Fc treatment , * * * P = 0 . 0009 . ( C ) Vessel density was quantified by CD31 - stained vessels and normalized to the total vessel area , * * * P = 0 . 0002 . ( Mean ± s . e . m ; n = 23 retinae in each group from four independent experiments ; unpaired Student ' s t - test . ) D , E ( D ) Number of sprouting tips per retina , * P < 0 . 0001 . ( E ) Branch points in the capillary plexus represented as the ratio to the control IgG - treated retinae , * * * P < 0 . 0001 . ( Mean ± s . e . m ; n = 18 retinae in each group from three independent experiments ; unpaired Student ' s t - test . ) F Pups received same treatment as described in ( A ) . The astrocyte layer was examined at P5 by GFAP staining . Scale bar , 100 μm ( upper panel ) and 20 μm ( lower panel ) . Representative images of six retinae per group . G Six - week - old C57BL / 6N male mice were intravitreally injected with Sema3C - Fc ( right eye ) or control IgG - Fc ( left eye ) , and retinae were collected 2 days later . The retinal vessel network was examined with CD31immunostaining . Scale bar , 100 μm ( upper panel ) and 20 μm ( lower panel ) . Representative images of four retinae per group .

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

Figure 3A C57BL/6N mice pups at P3 received 1 μg of mouse Sema3C-Fc (right eye) or control IgG-Fc (left eye). The retinal vasculature was examined at P5 by whole-mount CD31immunofluorescence staining. Representative images show the total retinal vasculature and sprouting tips at the vascular front (iii and iv, arrows) and the enlarged intercapillary space (ii and iv, asterisks). Scale bar, 100 μm and 20 μm (iii and iv).B,C (B) Retinal vessel outgrowth measured as the total area from the central optic disk to the vascular front normalized to control IgG-Fc treatment, ***P = 0.0009. (C) Vessel density was quantified by CD31-stained vessels and normalized to the total vessel area, ***P = 0.0002. (Mean ± s.e.m; n = 23 retinae in each group from four independent experiments; unpaired Student's t-test.)D,E (D) Number of sprouting tips per retina, *P < 0.0001. (E) Branch points in the capillary plexus represented as the ratio to the control IgG-treated retinae, ***P < 0.0001. (Mean ± s.e.m; n = 18 retinae in each group from three independent experiments; unpaired Student's t-test.)F Pups received same treatment as described in (A). The astrocyte layer was examined at P5 by GFAP staining. Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Representative images of six retinae per group.G Six-week-old C57BL/6N male mice were intravitreally injected with Sema3C-Fc (right eye) or control IgG-Fc (left eye), and retinae were collected 2 days later. The retinal vessel network was examined with CD31immunostaining. Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Representative images of four retinae per group.


Figure 4HUVECs were treated with Sema3C - or GFP - conditioned medium for 24 h . Cell proliferation was quantified by BrdU incorporation ( mean ± s . e . m ; n = 3 ; n . s . , non - significant ) . Apoptosis of HUVEC was measured by caspase - 3 and caspase - 7 activities after 3 h of incubation with Sema3C - or control - conditioned medium ( mean ± s . e . m ; n = 3 , * P = 0 . 018 using unpaired Student ' s t - test ) . Serum - starved HUVECs were incubated with conditioned medium or conditioned medium + VEGF ( 50 ng / ml ) for 15 min . Cell lysates were subjected to SDS - PAGE and immunoblotted with anti - phospho - AKT antibody . Quantification of phospho - AKT normalized to total AKT levels ( mean ± s . e . m ; n = 4 ; control vs . Sema3C ( − VEGF ) , * P = 0 . 034 and control vs . Sema3C ( + VEGF ) , * P = 0 . 024 using unpaired Student ' s t - test ) . HUVECs were adenovirally transduced with VE - cadherin - GFP and plated on ibidi slides ( n = 4 ; control vs . Sema3C ( − VEGF ) at 20 × magnification ( Supplementary Movies S1 and S2 ) ) . Representative pictures are shown at time points 0 and 1 h . Arrowheads indicate gaps between endothelial cells . Scale bar , 100 μm . C57BL / 6Nmicepups at P5 received 1 μg of recombinant Sema3C or IgG - Fc proteins . Endothelial cell junctions were examined by whole - mount VE - cadherinimmunofluorescence staining 6 h later . Representative images show VE - cadherin at cell junctions ( arrows ) . The linear structure ( i ) was disrupted in Sema3C - treated retinae ( ii ) . Scale bar , 100 μm ( upper panel ) and 20 μm ( lower panel ) . Whole - mount immunostaining of the vascular bed by CD31 and apoptotic cells by cleaved caspase - 3 in the retinae at P5 8 h after intravitreal injection of Sema3C or IgG - Fc . Representative images show active caspase - 3 signals that co - localize with endothelial cells ( arrows ) . Scale bar , 100 μm . Quantification of cleaved caspase - 3 normalized to area of CD31 - stained vessels ( mean ± s . e . m ; n = 5 ; * * P = 0 . 007 using paired 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 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 O O 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 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

Figure 4HUVECs were treated with Sema3C- or GFP-conditioned medium for 24 h. Cell proliferation was quantified by BrdU incorporation (mean ± s.e.m; n = 3; n.s., non-significant).Apoptosis of HUVEC was measured by caspase-3 and caspase-7 activities after 3 h of incubation with Sema3C- or control-conditioned medium (mean ± s.e.m; n = 3, *P = 0.018 using unpaired Student's t-test).Serum-starved HUVECs were incubated with conditioned medium or conditioned medium + VEGF (50 ng/ml) for 15 min. Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-AKT antibody.Quantification of phospho-AKT normalized to total AKT levels (mean ± s.e.m; n = 4; control vs. Sema3C (− VEGF), *P = 0.034 and control vs. Sema3C (+ VEGF), *P = 0.024 using unpaired Student's t-test).HUVECs were adenovirally transduced with VE-cadherin-GFP and plated on ibidi slides (n = 4; control vs. Sema3C (− VEGF) at 20× magnification (Supplementary Movies S1 and S2)). Representative pictures are shown at time points 0 and 1 h. Arrowheads indicate gaps between endothelial cells. Scale bar, 100 μm.C57BL/6Nmicepups at P5 received 1 μg of recombinant Sema3C or IgG-Fc proteins. Endothelial cell junctions were examined by whole-mount VE-cadherinimmunofluorescence staining 6 h later. Representative images show VE-cadherin at cell junctions (arrows). The linear structure (i) was disrupted in Sema3C-treated retinae (ii). Scale bar, 100 μm (upper panel) and 20 μm (lower panel).Whole-mount immunostaining of the vascular bed by CD31 and apoptotic cells by cleaved caspase-3 in the retinae at P5 8 h after intravitreal injection of Sema3C or IgG-Fc. Representative images show active caspase-3 signals that co-localize with endothelial cells (arrows). Scale bar, 100 μm.Quantification of cleaved caspase-3 normalized to area of CD31-stained vessels (mean ± s.e.m; n = 5; **P = 0.007 using paired Student's t-test).


Figure 5A HUVECs were transduced with adenoviral vector expressing RFP - conjugated F - actin binding peptide ( LifeAct ) . Time - lapse imaging of F - actin in HUVEC spheroids was obtained at 20 × magnification ( Supplementary Movies S3 and S4 ) . Representative images are shown at 0 and 2 h . Arrowheads indicate detached cells after Sema3C treatment . Scale bar , 100 μm . B Endothelial cell migration was determined using a scratch wound assay with a co - culture model . A wound was generated in a confluent HUVEC monolayer , and cells were cultured in basal medium with or without VEGF ( 25 ng / ml ) in a 24 - well plate . At the same time , the transwells containing Sema3C or GFP - expressing pericytes . Representative pictures show remaining gaps within the HUVEC monolayer . Scale bar , 200 μm . C Migration distance was measured after 24 h and normalized to control treatment ( mean ± s . e . m ; n = 3 independent experiments , control vs . Sema3C ( − VEGF ) , * P = 0 . 021 and control vs . Sema3C ( + VEGF ) , * P = 0 . 039 using unpaired Student ' s t - test ) . D , E ( D ) Serum - starved HUVECs were treated with Sema3C - or control - conditioned medium with or without VEGF ( 50 ng / ml ) for 10 min . Cell lysates were subjected to SDS - PAGE and probed with anti - phosphoT180 / Y182 p38MAPK antibody . Quantification of p38MAPK activation was normalized to total p38MAPK and presented as the ratio to the cells with control medium without VEGF treatment . Control vs . Sema3C ( − VEGF ) , * P = 0 . 046 and control vs . Sema3C ( + VEGF ) , * P = 0 . 043 . ( E ) HUVECs were treated as described before and the activation of FAK detected by anti - phosphoY397 FAK antibody and normalized to the total amount of FAK . Control vs . Sema3C ( − VEGF ) , * P = 0 . 033 and control vs . Sema3C ( + VEGF ) , * P = 0 . 047 . ( Mean ± s . e . m ; n = 3 independent experiments ; unpaired Student ' s t - test . ) F Serum - starved HUVECs were treated with conditioned medium for 15 min . Focal adhesion proteins were examined with anti - phosphoY397FAK and anti - vinculin antibodies . F - actin was stained with Alexa Fluor 546 - conjugated phalloidin . Merged images show the coupling of stress fibers with focal adhesion complexes in the cells treated with control medium . Inset , phospho - Y397FAK and vinculin formed focal adhesions that were stained in an elongated dot - like structure ( i ) . Lower panel ( ii ) , Sema3C treatment induced F - actin disassembly and membrane ruffling . Phospho - Y397FAK and vinculin were randomly distributed at the peripheral membrane . Scale bars , 10 μm .

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

Figure 5A HUVECs were transduced with adenoviral vector expressing RFP-conjugated F-actin binding peptide (LifeAct). Time-lapse imaging of F-actin in HUVEC spheroids was obtained at 20× magnification (Supplementary Movies S3 and S4). Representative images are shown at 0 and 2 h. Arrowheads indicate detached cells after Sema3C treatment. Scale bar, 100 μm.B Endothelial cell migration was determined using a scratch wound assay with a co-culture model. A wound was generated in a confluent HUVEC monolayer, and cells were cultured in basal medium with or without VEGF (25 ng/ml) in a 24-well plate. At the same time, the transwells containing Sema3C or GFP-expressing pericytes. Representative pictures show remaining gaps within the HUVEC monolayer. Scale bar, 200 μm.C Migration distance was measured after 24 h and normalized to control treatment (mean ± s.e.m; n = 3 independent experiments, control vs. Sema3C (− VEGF), *P = 0.021 and control vs. Sema3C (+ VEGF), *P = 0.039 using unpaired Student's t-test).D,E (D) Serum-starved HUVECs were treated with Sema3C- or control-conditioned medium with or without VEGF (50 ng/ml) for 10 min. Cell lysates were subjected to SDS-PAGE and probed with anti-phosphoT180/Y182 p38MAPK antibody. Quantification of p38MAPK activation was normalized to total p38MAPK and presented as the ratio to the cells with control medium without VEGF treatment. Control vs. Sema3C (− VEGF), *P = 0.046 and control vs. Sema3C (+ VEGF), *P = 0.043. (E) HUVECs were treated as described before and the activation of FAK detected by anti-phosphoY397 FAK antibody and normalized to the total amount of FAK. Control vs. Sema3C (− VEGF), *P = 0.033 and control vs. Sema3C (+ VEGF), *P = 0.047. (Mean ± s.e.m; n = 3 independent experiments; unpaired Student's t-test.)F Serum-starved HUVECs were treated with conditioned medium for 15 min. Focal adhesion proteins were examined with anti-phosphoY397FAK and anti-vinculin antibodies. F-actin was stained with Alexa Fluor 546-conjugated phalloidin. Merged images show the coupling of stress fibers with focal adhesion complexes in the cells treated with control medium. Inset, phospho-Y397FAK and vinculin formed focal adhesions that were stained in an elongated dot-like structure (i). Lower panel (ii), Sema3C treatment induced F-actin disassembly and membrane ruffling. Phospho-Y397FAK and vinculin were randomly distributed at the peripheral membrane. Scale bars, 10 μm.


Figure 6HUVECs were transfected with siRNA against PlexinD1 or control NS ( non - silence ) siRNA . qPCR analyses and Western blotting were performed after 48 h ( n = 3 ; paired Student ' s t - test , NS siRNA vs . PlexinD1 siRNA , * * * P = 0 . 0004 ) . Cell lysates were subjected to SDS - PAGE and probed with anti - PlexinD1 . HUVECs were transfected with siRNA against PlexinD1 and plated on gelatin - coated coverslips . Confluent monolayers were treated with Sema3C - , Sema3Cp60 - or control - conditioned medium for 30 min . Cells were fixed and stained with Alexa - Fluor 488 - conjugated phalloidin to visualize F - actin . Arrows indicate Sema3C - induced cell membrane ruffling . Scale bar , 20 μm . Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei ( n = 5 ; mean ± s . e . m . , paired Student ' s t - test , * * P = 0 . 001 ) . Silencing of Nrp - 1 by lentiviral particles expressing shRNA . After culturing 72 h in selection medium , RNA was harvested for qPCR analysis . NS shRNA vs . Nrp - 1 shRNA : # 1 , * P = 0 . 017 and # 2 , * P = 0 . 02 ; n = 3 ; mean ± s . e . m . Cell lysates were subjected to SDS - PAGE and probed with anti - Nrp - 1 . Depletion of Nrp - 1 abolished the response to Sema3C . Sema3Cp60 had no effect on HUVEC adhesion . Arrows indicate membrane ruffling . Scale bar , 20 μm . Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei ( n = 5 ; mean ± s . e . m . ; paired Student ' s t - test , * * * P = 0 . 0003 ) . HUVECs were treated with Sema3C - conditioned medium for 10 min and lysed , and Nrp - 1 was immunoprecipitated . Bound Sema3C was detected by Western blotting .

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

Figure 6HUVECs were transfected with siRNA against PlexinD1 or control NS (non-silence) siRNA. qPCR analyses and Western blotting were performed after 48 h (n = 3; paired Student's t-test, NS siRNA vs. PlexinD1 siRNA, ***P = 0.0004). Cell lysates were subjected to SDS-PAGE and probed with anti-PlexinD1.HUVECs were transfected with siRNA against PlexinD1 and plated on gelatin-coated coverslips. Confluent monolayers were treated with Sema3C-, Sema3Cp60- or control-conditioned medium for 30 min. Cells were fixed and stained with Alexa-Fluor 488-conjugated phalloidin to visualize F-actin. Arrows indicate Sema3C-induced cell membrane ruffling. Scale bar, 20 μm. Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei (n = 5; mean ± s.e.m., paired Student's t-test, **P = 0.001).Silencing of Nrp-1 by lentiviral particles expressing shRNA. After culturing 72 h in selection medium, RNA was harvested for qPCR analysis. NS shRNA vs. Nrp-1 shRNA: #1, *P = 0.017 and #2, *P = 0.02; n = 3; mean ± s.e.m. Cell lysates were subjected to SDS-PAGE and probed with anti-Nrp-1.Depletion of Nrp-1 abolished the response to Sema3C. Sema3Cp60 had no effect on HUVEC adhesion. Arrows indicate membrane ruffling. Scale bar, 20 μm. Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei (n = 5; mean ± s.e.m.; paired Student's t-test, ***P = 0.0003).HUVECs were treated with Sema3C-conditioned medium for 10 min and lysed, and Nrp-1 was immunoprecipitated. Bound Sema3C was detected by Western blotting.


Figure 7RNA scope analyses showed Sema3C and PlexinD1 expression in pre - retinal tufts ( OIR model , P19 ) . The retinae were subsequently stained against collagen IV . Sema3CmRNA was detected in peri - endothelial cells . Scale bar : 20 μm . Nrp - 1 expression in pre - retinal tufts ( OIR model , P17 ) . Abundant expression of Nrp1 in endothelial cells . Pericytes at vascular tufts were immunostained with anti - NG2 . Scale bar , 20 μm . H & amp ; amp ; E staining shows neovascular tufts ( arrows ) . Representative of five retinae per group in two independent experiments . Scale bar , 50 μm . Whole - mount CD31 immunostaining . The white line defines the whole retinal area , and red line outlines the vaso - obliterative zone . Inset image shows large numbers of peri - retinal tufts ( i , arrow heads ) in control IgG - Fc - treated retinae compared to Sema3C - treated retinae ( ii ) . Scale bar , 500 μm ( upper panel ) and 100 μm ( lower panel ) . The avascular area is presented as percentage of the whole retinal area ( n = 10 retinae in each group ; n . s . , non - significant using paired Student ' s t - test ) . The formation of pre - retinal tufts was quantified in the superficial layer of the retinal vasculature and normalized to whole retinal area ( n = 6 retinae in each group ; * P = 0 . 033 using paired 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 7RNA scope analyses showed Sema3C and PlexinD1 expression in pre-retinal tufts (OIR model, P19). The retinae were subsequently stained against collagen IV. Sema3CmRNA was detected in peri-endothelial cells. Scale bar: 20 μm.Nrp-1 expression in pre-retinal tufts (OIR model, P17). Abundant expression of Nrp1 in endothelial cells. Pericytes at vascular tufts were immunostained with anti-NG2. Scale bar, 20 μm.H&amp;E staining shows neovascular tufts (arrows). Representative of five retinae per group in two independent experiments. Scale bar, 50 μm.Whole-mount CD31 immunostaining. The white line defines the whole retinal area, and red line outlines the vaso-obliterative zone. Inset image shows large numbers of peri-retinal tufts (i, arrow heads) in control IgG-Fc-treated retinae compared to Sema3C-treated retinae (ii). Scale bar, 500 μm (upper panel) and 100 μm (lower panel).The avascular area is presented as percentage of the whole retinal area (n = 10 retinae in each group; n.s., non-significant using paired Student's t-test).The formation of pre-retinal tufts was quantified in the superficial layer of the retinal vasculature and normalized to whole retinal area (n = 6 retinae in each group; *P = 0.033 using paired Student's t-test).


Figure 1Confocal images of mPS - PI stained 6 - day - old seedlings of Col - 0 ( A ) , bravo - 2 ( B ) , wox5 - 1 ( C ) and bravo - 2 wox5 - 1 ( D ) mutants . Left black arrows indicate QC cells and right white arrows indicate CSC . Scale bar : 50 µm . Quantification of the QC divisions in 6 - day - old roots expressed in percentage ( n > 50 , 3 replicates ) . D : QC divided ; ND : QC non divided . Quantification of CSC layers in 6 - day - old roots expressed in percentage ( n > 50 , 3 replicates ) . Lateral root density ( number of lateral roots per mm of root length ) of 7 - day - olf WT , bravo - 2 , wox5 - 1 and bravo - 2 wox5 - 1 mutants ( n > 40 , 2 replicates ) . Different letters indicate statistically significant differences ( p - value < 0 . 05 Student ´ s t - test ) . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers .

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

Figure 1Confocal images of mPS-PI stained 6-day-old seedlings of Col-0 (A), bravo-2 (B), wox5-1 (C) and bravo-2 wox5-1 (D) mutants. Left black arrows indicate QC cells and right white arrows indicate CSC. Scale bar: 50 µm.Quantification of the QC divisions in 6-day-old roots expressed in percentage (n>50, 3 replicates). D: QC divided; ND: QC non divided.Quantification of CSC layers in 6-day-old roots expressed in percentage (n>50, 3 replicates).Lateral root density (number of lateral roots per mm of root length) of 7-day-olf WT, bravo-2, wox5-1 and bravo-2 wox5-1 mutants (n>40, 2 replicates). Different letters indicate statistically significant differences (p- value < 0.05 Student´s t-test). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.


Figure 2Confocal images of PI - stained 6 - day - old roots . GFP - tagged expression is shown in yellow . A - C ) pBRAVO : GFP in WT ( A ) , bravo - 2 ( B ) and wox5 - 1 ( C ) knockout backgrounds . D - G ) pWOX5 : GFP in the WT ( D ) , bravo - 2 ( E ) , wox5 - 1 ( F ) and bravo - 2 wox5 - 1 ( G ) knockout backgrounds . Scale bar : 50 µm . Quantification of the GFP fluorescent signal of the roots in A - C ( H ) and D - G ( I ) . Boxplot indicating the average pixel intensity of the GFP in the stem cell niche ( n > 25 , 3 biological replicates , * p - value < 0 . 05 Student ´ s t - test for each genotype versus the WT in the same condition ) . Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN ( Appendix Figure S7 ) . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers and black dots are the experimental observations .

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Figure 2Confocal images of PI-stained 6-day-old roots. GFP-tagged expression is shown in yellow. A-C) pBRAVO:GFP in WT (A), bravo-2 (B) and wox5-1 (C) knockout backgrounds. D-G) pWOX5:GFP in the WT (D), bravo-2 (E), wox5-1 (F) and bravo-2 wox5-1 (G) knockout backgrounds. Scale bar: 50 µm. Quantification of the GFP fluorescent signal of the roots in A-C (H) and D-G (I). Boxplot indicating the average pixel intensity of the GFP in the stem cell niche (n>25, 3 biological replicates, *p-value < 0.05 Student´s t- test for each genotype versus the WT in the same condition). Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN (Appendix Figure S7). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers and black dots are the experimental observations.


Figure 3Parameter space exploration of ( C ) the alleviation and the ( D ) activation models . Boxplots showing the distribution of stationary promoter pBRAVO ( pB ) and pWOX5 ( pW ) fold - changes in each mutant and overexpression lines over the WT obtained for different parameter values . Superindexes denote the genotype , standing bravo for bravo mutant , wox5 for wox5 mutant , dm for the bravo wox5 double mutant , Woe for WOX5 overexpression and Boe for BRAVO overexpression . Box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . The results are obtained by solving N = 1000 different runs of each model at the stationary state ( red and blue stripplots ) for the WT , mutants and overexpressor scenarios . These N runs differ in the parameter values , which are chosen at random from a uniform distribution

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Figure 3Parameter space exploration of (C) the alleviation and the (D) activation models. Boxplots showing the distribution of stationary promoter pBRAVO (pB) and pWOX5 (pW) fold-changes in each mutant and overexpression lines over the WT obtained for different parameter values. Superindexes denote the genotype, standing bravo for bravo mutant, wox5 for wox5 mutant, dm for the bravo wox5 double mutant, Woe for WOX5 overexpression and Boe for BRAVO overexpression. Box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. The results are obtained by solving N=1000 different runs of each model at the stationary state (red and blue stripplots) for the WT, mutants and overexpressor scenarios. These N runs differ in the parameter values, which are chosen at random from a uniform distribution between


Figure 4Interaction of BRAVO with WOX5 ( B ) , BES1 ( C ) , BES1D ( D ) and TPL ( E ) ; and interaction of WOX5 with BRAVO ( G ) , BES1 ( H ) , BES1D ( I ) and TPL ( J ) measured by FRET - FLIM . GFP fluorescence lifetime τ [ ns ] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells . GFP fluorescence lifetime fitted pixel - wise with a mono - exponential model of BRAVO and WOX5 interactions . mV , mVenus ; mCh , mCherry . Scale bar : 5 µm . Fluorescence - weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double - exponential model of the indicated samples are summarized in box plots . Statistical significance was tested by one - way ANOVA with a Sidakholm post - hoc test . Different letters indicate statistically significant differences ( p - value < 0 . 01 ) . For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers . Yeast two - hybrid assay showing BRAVO interacting with WOX5 , BES1 - D and TPL ; and BES1 - D interacting with TPL . In the left column yeast cells were grown on control media , and in the right column yeast cells were grown on control media lacking Leu , Trp and His , indicating an interaction between the proteins .

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Figure 4Interaction of BRAVO with WOX5 (B), BES1 (C), BES1D (D) and TPL (E); and interaction of WOX5 with BRAVO (G), BES1 (H), BES1D (I) and TPL (J) measured by FRET-FLIM. GFP fluorescence lifetime τ [ns] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells. GFP fluorescence lifetime fitted pixel-wise with a mono-exponential model of BRAVO and WOX5 interactions. mV, mVenus; mCh, mCherry. Scale bar: 5 µm.Fluorescence-weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double-exponential model of the indicated samples are summarized in box plots. Statistical significance was tested by one-way ANOVA with a Sidakholm post-hoc test. Different letters indicate statistically significant differences (p-value < 0.01). For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured. In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.Yeast two-hybrid assay showing BRAVO interacting with WOX5, BES1-D and TPL; and BES1-D interacting with TPL. In the left column yeast cells were grown on control media, and in the right column yeast cells were grown on control media lacking Leu, Trp and His, indicating an interaction between the proteins.


Figure 6Expression of the 380 common BRAVO and WOX5 regulated genes from ( B ) . Expression of the 53 genes from ( C ) showing similar expression in bravo and wox5 mutants . Expression of the 41 genes from ( C ) showing opposite expression levels in bravo and wox5 mutants .

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Figure 6Expression of the 380 common BRAVO and WOX5 regulated genes from (B). Expression of the 53 genes from (C) showing similar expression in bravo and wox5 mutants. Expression of the 41 genes from (C) showing opposite expression levels in bravo and wox5 mutants.


Figure 1 . Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Time course of antibody response . Serum samples were diluted 40 - fold prior to ELISA analysis . Animals in the high - dose and low - dose group were immunized with 1 × 109 and 1 × 108 pfu virus each time , respectively . Arrows indicate the time point for immunization . Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Determination of virus neutralization antibody titer using 96 - well microneutralization assay . An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples . Statistical significance between different data sets was calculated using t - test . Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . ELISPOT analysis of IFN - γ response in PBMC samples . Pool A and B contain peptides corresponding to S1 - orf8 fusion and N , respectively . Data represent two animals from each group . Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Average food consumption after live virus challenge .

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Figure 1.Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Time course of antibody response. Serum samples were diluted 40-fold prior to ELISA analysis. Animals in the high-dose and low-dose group were immunized with 1×109 and 1×108 pfu virus each time, respectively. Arrows indicate the time point for immunization.Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Determination of virus neutralization antibody titer using 96-well microneutralization assay. An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples. Statistical significance between different data sets was calculated using t-test.Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. ELISPOT analysis of IFN-γ response in PBMC samples. Pool A and B contain peptides corresponding to S1-orf8 fusion and N, respectively. Data represent two animals from each group.Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Average food consumption after live virus challenge.


Figure 2 . Histology and IHC study on postmortem lung sections from rhesus macaques infected with live SARS - CoV . All animals were euthanized 7 days after viral challenge , and anti - SARS - CoV monoclonal antibody was used to identify SARS antigens in the tissue . Representative data from lung sections were shown . Histology samples include : a . Control group . b . High - dose group . c . Placebo group . The arrowheads point to alveoli space filled with proteinaceous fluid . IHC samples include : d . Control group . e . High - dose group . f . Placebo group . Arrows point to cells that were positive for SARS antigen expression .

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Figure 2. Histology and IHC study on postmortem lung sections from rhesus macaques infected with live SARS-CoV. All animals were euthanized 7 days after viral challenge, and anti-SARS-CoV monoclonal antibody was used to identify SARS antigens in the tissue. Representative data from lung sections were shown. Histology samples include: a. Control group. b. High-dose group. c. Placebo group. The arrowheads point to alveoli space filled with proteinaceous fluid. IHC samples include: d. Control group. e. High-dose group. f. Placebo group. Arrows point to cells that were positive for SARS antigen expression.


Figure 1mRNA levels of TMX1 ( A ) and NFAT1 ( B ) in primary melanocytes , keratinocytes and melanoma cell lines . data are are normalized to the expression of TBP and are presented as mean ± SEM ( n ≥ 3 ) . Representative immunoblot ( from 3 ) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines ; actin was used as a loading control . Indicated band intensities were normalized to actin and background signal was subtracted . Melan - A ( brown ) , TMX1 ( red - brown ) and NFAT1 ( deep red ) staining ( IHC ) in a primary stage 4 high risk nodular melanoma patient I ( out of four ) , the enlarged regions show healthy tissue ( panels 2 , 5 and 8 ) and tumor tissue ( panels 3 , 6 and 9 ) . Arrows indicate Melan - A positive melanocytes . TMX1 ( red - brown ) and NFAT1 ( deep red ) staining of healthy human skin and increasing melanoma stages ; P1 - P13 refer to the donor patient number .

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Figure 1mRNA levels of TMX1 (A) and NFAT1 (B) in primary melanocytes, keratinocytes and melanoma cell lines. data are are normalized to the expression of TBP and are presented as mean ± SEM (n≥3).Representative immunoblot (from 3) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines; actin was used as a loading control. Indicated band intensities were normalized to actin and background signal was subtracted.Melan-A (brown), TMX1 (red-brown) and NFAT1 (deep red) staining (IHC) in a primary stage 4 high risk nodular melanoma patient I (out of four), the enlarged regions show healthy tissue (panels 2, 5 and 8) and tumor tissue (panels 3, 6 and 9). Arrows indicate Melan-A positive melanocytes.TMX1 (red-brown) and NFAT1 (deep red) staining of healthy human skin and increasing melanoma stages; P1-P13 refer to the donor patient number.


Figure 2Insulin ( 50 µg / mL ) induced the translocation of NFAT1 - GFP to the nucleus of WM3734 cells . ( A ) Ratio images ( F / F0 ) , ( B ) corresponding endpoint quantification ( n = 5 ) . Reduced nuclear translocation of NFAT1 - GFP in TMX1 or TMX3 silenced ( siRNA ) melanoma cells . ( C ) Images show NFAT1 - GFP fluorescence intensity before and after stimulation with thapsigargin ( Tg ; 1 µM ) in WM3734 cells . ( D ) Corresponding time - dependent nuclear import of NFAT1 as a change of F / F0 . ( E ) Normalized endpoint quantification . Same analysis was performed with Mel Juso cells with ( F ) images , ( G ) time - dependent nuclear import and ( H ) normalized endpoint quantification . data are presented as mean ± SEM ( n values : WM3734 , control = 142 , TMX1 kd = 116 , TMX3 kd = 148 ; Mel Juso , control = 75 , TMX1 kd = 47 , TMX3 kd = 67 ) .

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Figure 2Insulin (50 µg/mL) induced the translocation of NFAT1-GFP to the nucleus of WM3734 cells. (A) Ratio images (F/F0), (B) corresponding endpoint quantification (n=5).Reduced nuclear translocation of NFAT1-GFP in TMX1 or TMX3 silenced (siRNA) melanoma cells. (C) Images show NFAT1-GFP fluorescence intensity before and after stimulation with thapsigargin (Tg; 1 µM) in WM3734 cells. (D) Corresponding time-dependent nuclear import of NFAT1 as a change of F/F0. (E) Normalized endpoint quantification. Same analysis was performed with Mel Juso cells with (F) images, (G) time-dependent nuclear import and (H) normalized endpoint quantification. data are presented as mean ± SEM (n values: WM3734, control=142, TMX1 kd=116, TMX3 kd=148; Mel Juso, control=75, TMX1 kd=47, TMX3 kd=67).


Figure 3Thapsigargin ( Tg ) induced Fura - 2 based cytosolic Ca2 + imaging in Ringer ' s buffer containing 0 . 25 mM Ca2 + . ( B ) Quantification of basal cytosolic calcium levels and ( C ) SOCE quantification ( plateau - basal ) for WM3734 after stable silencing of TMX1 ( two clones ) . : In ( B - C ) , data are presented as mean ± SEM ( n values : WM3734 , control = 939 , TMX1 kds 1 = 988 , TMX1 kds 2 = 508 ) . Cytosolic Ca2 + imaging ( Fura - 2 ) and ( E ) SOCE quantification ( plateau - basal ) for WM3734 after transient silencing of TMX1 or TMX3 . In ( E ) , data are presented as mean ± SEM ( n values : WM3734 , control = 30 , TMX1 kd = 49 , TMX3 kd = 52 ) . Cellular H2O2 ( HyPer ) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd . ( F ) Exemplary ratiometric images ( F505 nm / F420 nm ) are shown for WM3734 and Mel Juso . Quantification of basal cellular H2O2 in WM3734 ( G ) and Mel Juso ( H ) cells . In ( G - H ) , data are presented as mean ± SEM ( n values : WM3734 : control = 168 , TMX1 kd = 209 , TMX3 kd = 192 ; Mel Juso : control = 297 , TMX1 kd = 343 , TMX3 kd = 440 ) . In ( F ) scale bars : 10 µm ; color code : WM3734 : color code : blue = 0 , red = 3 ; Mel Juso : color code : blue = 0 , red = 1 . 5 .

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Figure 3Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.25 mM Ca2+. (B) Quantification of basal cytosolic calcium levels and (C) SOCE quantification (plateau-basal) for WM3734 after stable silencing of TMX1 (two clones). : In (B-C), data are presented as mean ± SEM (n values: WM3734, control=939, TMX1 kds 1=988, TMX1 kds 2=508).Cytosolic Ca2+ imaging (Fura-2) and (E) SOCE quantification (plateau-basal) for WM3734 after transient silencing of TMX1 or TMX3. In (E), data are presented as mean ± SEM (n values: WM3734, control=30, TMX1 kd=49, TMX3 kd=52).Cellular H2O2 (HyPer) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd. (F) Exemplary ratiometric images (F505 nm / F420 nm) are shown for WM3734 and Mel Juso. Quantification of basal cellular H2O2 in WM3734 (G) and Mel Juso (H) cells. In (G-H), data are presented as mean ± SEM (n values: WM3734: control=168, TMX1 kd=209, TMX3 kd=192; Mel Juso: control=297, TMX1 kd=343, TMX3 kd=440). In (F) scale bars: 10 µm; color code: WM3734: color code: blue=0, red=3; Mel Juso: color code: blue=0, red=1.5.


Figure 4WM3734 cells were pre - incubated for 5 min with extracellular H2O2 ( 0 - 500 µM ) . Tg - stimulated NFAT1 - GFP nuclear translocation was measured as F / F0 . The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13 . 2 µM ( red mark ) . Tg - induced NFAT1 - GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre - incubation with 100 µM N - acetylcysteine ( NAC ) for 48 h . Endpoint quantification of the data from ( C ) and upon treatment with NAC , PEG - catalase ( 50 U / mL ) , or DTT ( 1 mM ) . data are presented as mean ± SEM ( n values : control = 73 , control + NAC = 19 , TMX1 kd = 57 , TMX1 kd + NAC = 63 , TMX1 kd + catalase = 58 , TMX1 kd + DTT = 22 ) . Cytosolic calcineurin activity ( CaNAR2 ) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation . Quantification of the basal calcineurin activity and the maximal calcineurin activity ( plateau - basal ) . data are presented as mean ± SEM ( n values : control = 49 , TMX1 kd = 48 , TMX3 kd = 63 ) . Normalized Tg - induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre - incubation with extracellular H2O2 ( 0 - 200 µM ) . Quantification of the maximum cytosolic calcineurin activity ( plateau - basal ) . Quantification of the basal calcineurin activity and the maximal calcineurin activity ( plateau - basal ) 8 h after treatment with the antioxidant NAC ( 100 µM ) in WM3734 with transient knockdown of TMX1 . data are presented as mean ± SEM ( n values : control = 15 , TMX1 kd = 24 , TMX1 kd + NAC = 19 ) .

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Figure 4WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM). Tg-stimulated NFAT1-GFP nuclear translocation was measured as F/F0.The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13.2 µM (red mark).Tg-induced NFAT1-GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre-incubation with 100 µM N-acetylcysteine (NAC) for 48 h.Endpoint quantification of the data from (C) and upon treatment with NAC, PEG-catalase (50 U/mL), or DTT (1 mM). data are presented as mean ± SEM (n values: control=73, control+NAC= 19, TMX1 kd=57, TMX1 kd+NAC=63, TMX1 kd+catalase=58, TMX1 kd+DTT=22).Cytosolic calcineurin activity (CaNAR2) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation.Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal). data are presented as mean ± SEM (n values: control=49, TMX1 kd=48, TMX3 kd=63).Normalized Tg-induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre-incubation with extracellular H2O2 (0 - 200 µM). Quantification of the maximum cytosolic calcineurin activity (plateau-basal).Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal) 8 h after treatment with the antioxidant NAC (100 µM) in WM3734 with transient knockdown of TMX1. data are presented as mean ± SEM (n values: control=15, TMX1 kd=24, TMX1 kd+NAC=19).


Figure 5Mitochondrial H2O2 ( mito - HyPer ) was measured in two cell lines upon transient TMX knockdown . Exemplary ratiometric images ( F505 nm / F420 nm ) are shown ( A ) ; corresponding quantification for ( B ) WM3734 and ( C ) Mel Juso . , data are presented as mean ± SEM ( n values : WM3734 : control = 546 , TMX1 kd = 510 , TMX3 kd = 621 ; Mel Juso : control = 416 , TMX1 kd = 418 , TMX3 kd = 442 ) . Mitochondrial Ca2 + uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria - targeted calcium sensor ( 4mt - D3cpV ) ( D ) . Cells were exposed to 0 . 25 mM Ca2 + containing Ringer ' s buffer and Tg . ( E ) Quantification of basal mitochondrial Ca2 + levels and ( F ) mitochondrial Ca2 + uptake ( basal - plateau ) . , data are presented as mean ± SEM ( n values : control = 62 , TMX1 kds = 45 ) . Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1 . The mean mitochondrial volume was modeled based on microscopy , the quantification of mean volume ( µm3 ) and mean surface ( µm2 ) is depicted in ( G ) and ( H ) respectively . data are presented as boxplots ( n values : control = 101 , TMX1 kds = 90 ; center line : median ; box : 25 % and 75 % percentile ; whiskers : 1 . 5 times interquartile range ; outliers are shown as dots ) . Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1 . A peripheral mask was applied based on the membrane staining ( CellMask Green ) and mitochondria covered area ( MitoTracker DeepRed ) was evaluated ( see Fig . 9 ) . Representative images ( I ) and quantification ( J ) . In ( J ) , data are presented as mean ± SEM ( n values : control = 163 , TMX1 kds = 116 ) . Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown ( K ) . Corresponding quantification of the distance between ( L ) mitochondria and plasma membrane , ( M ) mitochondria and ER , and ( N ) the length of the MAM ( mitochondria - ER contact site ) . In ( L ) , data are presented as mean ± SEM ( n values : control = 100 , TMX1 kds = 100 ) . In ( M - N ) , data are presented as mean ± SEM ( n values : control = 60 , TMX1 kds = 60 ) . Cellular H2O2 concentration ( HyPer ) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 ( 140 nM ) ( O ) Representative ratiometric images ( F505 nm / F420 nm ) and ( P ) quantification . In ( P ) , data are presented as mean ± SEM ( n values : control = 837 , TMX1 kd = 888 , TMX1 kd + NOX4 kd = 793 , TMX1 kd + GKT = 844 ) .

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

Figure 5Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown. Exemplary ratiometric images (F505 nm / F420 nm) are shown (A); corresponding quantification for (B) WM3734 and (C) Mel Juso. , data are presented as mean ± SEM (n values: WM3734: control=546, TMX1 kd=510, TMX3 kd=621; Mel Juso: control=416, TMX1 kd=418, TMX3 kd=442).Mitochondrial Ca2+ uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria-targeted calcium sensor (4mt-D3cpV) (D). Cells were exposed to 0.25 mM Ca2+ containing Ringer's buffer and Tg. (E) Quantification of basal mitochondrial Ca2+ levels and (F) mitochondrial Ca2+ uptake (basal-plateau). , data are presented as mean ± SEM (n values: control=62, TMX1 kds=45).Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1. The mean mitochondrial volume was modeled based on microscopy, the quantification of mean volume (µm3) and mean surface (µm2) is depicted in (G) and (H) respectively. data are presented as boxplots (n values: control=101, TMX1 kds=90; center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range; outliers are shown as dots).Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1. A peripheral mask was applied based on the membrane staining (CellMask Green) and mitochondria covered area (MitoTracker DeepRed) was evaluated (see Fig. 9). Representative images (I) and quantification (J). In (J), data are presented as mean ± SEM (n values: control=163, TMX1 kds=116).Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown (K). Corresponding quantification of the distance between (L) mitochondria and plasma membrane, (M) mitochondria and ER, and (N) the length of the MAM (mitochondria-ER contact site). In (L), data are presented as mean ± SEM (n values: control=100, TMX1 kds= 100). In (M-N), data are presented as mean ± SEM (n values: control=60, TMX1 kds= 60).Cellular H2O2 concentration (HyPer) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 (140 nM) (O) Representative ratiometric images (F505 nm / F420 nm) and (P) quantification. In (P), data are presented as mean ± SEM (n values: control=837, TMX1 kd=888, TMX1 kd + NOX4 kd=793, TMX1 kd + GKT=844).


Figure 6Proliferation ( 48 h ) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3 . Proliferation ( 48 h ) of melanoma cell lines with stable TMX1 knockdown ( two clones ) . Proliferation ( 24 h ) of melanoma cell lines after transient knockdown of NFAT1 . Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC ( 100 µM ) or mTEMPO ( 100 nM ) . Transwell migration ( 48 h ) of WM3734 after transient knockdown of TMX1 or TMX3 . Transwell migration ( 48 h ) of WM3734 after transient knockdown of TMX1 and treatment with NAC ( 100 µM ) or mTEMPO ( 1 µM ) . Transwell migration ( 48 h ) of melanoma cell lines after preventing NFAT - calcineurin interaction with dipyridamole ( 40 µM ) or inhibition of calcineurin with cyclosporine A ( CsA , 2 µM ) . Invasion of WM3734 with stable TMX1 knockdown ( two clones ) through Matrigel for 96 h . Mouse xenograft model to assess tumor growth of WM3734 with stable TMX1 knockdown ( TMX1 kds ; two clones ) in immunodeficient mice . ( I ) Tumor growth in the first 19 days after injection ; ( J ) the corresponding tumor volume quantified after 19 days and ( K ) after 45 days .

O B-PANEL_START 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 B-PANEL_START 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 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 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 B-PANEL_START 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

Figure 6Proliferation (48 h) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3.Proliferation (48 h) of melanoma cell lines with stable TMX1 knockdown (two clones).Proliferation (24 h) of melanoma cell lines after transient knockdown of NFAT1.Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (100 nM).Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 or TMX3.Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (1 µM).Transwell migration (48 h) of melanoma cell lines after preventing NFAT-calcineurin interaction with dipyridamole (40 µM) or inhibition of calcineurin with cyclosporine A (CsA, 2 µM).Invasion of WM3734 with stable TMX1 knockdown (two clones) through Matrigel for 96 h.Mouse xenograft model to assess tumor growth of WM3734 with stable TMX1 knockdown (TMX1 kds; two clones) in immunodeficient mice. (I) Tumor growth in the first 19 days after injection; (J) the corresponding tumor volume quantified after 19 days and (K) after 45 days.


Figure 7Kaplan - Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients , separated in groups with high or low expression of any of the three genes of interest ( GOI ) , TMX1 , TMX3 and NFAT1 . mRNA expression levels of NFAT1 , TMX1 and TMX3 in BRAF WT and BRAF V600E melanoma patients . In ( B ) , expression levels are based on the FPKM values of the TCGA samples ( center line : median ; box : 25 % and 75 % percentile ; whiskers : 1 . 5 times interquartile range ) . Kaplan - Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients , separated in groups with ( C ) BRAF WT and ( D ) BRAF V600E and with high or low expression of NFAT1 .

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 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 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 7Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with high or low expression of any of the three genes of interest (GOI), TMX1, TMX3 and NFAT1.mRNA expression levels of NFAT1, TMX1 and TMX3 in BRAF WT and BRAF V600E melanoma patients. In (B), expression levels are based on the FPKM values of the TCGA samples (center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range).Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with (C) BRAF WT and (D) BRAF V600E and with high or low expression of NFAT1.


Figure 1 ( A and B ) Tau increased β - catenin acetylation ( Ace - β - cat ) measured by immunoprecipitation using anti - β - cat , or Western blotting using anti - β - cat , anti - acetylated lysine ( Ace - lys ) and HT7 ( probes specifically human total Tau ) . HEK293 cells transiently transfected with wildtype Tau40 ( Tau ) or the empty vector ( Vec ) ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec Data were analyzed by Student ' s t test ( C and D ) β - cat mutation at K49 ( K49R ) but not K19 ( K19R ) abolished Tau - induced acetylation . HEK293 cells were co - transfected with eGFP - β - cat ( WT , or K19R , or K49R , or K19R / K49R ) and Tau or the empty vector for 48 h , and then immunoprecipitated using anti - GFP and Western blotting using anti - β - cat , anti - Ace - lys and HT7 ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs β - cat WT ( D ) ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs β - cat WT plus Tau ( D ) ; & & , P < 0 . 01 , & & & , P < 0 . 001 vs β - cat K19R plus Tau ( D ) ; the absence of asterisk indicates that the difference is not significant . Data were analyzed by one - way ANOVA ( E and F ) The increased β - cat ( K49 ) acetylation was detected in the hippocampal extracts of human Tau transgenic mice ( Tau + ) compared with the endogenous Tau knockout mice ( Tau - ) , measured by Western blotting using acetylation site - specific antibody ( Ace - β - cat - K49 ) ; arrowhead in panel E denoted nonspecific band identified by molecular weight . The remarkably increased levels of phosphorylated Tau at Ser199 ( pS199 ) and AT8 epitopes were detected in Tau + mice ( at least 3 mice per group were used ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs vs Tau - ( F ) Data were analyzed by Student ' s t test ( G and H ) K18 deletion of Tau ( Tau - k18 ( - ) ) attenuated Tau - induced β - cat acetylation . HEK293 cells were transfected with empty Vec , or Tau40 or Tau - k18 ( - ) for 48 h , and the ace - β - cat was measured by immunoprecipitation using anti - β - cat and Western blotting using anti - ace - lys , anti - β - cat , and Tau5 ( detecting total Tau ; n = 3 from three independent experiments ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Tau ( H ) Data were analyzed by one - way ANOVAThe increased total and Ace - β - cat ( K49 ) levels positively correlated with increased phosphorylated Tau pS199 and AT8 as detected by Western blotting in the hippocampal extracts of AD patients compared with age - matched controls ( n = 10 each group , Pearson analysis was used for correlation analysis Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec vs Ctrl ( J ) Data were analyzed by Student ' s t testThe increased total and Ace - β - cat ( K49 ) levels positively correlated with increased phosphorylated Tau pS199 and AT8 as detected by Western blotting in the hippocampal extracts of AD patients compared with age - matched controls ( n = 10 each group Pearson analysis was used for correlation analysis ( M and N ) Expressing pseudo - phosphorylated Tau ( TauS199E ) further increased the K49 - acetylation of β - catenin . N2a cells were transiently transfected with Vec , Tau40 or TauS199E for 48 h , levels of K49 - acetylated β - cat , total β - cat and total Tau were detected by Western blotting ( n = 3 biological replicates each group ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs β - cat WT plus vs Tau ( N ) Data were analyzed by one - way ANOVA ( O and P ) Inhibiting acetyltransferases CBP / P300 by TPOP146 ( 134 nM ) or PCAF by L - 45 ( 126 nM ) for 24 h did not significantly decrease the K49 - acetylated β - cat level in Tau - overexpressing N2a cells ( n = 3 biological replicates each group ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec Data were analyzed by one - way

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

Figure 1(A and B) Tau increased β-catenin acetylation (Ace-β-cat) measured by immunoprecipitation using anti-β-cat, or Western blotting using anti-β-cat, anti-acetylated lysine (Ace-lys) and HT7 (probes specifically human total Tau). HEK293 cells transiently transfected with wildtype Tau40 (Tau) or the empty vector (Vec) (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec Data were analyzed by Student's t test(C and D) β-cat mutation at K49 (K49R) but not K19 (K19R) abolished Tau-induced acetylation. HEK293 cells were co-transfected with eGFP-β-cat (WT, or K19R, or K49R, or K19R/K49R) and Tau or the empty vector for 48 h, and then immunoprecipitated using anti-GFP and Western blotting using anti-β-cat, anti-Ace-lys and HT7 (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT (D) ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs β-cat WT plus Tau (D) ; &&, P<0.01, &&&, P<0.001 vs β-cat K19R plus Tau (D); the absence of asterisk indicates that the difference is not significant. Data were analyzed by one-way ANOVA(E and F) The increased β-cat (K49) acetylation was detected in the hippocampal extracts of human Tau transgenic mice (Tau+) compared with the endogenous Tau knockout mice (Tau-), measured by Western blotting using acetylation site-specific antibody (Ace-β-cat-K49); arrowhead in panel E denoted nonspecific band identified by molecular weight. The remarkably increased levels of phosphorylated Tau at Ser199 (pS199) and AT8 epitopes were detected in Tau+ mice (at least 3 mice per group were used). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs vs Tau- (F) Data were analyzed by Student's t test(G and H) K18 deletion of Tau (Tau-k18(-)) attenuated Tau-induced β-cat acetylation. HEK293 cells were transfected with empty Vec, or Tau40 or Tau-k18(-) for 48 h, and the ace-β-cat was measured by immunoprecipitation using anti-β-cat and Western blotting using anti-ace-lys, anti-β-cat, and Tau5 (detecting total Tau; n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau (H) Data were analyzed by one-way ANOVAThe increased total and Ace-β-cat (K49) levels positively correlated with increased phosphorylated Tau pS199 and AT8 as detected by Western blotting in the hippocampal extracts of AD patients compared with age-matched controls (n=10 each group, Pearson analysis was used for correlation analysis Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec vs Ctrl (J) Data were analyzed by Student's t testThe increased total and Ace-β-cat (K49) levels positively correlated with increased phosphorylated Tau pS199 and AT8 as detected by Western blotting in the hippocampal extracts of AD patients compared with age-matched controls (n=10 each group Pearson analysis was used for correlation analysis(M and N) Expressing pseudo-phosphorylated Tau (TauS199E) further increased the K49-acetylation of β-catenin. N2a cells were transiently transfected with Vec, Tau40 or TauS199E for 48 h, levels of K49-acetylated β-cat, total β-cat and total Tau were detected by Western blotting (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs β-cat WT plus vs Tau (N) Data were analyzed by one-way ANOVA(O and P) Inhibiting acetyltransferases CBP/P300 by TPOP146 (134 nM) or PCAF by L-45 (126 nM) for 24 h did not significantly decrease the K49-acetylated β-cat level in Tau-overexpressing N2a cells (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec Data were analyzed by one-way ANOVA


Figure 2 ( A ) Tau directly acetylates β - cat in test - tube . Both Tau40 and β - cat were purified from E . coli using Ni - NTA resin ( n = 3 from three independent experiments ) . ( B and C ) Mutation of Tau40 at acetyltransferase activity region abolishes its acetylation activity on β - cat . eGFP - Tau40 , eGFP - Tau - 2CA and eGFP - Tau - k18 ( - ) were purified from HEK293 cells ( using anti - GFP beads ) and GST - β - cat was from E . coli ( B ) , or both Tau40 and β - cat were from E . coli ( C ) ( n = 3 from three independent experiments ) . ( D and E ) Mutation of β - cat K49 abolishes its acetylation by Tau . Both myc - Tau40 and eGFP - β - cat were purified from HEK293 cells ( using anti - GFP or anti - Myc beads ) ( D ) , or both from E . coli ( E ) ( n = 3 from three independent experiments ) . Kinetics of β - cat acetylation by Tau . Tau acetylates β - cat in concentration - ( F ) and its acetyltransferase activity domain - dependent manner . Michaelis - Menten parameters were determined using Graphpad Prism 6 ( n = 3 from three independent experiments ) . Kinetics of β - cat acetylation by Tau . Tau acetylates β - cat in time - ( G ) and its acetyltransferase activity domain - dependent manner . Michaelis - Menten parameters were determined using Graphpad Prism 6 ( n = 3 from three independent experiments ) . Kinetics of β - cat acetylation by Tau . Tau acetylates β - cat in pH - ( H ) , and its acetyltransferase activity domain - dependent manner . Michaelis - Menten parameters were determined using Graphpad Prism 6 ( n = 3 from three independent experiments ) . Kinetics of β - cat acetylation by Tau . The affinity ( Km ) of Tau to β - cat was measured by the initial rate of Tau as a function of the β - cat concentrations ( I , J ) . Michaelis - Menten parameters were determined using Graphpad Prism 6 ( n = 3 from three independent experiments ) .

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 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 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 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 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

Figure 2(A) Tau directly acetylates β-cat in test-tube. Both Tau40 and β-cat were purified from E. coli using Ni-NTA resin (n = 3 from three independent experiments).(B and C) Mutation of Tau40 at acetyltransferase activity region abolishes its acetylation activity on β-cat. eGFP-Tau40, eGFP-Tau-2CA and eGFP-Tau-k18(-) were purified from HEK293 cells (using anti-GFP beads) and GST-β-cat was from E. coli (B), or both Tau40 and β-cat were from E. coli (C) (n = 3 from three independent experiments).(D and E) Mutation of β-cat K49 abolishes its acetylation by Tau. Both myc-Tau40 and eGFP-β-cat were purified from HEK293 cells (using anti-GFP or anti-Myc beads) (D), or both from E. coli (E) (n = 3 from three independent experiments).Kinetics of β-cat acetylation by Tau. Tau acetylates β-cat in concentration- (F) and its acetyltransferase activity domain-dependent manner. Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).Kinetics of β-cat acetylation by Tau. Tau acetylates β-cat in time- (G) and its acetyltransferase activity domain-dependent manner. Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).Kinetics of β-cat acetylation by Tau. Tau acetylates β-cat in pH- (H), and its acetyltransferase activity domain-dependent manner. Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).Kinetics of β-cat acetylation by Tau. The affinity (Km) of Tau to β-cat was measured by the initial rate of Tau as a function of the β-cat concentrations (I, J). Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).


Figure 3 ( A and B ) Expressing Tau inhibited β - cat ubiquitination in HEK293 cells , measured by immunoprecipitation using antibody against β - cat and Western blotting using anti - ubiquitination ( anti - Ub ) and anti - β - cat , respectively ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec ( B ) Data were analyzed by Student ' s t test . ( C and D ) Overexpressing Tau inhibited β - cat ubiquitination in hippocampi of Tau transgenic mice , measured by immunoprecipitation using anti - Ub and Western blotting using anti - β - cat , respectively ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Tau - ( D ) Data were analyzed by Student ' s t test . ( E and F ) Expressing Tau - k18 ( - ) attenuated Tau - induced β - cat elevation . HEK293 cells were transfected with Vec , or Tau - 40 , or Tau - k18 ( - ) for 24 h , and then were treated with Chx for 12 h or 24 h , followed by Western blotting . β - cat protein level was normalized to β - actin ( n = 4 biological replicates each group ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Tau ( F ) Data were analyzed by Two - way repeated - measures ANOVA ( G and H ) K49 acetylation of β - cat inhibited its ubiquitination . HEK293 cells were co - transfected with HA - ubiquitin and GFP - β - cat or pseudo - acetylated K49 - β - catenin ( K49Q ) , and then β - cat was immunoprecipitated by anti - GFP and blotted by anti - Ub and anti - β - cat ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs β - cat WT Data were analyzed by Student ' s t test . ( I and J ) K49 acetylation of β - cat inhibited its degradation . HEK293 cells were transfected with β - cat WT or K49Q for 24 h , and then treated with cycloheximide ( Chx , 100 μg / ml ) for 12 h or 24 h ( n = 4 biological replicates each group ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs β - cat WT Data were analyzed by Two - way repeated - measures ANOVAβ - cat mutation at K49 ( K49R ) restored the degradation of β - catenin . HEK293 cells were transfected with β - cat WT or co - transfected with β - cat WT and Tau40 , or co - transfected with β - cat K49R and Tau40 for 24 h , and then treated with cycloheximide ( Chx , 100 μg / ml ) for 12 h or 24 h ( n = 4 biological replicates each group ) . β - cat mutation at K49 ( K49R ) restored the degradation of β - catenin . HEK293 cells were transfected with β - cat WT or co - transfected with β - cat WT and Tau40 , or co - transfected with β - cat K49R and Tau40 for 24 h , and then treated with cycloheximide ( Chx , 100 μg / ml ) for 12 h or 24 h ( n = 4 biological replicates each group ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Tau + β - cat WT ( L ) Data were analyzed by Two - way repeated - measures ANOVA ( M and N ) K49 acetylation of β - cat inhibited its phosphorylation at N - terminal domain . HEK293 cells were transfected with GFP - vec , or eGFP - β - cat ( WT ) or K49Q for 48 h , then treated with wortmannin ( WO , 1 μM ) plus GF109203X ( GFX , 1 μM ) to activate GSK - 3β for 1 h followed by Western blotting ( n = 3 biological replicates each group ) . Data information : Data are presented as mean ± SEM ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs β - cat WT plus WO / GFX ( N ) . data in ( N ) was analyzed by one - way ANOVA .

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 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 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 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 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 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 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

Figure 3(A and B) Expressing Tau inhibited β-cat ubiquitination in HEK293 cells, measured by immunoprecipitation using antibody against β-cat and Western blotting using anti- ubiquitination (anti-Ub) and anti-β-cat, respectively (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec (B) Data were analyzed by Student's t test.(C and D) Overexpressing Tau inhibited β-cat ubiquitination in hippocampi of Tau transgenic mice, measured by immunoprecipitation using anti-Ub and Western blotting using anti-β-cat, respectively (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau- (D) Data were analyzed by Student's t test.(E and F) Expressing Tau-k18(-) attenuated Tau-induced β-cat elevation. HEK293 cells were transfected with Vec, or Tau-40, or Tau-k18(-) for 24 h, and then were treated with Chx for 12 h or 24 h, followed by Western blotting. β-cat protein level was normalized to β-actin (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau (F) Data were analyzed by Two-way repeated-measures ANOVA(G and H) K49 acetylation of β-cat inhibited its ubiquitination. HEK293 cells were co-transfected with HA-ubiquitin and GFP-β-cat or pseudo-acetylated K49-β-catenin (K49Q), and then β-cat was immunoprecipitated by anti-GFP and blotted by anti-Ub and anti-β-cat (n=3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT Data were analyzed by Student's t test.(I and J) K49 acetylation of β-cat inhibited its degradation. HEK293 cells were transfected with β-cat WT or K49Q for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT Data were analyzed by Two-way repeated-measures ANOVAβ-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group).β-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau+β-cat WT (L) Data were analyzed by Two-way repeated-measures ANOVA(M and N) K49 acetylation of β-cat inhibited its phosphorylation at N-terminal domain. HEK293 cells were transfected with GFP-vec, or eGFP-β-cat (WT) or K49Q for 48 h, then treated with wortmannin (WO, 1 μM) plus GF109203X (GFX, 1 μM) to activate GSK-3β for 1 h followed by Western blotting (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT plus WO/GFX (N). data in (N) was analyzed by one-way ANOVA.


Figure 4 ( A and B ) Expressing Tau - k18 ( - ) abolished its effect on β - cat K49 - acetylation and c - cas - 3 ( cleaved caspase - 3 ) . N2a cells were transfected with Vec , or Tau40 , or Tau - k18 ( - ) for 48 h , and then treated with 0 . 5 μM staurosporine ( STP , an apoptotic inducer ) for 4 h , followed by Western blotting ( n = 3 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs vec ( B ) # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs Vec plus STP ( B ) ; $ $ $ , P < 0 . 001 vs Tau plus STP ( B ) Data were analyzed by one - way ANOVA . Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . ; N2a cells , transfected with myc - Vec , or myc - Tau40 , or myc - Tau - k18 ( - ) , or myc - Tau - 2CA for 48 h , were then treated with 0 . 5 μM STP for 4 h followed by flow cytometry using Annexin - FITC ( C ) Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau plus STP Data were analyzed by one - way ANOVA . Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . N2a cells , transfected with eGFP - Vec , or eGFP - Tau40 , or eGFP - Tau - k18 ( - ) , or Tau - 2CA for 48 h , were then treated with 0 . 5 μM STP for 4 h followed by co - staining with c - cas - 3 ( red ) ( D In panel D ( middle row ) , the Tau expression ( white arrowheads ) was largely not co - localized with c - casp - 3 ( yellow arrowheads ) in the same visual field ( n = 4 ~ 8 biological replicates each group ; scale bar , 10 μm ) . Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . N2a cells , transfected with eGFP - Vec , or eGFP - Tau40 , or eGFP - Tau - k18 ( - ) , or Tau - 2CA for 48 h , were then treated with 0 . 5 μM STP for 4 h followed by co - staining with c - cas - 3 ( red ) The apoptotic rate ( E ) was presented as the ratio of yellow ( c - casp - 3 and GFP co - stained ) to green ( the total GFP - expressing cells ) staining . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau plus STP Data were analyzed by one - way ANOVA . Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . N2a cells , transfected with myc - Vec , or myc - Tau40 , or myc - Tau - k18 ( - ) , or myc - Tau - 2CA for 48 h , were then treated with 0 . 5 μM STP for 4 h followed by CCK8 assay ( F ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau plus STP ♧ ♧ ♧ , P < 0 . 001 vs vec Data were analyzed by one - way ANOVA . ( G ) Non - acetylation modifiable mutation of β - cat at K49 ( K49R ) , not K19 ( K19R ) , abolished the anti - apoptotic effect of Tau . N2a cells were co - transfected with Tau40 and β - cat WT , or K49R , or K19R for 48 h , and then treated with 0 . 5 μM STP for 4 h . The cell viability was measured by CCK8 assay ( n = 8 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec plus STP ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs vs β - cat + Vec plus STP ( G ) ; $ $ $ , P < 0 . 001 vs β - cat + Tau plus STP ( G ) ; & & & , P < 0 . 001 vs β - cat K49R + Tau plus STP ( G ) . ♧ ♧ ♧ , P < 0 . 001 vs vec Data were analyzed by one - way ANOVA . ( H ) Expressing β - cat K49Q most significantly increased cell viability . N2a cells were transfected with β - cat WT or K49R or K49Q or the empty Vec for 48 h , and then treated with 0 . 5 μM STP for 4 h . The cell viability was measured by CCK8 assay ( n = 8 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec plus STP ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs β - cat plus STP ( H ) , ; $ $ $ , P < 0 . 001 vs β - cat K49Q plus STP ( H ) ♧ ♧ ♧ , P < 0 . 001 vs vec Data were analyzed by one - way ANOVA . ( I ) Overexpressing Ser199 - phosphorylation mimic Tau ( TauS199E ) further augmented cell viability . N2a cells were transfected with empty vec , Tau40 or TauS199E for 48h and then treated with 0 . 5 μM STP for 4 h . The cell viability was measured by CCK8 assay ( n = 8 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec plus STP ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs vs Tau plus STP ( I ) Data were analyzed by one - way ANOVA .

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 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 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 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 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 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 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

Figure 4(A and B) Expressing Tau-k18(-) abolished its effect on β-cat K49-acetylation and c-cas-3 (cleaved caspase-3). N2a cells were transfected with Vec, or Tau40, or Tau-k18(-) for 48 h, and then treated with 0.5 μM staurosporine (STP, an apoptotic inducer) for 4 h, followed by Western blotting (n=3 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs vec (B) #, P<0.05, ##, P<0.01, ###, P<0.001 vs Vec plus STP (B) ; $$$, P<0.001 vs Tau plus STP (B) Data were analyzed by one-way ANOVA.Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. ; N2a cells, transfected with myc-Vec, or myc-Tau40, or myc-Tau-k18(-), or myc-Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by flow cytometry using Annexin-FITC (C) Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP Data were analyzed by one-way ANOVA.Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with eGFP-Vec, or eGFP-Tau40, or eGFP-Tau-k18(-), or Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by co-staining with c-cas-3 (red) (D In panel D (middle row), the Tau expression (white arrowheads) was largely not co-localized with c-casp-3 (yellow arrowheads) in the same visual field (n=4~8 biological replicates each group; scale bar, 10 μm).Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with eGFP-Vec, or eGFP-Tau40, or eGFP-Tau-k18(-), or Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by co-staining with c-cas-3 (red) The apoptotic rate (E) was presented as the ratio of yellow (c-casp-3 and GFP co-stained) to green (the total GFP-expressing cells) staining. Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP Data were analyzed by one-way ANOVA.Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with myc-Vec, or myc-Tau40, or myc-Tau-k18(-), or myc-Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by CCK8 assay (F). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.(G) Non-acetylation modifiable mutation of β-cat at K49 (K49R), not K19 (K19R), abolished the anti-apoptotic effect of Tau. N2a cells were co-transfected with Tau40 and β-cat WT, or K49R, or K19R for 48 h, and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs vs β-cat + Vec plus STP (G) ; $$$, P<0.001 vs β-cat + Tau plus STP (G) ; &&&, P<0.001 vs β-cat K49R+ Tau plus STP (G). ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.(H) Expressing β-cat K49Q most significantly increased cell viability. N2a cells were transfected with β-cat WT or K49R or K49Q or the empty Vec for 48 h, and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs β-cat plus STP (H), ; $$$, P<0.001 vs β-cat K49Q plus STP (H) ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.(I) Overexpressing Ser199-phosphorylation mimic Tau (TauS199E) further augmented cell viability. N2a cells were transfected with empty vec, Tau40 or TauS199E for 48h and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs vs Tau plus STP (I) Data were analyzed by one-way ANOVA.