G , H . Representative ( G ) ( H ) of hindlimb intermuscular fat in Sham - R CtM - R mice . were obtained from a longitudinal section of hind leg . fat data are presented as a of .

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G, H. Representative images (G) and weight (H) of the hindlimb intermuscular fat in the Sham-R and CtM-R mice. Images were obtained from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum hindlimb muscle ketone , quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


B . gain in response to different of dietary BCAAs .

B. Body weight gain in response to the different levels of dietary BCAAs.


C. Representative images of gross anatomy.


D . total of extramuscular fat ( posterior subcutaneous fat , epididymal fat , mesenteric fat , retroperitoneal fat ) in mice fed high - fat diet ( HFD ) with or without BCAAs .

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D. The total weight of the extramuscular fat (posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the mice fed high-fat diet (HFD) with or without BCAAs.


E . serum BCAA in mice fed HFD with or without BCAAs .

E. The serum BCAA levels in the mice fed HFD with or without BCAAs.


F . PCoA , on weighted UniFrac matrix , of 16S rRNA gene data for luminal contents of ileum colon are shown . gain data are presented as a of initial . fat data are presented as a of .

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F. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown. The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight.


G . Serum hindlimb muscle ketone , quantified enzymatically .

G. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


( B , Sequential dissociation of EI24 BiP from IRE1 elicits a two - phase activation of IRE1 in response to ER stress . were treated with 4 μM tunicamycin ( TM ) for indicated times . interactions of EI24 BiP with IRE1 were analyzed by

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(B, Sequential dissociation of EI24 and BiP from IRE1 elicits a two-phase activation of IRE1 in response to ER stress. Cells were treated with 4 μM tunicamycin (TM) for the indicated times. The interactions of EI24 and BiP with IRE1 were analyzed by immunoprecipitation


( D ) EI24 knockout promotes activation of IRE1 , but not PERK or ATF6 . Control ( sgGFP ) EI24 knockout ( sgEI24 ) HeLa were treated with 4 μM TM for indicated times . expression was analyzed by immunoblotting .

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(D) EI24 knockout promotes the activation of IRE1, but not PERK or ATF6. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were treated with 4 μM TM for the indicated times. Protein expression was analyzed by immunoblotting.


( G ) Expression of Flag - EI24 in EI24 knockout inhibits IRE1 activity . Flag - Metap2 served as control . transfected with indicated cDNAs were treated with or without 4 μM TM for 2 hr . expression was analyzed by immunoblotting .

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(G) Expression of Flag-EI24 in EI24 knockout cells inhibits IRE1 activity. Flag-Metap2 served as the control. Cells transfected with the indicated cDNAs were treated with or without 4 μM TM for 2 hr. Protein expression was analyzed by immunoblotting.


( B ) EI24 persistently localizes on ER regardless of presence or absence of ER stress . HeLa stably expressing Flag - EI24 were treated with or without 4 μM tunicamycin ( TM ) for 2 hr . Colocalization of EI24 with ER was analyzed . KDEL - mCherry , ER marker . Scale bar , 10 μm .

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(B) EI24 persistently localizes on the ER regardless of the presence or absence of ER stress. HeLa cells stably expressing Flag-EI24 were treated with or without 4 μM tunicamycin (TM) for 2 hr. Colocalization of EI24 with ER was analyzed. KDEL-mCherry, ER marker. Scale bar, 10 μm.


( D ) C - terminus of EI24 is required for IRE1 binding . HeLa transfected with indicated EI24 mutants were treated as in ( B ) , after which - interactions were analyzed by immunoprecipitation ( IP ) . star represents IgG chains . ( E ) C - terminus of EI24 binds to IRE1 in a persistent fashion . HeLa transfected with indicated EI24 domains were treated as in ( B ) . - interactions were analyzed by IP . star represents IgG chains . (

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(D) The C-terminus of EI24 is required for IRE1 binding. HeLa cells transfected with the indicated EI24 mutants were treated as in (B), after which protein-protein interactions were analyzed by immunoprecipitation (IP). The star represents IgG light chains. (E) The C-terminus of EI24 binds to IRE1 in a persistent fashion. HeLa cells transfected with the indicated EI24 domains were treated as in (B). Protein-protein interactions were analyzed by IP. The star represents IgG light chains. (


( I ) EI24 overexpression cannot prevent activation of phospho - mimicking ( 3SE ) IRE1 mutant . Wild - type IRE1 3SE knock - in HeLa were transfected with empty vector or Flag - EI24 . Transfected were treated as in ( B ) . expressions were analyzed by immunoblotting .

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(I) EI24 overexpression cannot prevent the activation of the phospho-mimicking (3SE) IRE1 mutant. Wild-type and IRE1 3SE knock-in HeLa cells were transfected with empty vector or Flag-EI24. Transfected cells were treated as in (B). Protein expressions were analyzed by immunoblotting.


( L ) Expression of full - EI24 or △ TM34 or △ C mutant in EI24 knockout HeLa for comparison of their functions . HeLa transfected with indicated EI24 mutants were treated as in ( B ) . expressions were analyzed by immunoblotting .

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(L) Expression of full-length EI24 or the △TM34 or △C mutant in EI24 knockout HeLa cells for comparison of their functions. HeLa cells transfected with the indicated EI24 mutants were treated as in (B). Protein expressions were analyzed by immunoblotting.


( B , C ) EI24 knockout causes resting - state ER calcium reduction ( B ) calcium elevation ( C ) under ER stress . Control ( sgGFP ) EI24 knockout ( sgEI24 ) HeLa were first perfused in TM - free buffer , switched to buffer containing 4 μM TM to induce ER stress where indicated . Resting state calcium in ER ( B ) cytosol ( C ) with or without TM are displayed in columns . Data information : N = 20 for one replicate . Data are mean ± ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - Student ' s t - .

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(B, C) EI24 knockout causes resting-state ER calcium reduction (B) and cytosolic calcium elevation (C) under ER stress. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were first perfused in TM-free buffer, and switched to the buffer containing 4 μM TM to induce ER stress where indicated. Resting state calcium levels in the ER (B) and cytosol (C) with or without TM are displayed in columns. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( G ) Knockdown of IP3R1 prevents calcium mobilization from ER to cytosol . measurements were similarly performed as in ( E ) , were from same batch of . Data information : N = 20 for one replicate . Data are mean ± ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - Student ' s t - .

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(G) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( I ) Wild - type EI24 , but not TM34 - or C - terminus - deleted mutant , preserves ER calcium stores in EI24 knockout . measurements were similarly performed as in ( E ) , were from same batch of . Data information : N = 20 for one replicate . Data are mean ± ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - Student ' s t - .

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(I) Wild-type EI24, but not the TM34- or C-terminus-deleted mutant, preserves ER calcium stores in EI24 knockout cells. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( B ) Knockdown of IP3R1 prevents ER stress - induced apoptosis in EI24 knockout . were cultured with or without 4 μM TM for 8 hr . Percentages of early apoptotic were measured by flow cytometry . Data information : N ≈ 10000 for one biological replicate . Data are mean ± ( n = 3 or 4 biologically independent replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - Student ' s t -

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(B) Knockdown of IP3R1 prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test


( D ) Knockdown of IP3R1 prevents ER stress - induced apoptosis . were treated with 4 μM TM for indicated times . expressions were analyzed by immunoblotting .

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(D) Knockdown of IP3R1 prevents ER stress-induced apoptosis. Cells were treated with 4 μM TM for the indicated times. Protein expressions were analyzed by immunoblotting.


( F - H ) Reintroduction of full - EI24 , but not TM34 - or C - terminus - deleted mutant ( F , G ) or C - terminus alone ( H ) , inhibited ER stress - induced apoptosis in EI24 knockout . treated with or without TM were analyzed for apoptosis by flow cytometry ( F , H ) or by immunoblotting ( G ) . Arrows in ( H ) denote decreased or increased apoptotic . Data information : N ≈ 10000 for one biological replicate . Data are mean ± ( n = 3 or 4 biologically independent replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - Student ' s t - for F ) , one - Student ' s t - for ( H ) .

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(F-H) Reintroduction of full-length EI24, but not the TM34- or C-terminus-deleted mutant (F, G) or the C-terminus alone (H), inhibited ER stress-induced apoptosis in EI24 knockout cells. Cells treated with or without TM were analyzed for apoptosis by flow cytometry (F, H) or by immunoblotting (G). Arrows in (H) denote decreased or increased apoptotic levels. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test for F), one-tailed Student's t-test for (H).


C . Kinesin family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm , identified by mass - spectrometry ( schematics are on ( Hirokawa et al , 2009 ) . Biotinylated A3 peptide served as a control . Mean spectral ( normalized plus pseudocounts ) ± , n = 3 independent biological repeats . shown were found to significantly purify with R3 , but not A3 , with probability > 0 . 95 a mean fold change > 2 by SAINTexpress

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C. Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm, identified by mass-spectrometry (schematics are based on (Hirokawa et al, 2009). Biotinylated A3 peptide served as a control. Mean spectral counts (normalized plus pseudocounts) ± SEM, n = 3 independent biological repeats. All proteins shown were found to significantly purify with R3, but not A3, with probability > 0.95 and a mean fold change > 2 by SAINTexpress analysis


A . GAR domain deletion or mutation perturbs binding to Kif5a . Co - immunoprecipitation of Kif5a HA - Dendra2 - tagged nucleolin ( both overexpressed in HEK - 293 ) . IP was performed with HA antibody probed in Western blot with anti - Kif5a anti - HA antibodies . Ncl FL - HA - Dendra2 - full nucleolin ; Ncl ΔGAR - HA - Dendra2 - nucleolin with a GAR domain deletion ; Ncl GAR ( N ) - HA - Dendra2 - nucleolin with 10 arginines in GAR domain mutated to asparagines ; DEN - Dendra2 . Note that HA input blots for HA - Dendra2 ( Dendra ) other three constructs are from same , but shown discontinuously owing to different migration of these in PAGE .

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A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA -Dendra2- full length nucleolin; Ncl ΔGAR - HA-Dendra2- nucleolin with a GAR domain deletion; Ncl GAR(N) - HA-Dendra2- nucleolin with all 10 arginines in the GAR domain mutated to asparagines; DEN - Dendra2. Note that HA input blots for HA-Dendra2 (Dendra) and the other three constructs are from the same membrane, but shown discontinuously owing to the different migration of these proteins in PAGE.


A . GAR domain deletion or mutation perturbs binding to Kif5a . Co - immunoprecipitation of Kif5a HA - Dendra2 - tagged nucleolin ( both overexpressed in HEK - 293 ) . IP was performed with HA antibody probed in Western blot with anti - Kif5a anti - HA antibodies . Ncl FL -

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A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA


D . Uptake of TAMRA - labeled R4 N4 peptides into cultured DRG neurons . were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4 ° C , washed , fixed analyzed by confocal microscopy . PC - phase contrast . Scale bar - 10 µm . E . of ( D ) . n = 3 independent biological repeats ; means ± ; * p < 0 . 05 , paired Student ' s t - a . u . - arbitrary units .

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D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm. E. Quantification of (D). n = 3 independent biological repeats; means ± SEM; * p<0.05, paired Student's t-test a. u. - arbitrary units.


D . Uptake of TAMRA - labeled R4 N4 peptides into cultured DRG neurons . were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4 ° C , washed , fixed analyzed by confocal microscopy . PC - phase contrast . Scale bar - 10 µm .

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D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm.


G . HEK - 293 were transfected with plasmids expressing HA - Dendra2 - full nucleolin ( Ncl FL ) , HA - Dendra2 - nucleolin with a GAR domain deletion ( Ncl ΔGAR ) or HA - Dendra2 - nucleolin with 10 arginines in GAR domain mutated to asparagines ( Ncl GAR ( N ) ) processed as in ( F ) .

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G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl FL), HA-Dendra2-nucleolin with a GAR domain deletion (Ncl ΔGAR) or HA-Dendra2-nucleolin with all 10 arginines in the GAR domain mutated to asparagines (Ncl GAR(N)) and processed as in (F).


G . HEK - 293 were transfected with plasmids expressing HA - Dendra2 - full nucleolin (

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G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl


A . GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons , transduced with peripheral neuron specific AAV - PHP . S vector expressing full HA - Dendra2 - nucleolin ( Ncl FL ) or respective ΔGAR mutant ( Ncl ΔGAR ) upon seeding analyzed by epifluorescence microscopy 9 - 10 days later . Shown are representative Dendra2 collected from non - activated ( green ) emission . Hoechst 33342 ( 10 µM ) was used to outline nuclei . Superimposed phase contrast Dendra2 are shown on right . Scale bar - 25 µm . B . of nucleolar / ratios in DRG shown in ( A ) . n = 38 - 54 per sample in 3 independent biological repeats ; means ± ; * * * * p < 0 . 0001 , Mann - Whitney

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A. GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons, transduced with the peripheral neuron specific AAV-PHP.S vector expressing full length HA-Dendra2-nucleolin (Ncl FL) or respective ΔGAR mutant (Ncl ΔGAR) upon seeding and analyzed by epifluorescence microscopy 9-10 days later. Shown are representative Dendra2 fluorescence images collected from the non-activated (green) emission line. Hoechst 33342 (10 µM) was used to outline nuclei. Superimposed phase contrast and Dendra2 fluorescence images are shown on the right. Scale bar - 25 µm. B. Quantification of the nucleolar/nuclear intensity ratios in DRG cells shown in (A). n = 38-54 cells per sample in 3 independent biological repeats; means ± SEM; **** p<0.0001, Mann-Whitney test


C . Time - lapse live imaging of nuclei in DRG neurons transduced with AAV - PHP . S HA - Dendra2 - nucleolin ( full ΔGAR mutant ) , after a hypotonic challenge with double - distilled water ( ddH20 ) . Shown are epifluorescence ( Dendra2 green emission ) taken at indicated times after medium replacement with ddH20 . Scale bar - 10 µm . D . of nucleolar Dendra2 signal in ( C ) with values immediately before medium replacement set as 100 % . n = 17 - 24 per sample in 3 biological repeats ; means ± ; Two - way ANOVA with Sidák ' s post - detected a significant time - dependent reduction of nucleolar signal ( p < 0 . 0001 ) no difference between full ΔGAR mutant nucleolin ( p > 0 . 05 )

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C. Time-lapse live imaging of nuclei in DRG neurons transduced with AAV-PHP.S HA-Dendra2-nucleolin (full length and ΔGAR mutant), after a hypotonic challenge with double-distilled water (ddH20). Shown are epifluorescence images (Dendra2 green emission line) taken at indicated times after medium replacement with ddH20. Scale bar - 10 µm. D. Quantification of nucleolar Dendra2 signal in (C) with intensity values immediately before medium replacement set as 100%. n = 17-24 cells per sample in 3 biological repeats; means ± SEM; Two-way ANOVA with Sidák's post-test detected a significant time-dependent reduction of nucleolar signal (p < 0.0001) and no difference between full length and ΔGAR mutant nucleolin (p > 0.05)


B . Western blot of nucleolin in DRG neurons from wildtype ( WT ) GAR + / - mice cultured in Boyden chambers . Tuj1 was used as a loading control . higher nucleolin band in GAR + / - corresponds to WT nucleolin lower band to nucleolin with a 41 aa deletion ( corresponding to about 4 kDa reduction in ) . C . of ( B ) , total nucleolin ( sum of both bands where applicable ) were normalized to Tuj1 expressed as GAR + / - / WT ratios . n = 3 independent biological repeats ; means ± ; * p < 0 . 05 , 1 sample t - .

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B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size). C. Quantification of (B), total nucleolin levels (sum of both bands where applicable) were normalized to Tuj1 and expressed as GAR+/- / WT ratios. n = 3 independent biological repeats; means ± SEM; * p < 0.05, 1 sample t-test.


B . Western blot of nucleolin in DRG neurons from wildtype ( WT ) GAR + / - mice cultured in Boyden chambers . Tuj1 was used as a loading control . higher nucleolin band in GAR + / - corresponds to WT nucleolin lower band to nucleolin with a 41 aa deletion ( corresponding to about 4 kDa reduction in ) .

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B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size).


A , B . Representative maximum projections of exposure matched confocal of immunofluorescence for nucleolin neurofilament ( NF ) neuronal marker Tuj1 in sciatic nerve axons from WT GAR + / - mice shown in ( A ) . Upper panels show total nucleolin stain . Middle panels show merged image of nucleolin ( gray ) , NF Tuj1 ( magenta ) , DAPI ( blue ) . Lower panels show nucleolin overlaps with NF Tuj1 signals as " axon only " signal . B shows of nucleolin immunofluorescence with approximately a 50 % reduction in nucleolin in axons of GAR + / - mice in vivo . n = 3 WT , n = 4 GAR + / - ; means ± ; * p < 0 . 05 , unpaired Student ' s t - . Scale bar - 10 µm .

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A, B. Representative maximum projections of exposure matched confocal images of immunofluorescence for nucleolin and neurofilament (NF) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR+/- mice shown in (A). Upper panels show total nucleolin stain. Middle panels show merged image of nucleolin (gray), NF and Tuj1 (magenta), and DAPI (blue). Lower panels show nucleolin overlaps with NF and Tuj1 signals as the "axon only" signal. B shows quantification of nucleolin immunofluorescence with approximately a 50% reduction in nucleolin in the axons of GAR+/- mice in vivo. n = 3 WT, n = 4 GAR+/-; means ± SEM; * p < 0.05, unpaired Student's t-test. Scale bar - 10 µm.


C - F . Representative , exposure matched maximum projection confocal of FISH for Kpnb1 ( C ) or mTOR ( E ) mRNA NF plus Tuj1 immunostaining from sciatic nerve sections from WT ( left ) or GAR + / - mice . Upper panels for each show total mRNA signal . Middle panels show mRNA ( gray ) signals merged with NF plus Tuj1 ( magenta ) DAPI ( blue ) . Lower panels show mRNA signal that overlap with NF plus Tuj1 signal ( labeled " axon only " signal ) . of axonal Kpnb1 ( D ) mTOR ( F ) mRNA signals compared to negative control , DapB mRNA , show a significant reduction in these axonal mRNAs in GAR + / - mice . n = 3 WT , n = 3 GAR + / - ; means ± ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 unpaired Student ' s t - . Scale bar - 10 µm .

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C-F. Representative, exposure matched maximum projection confocal images of FISH for Kpnb1 (C) or mTOR (E) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT (left) or GAR+/- mice. Upper panels for each show total mRNA signal. Middle panels show mRNA (gray) signals merged with NF plus Tuj1 (magenta) and DAPI (blue). Lower panels show mRNA signal that overlap with NF plus Tuj1 signal (labeled "axon only" signal). Quantification of axonal Kpnb1 (D) and mTOR (F) mRNA signals compared to the negative control, DapB mRNA, show a significant reduction in these axonal mRNAs in the GAR+/- mice. n = 3 WT, n = 3 GAR+/-; means ± SEM; ** p < 0.01, *** p < 0.001, ****p < 0.0001 unpaired Student's t-test. Scale bar - 10 µm.


D . Representative for FISH of Inpp5f in DRG neurons treated for 48h with 10 µM AS1411 or 10 µM control aptamer , replated grown for 18h . FISH signal is shown in gray , somata ( left ) axons ( right ) are visualized by neurofilament immunostaining ( magenta ) . Scr - scrambled FISH probe served as a negative control . Scale bar - 10 µm .

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D. Representative images for FISH analysis of Inpp5f in DRG neurons treated for 48h with 10 µM AS1411 or 10 µM control aptamer, replated and grown for 18h. FISH signal is shown in gray, cell somata (left) and axons (right) are visualized by neurofilament immunostaining (magenta). Scr - scrambled FISH probe served as a negative control. Scale bar - 10 µm.


A , B . Representative exposure - matched FISH / IF for DRG neurons co - transfected with control vs . nucleolin ( Ncl ) siRNA plus siRNA - resistant wild type ( WT ; A ) or ∆ GAR ( B ) Ncl cDNAs . Axonal Kpnb1 mRNA is decreased with Ncl knockdown , this is not rescued by expression of ∆ GAR Ncl mutant ( scale bars - 10 µm ) .

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A, B. Representative exposure-matched FISH/IF images for DRG neurons co-transfected with control vs. nucleolin (Ncl) siRNA plus siRNA-resistant wild type (WT; A) or ∆GAR (B) Ncl cDNAs. Axonal Kpnb1 mRNA is decreased with the Ncl knockdown, and this is not rescued by expression of ∆GAR Ncl mutant (scale bars - 10 µm).


A . Cultured DRG neurons from wild type ( WT ) Thy1 - YFP mice Thy1 - YFP / GAR + / - mice ( YFP signal is shown in green ) . were imaged every hour for a period of 48 h . Scale bar - 100 μm . B . of time - lapse imaging experiment shown in ( A ) . Growth rate of longest neurite of each was calculated from time point of starting growth . n = 4 independent biological repeats ; means ± , * * * * p < 0 . 0001 , two - way ANOVA .

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A. Cultured DRG neurons from wild type (WT) Thy1-YFP mice and Thy1-YFP / GAR+/- mice (YFP signal is shown in green). Cells were imaged every hour for a period of 48 h. Scale bar - 100 μm. B. Quantification of the time-lapse imaging experiment shown in (A). Growth rate of the longest neurite of each cell was calculated from the time point of starting growth. n = 4 independent biological repeats; means ± SEM, **** p < 0.0001, two-way ANOVA.


A . Cultured DRG neurons from wild type ( WT ) Thy1 - YFP mice Thy1 - YFP / GAR + / - mice ( YFP signal is shown in green ) . were imaged every hour for a period of 48 h . Scale bar - 100 μm .

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A. Cultured DRG neurons from wild type (WT) Thy1-YFP mice and Thy1-YFP / GAR+/- mice (YFP signal is shown in green). Cells were imaged every hour for a period of 48 h. Scale bar - 100 μm.


C . of cultured DRG neurons from adult C57BL / 6 mice infected with AAV - PHP . s expressing HA - Dendra fused with wild type nucleolin GAR domain ( GAR WT ) or with GAR domain with 10 arginines substituted with asparagines , GAR ( N ) . Neurons were re - plated 9 - 10 days after AAV infection . 24h after re - plating were fixed stained with anti - HA ( grey ) anti - NFH ( magenta ) antibodies . Scale bar - 100 μm . D . of total axonal outgrowth mean process in HA - positive in ( C ) . Outgrowth measurements were on NFH . n = 6 independent biological repeats ; means ± ; * p < 0 . 05 , * * * p < 0 . 001 ( Student ' s - ) .

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C. Fluorescence images of cultured DRG neurons from adult C57BL/6 mice infected with AAV-PHP.s expressing HA-Dendra fused with the wild type nucleolin GAR domain (GAR WT) or with the GAR domain with all 10 arginines substituted with asparagines, GAR(N). Neurons were re-plated 9-10 days after AAV infection. 24h after re-plating cells were fixed and stained with anti-HA (grey) and anti-NFH (magenta) antibodies. Scale bar - 100 μm. D. Quantification of total axonal outgrowth and mean process length in HA-positive cells in (C). Outgrowth measurements were based on NFH staining. n = 6 independent biological repeats; means ± SEM; * p < 0.05, *** p < 0.001 (Student's-test).


C . of cultured DRG neurons from adult C57BL / 6 mice infected with AAV - PHP . s expressing HA - Dendra fused with wild type nucleolin GAR domain ( GAR WT ) or with GAR domain with 10 arginines substituted with asparagines , GAR ( N ) . Neurons were re - plated 9 - 10 days after AAV infection . 24h after re - plating were fixed stained with anti - HA ( grey ) anti - NFH ( magenta ) antibodies . Scale bar - 100 μm .

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C. Fluorescence images of cultured DRG neurons from adult C57BL/6 mice infected with AAV-PHP.s expressing HA-Dendra fused with the wild type nucleolin GAR domain (GAR WT) or with the GAR domain with all 10 arginines substituted with asparagines, GAR(N). Neurons were re-plated 9-10 days after AAV infection. 24h after re-plating cells were fixed and stained with anti-HA (grey) and anti-NFH (magenta) antibodies. Scale bar - 100 μm.


( C ) Confocal image of a constricting during ventral furrow formation showing an overlay between myosin - II probe Sqh : : GFP plasma marker GAP43 : : mCherry . Scale bar , 5

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(C) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin-II probe Sqh::GFP and the plasma membrane marker GAP43::mCherry. Scale bar, 5 μm


( D ) Movie ( still frames ) of a gastrulating Drosophila expressing endogenously tagged mVenus : : βH - spectrin imaged in a cross section using two - photon microscopy . βH - spectrin is enriched at apical surface of ventral mesodermal during tissue invagination ( arrowheads ) . Scale bar , 50 μm .

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(D) Movie (still frames) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin imaged in a cross section using two-photon microscopy. βH-spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination (arrowheads). Scale bar, 50 μm.


( E ) Z - Projections of confocal image stacks showing apical surface of a Drosophila expressing endogenously tagged mVenus : : βH - spectrin ( green ) plasma marker GAP43 : : mCherry at 5 min ( top ) , 7 min ( middle ) 9 min ( bottom ) after initial medio - apical accumulation of βH - spectrin . Scale bar , 20

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(E) Z-Projections of confocal image stacks showing the apical cell surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry at 5 min (top), 7 min (middle) and 9 min (bottom) after initial medio-apical accumulation of βH-spectrin. Scale bar, 20 μm


( F , G ) Immunostaining of βH - spectrin ( green ) visualized by STED nanoscopy revealed medio - apical βH - spectrin supra - in mesodermal ( F ) . In ectodermal , βH - spectrin localizes to apical junctions ( G ) . Note , in ( F ) junctional βH - spectrin signal is shown in magenta as a proxy for membranes . Scale bars , 2 . 5 μm .

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(F, G) Immunostaining of βH-spectrin (green) visualized by STED nanoscopy revealed medio-apical βH-spectrin supra-cellular fibers in mesodermal cells (F). In ectodermal cells, βH-spectrin localizes to apical cell junctions (G). Note, in (F) the junctional βH-spectrin signal is shown in magenta as a proxy for cell membranes. Scale bars, 2.5 μm.


( H - J ) Confocal of ventral surface of a Drosophila expressing endogenously tagged mVenus : : βH - spectrin ( H ) , myosin - II marker Sqh : : mCherry ( I ) a merge of two ( J ) with βH - spectrin in green Sqh : : mCherry in magenta . Scale bars : 20 μm .

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(H-J) Confocal images of the ventral surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (H), the myosin-II marker Sqh::mCherry (I) and a merge of the two (J) with βH-spectrin in green and Sqh::mCherry in magenta. Scale bars: 20 μm.


( K - L ) Co - of phalloidin ( F - actin reporter ; magenta ) βH - spectrin ( green ) of apical surface of a mesodermal at onset of ventral furrow formation ( K ) at a later time point ( L ) visualized by confocal microscopy . dashed lines indicate boundaries segmented on phalloidin - of sub - apical confocal sections . Scale bars : 5 μm .

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(K-L) Co-staining of phalloidin (F-actin reporter; magenta) and βH-spectrin (green) of the apical surface of a mesodermal cell at the onset of ventral furrow formation (K) and at a later time point (L) visualized by confocal microscopy. White dashed lines indicate the cell boundaries segmented based on the phalloidin-staining of sub-apical confocal sections. Scale bars: 5 μm.


( A ) Z - Projection of confocal image stacks spanning 5 μm of a control ( left ) co - expressing endogenously tagged mVenus : : βH - spectrin ( green ) plasma marker GAP43 : : mCherry ( magenta ) of an additionally expressing a maternally driven anti - GFP nanobody KD module to knock - down βH - spectrin ( right ) . Co - expression of anti - GFP nanobody KD system resulted in a drastic decrease of βH - spectrin as assessed by signal which dropped below detection limit .

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(A) Z-Projection of confocal image stacks spanning 5 μm of a control embryo (left) co-expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry (magenta) and of an embryo additionally expressing a maternally driven anti-GFP nanobody KD module to knock-down βH-spectrin (right). Co-expression of the anti-GFP nanobody KD system resulted in a drastic decrease of βH-spectrin protein levels as assessed by the fluorescent signal intensity which dropped below the detection limit.


( B ) Pie diagrams quantifying of control ( top , n = 50 ) βH - spectrin knock - down ( bottom , n = 40 ) that formed ventral furrow either normally or with minor ( green ) of that displayed severe abnormalities or did not at undergo tissue invagination ( red ) . assessment of was done in an unbiased randomized approach .

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(B) Pie diagrams quantifying the percentage of control embryos (top, n=50) and βH-spectrin knock-down embryos (bottom, n=40) that formed ventral furrow either normally or with minor phenotypes (green) and the percentage of embryos that displayed severe abnormalities or did not at all undergo tissue invagination (red). The assessment of phenotypes was done in an unbiased randomized approach.


( C - J ) Confocal of a control ( C , D ) βH - spectrin knock - down with a minor phenotype ( E , F ) , with a severe phenotype that underwent tissue invagination ( G , H ) with a severe phenotype that did not undergo tissue invagination ( I , J ) . Surface view is shown in ( C , E , G , I ) cross section through tissue at two different time points in ( D , F , H , J ) . expressed plasma marker GAP43 : : mCherry . Scale bars , 20 μm .

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(C-J) Confocal images of a control embryo (C,D) and βH-spectrin knock-down embryos with a minor phenotype (E,F), with a severe phenotype that underwent tissue invagination (G, H) and with a severe phenotype that did not undergo tissue invagination (I, J). Surface view is shown in (C,E,G,I) and the cross section through the tissue at two different time points in (D,F,H,J). All embryos expressed the plasma membrane marker GAP43::mCherry. Scale bars, 20 μm.


( K , L ) of apical surface ( K ) anisotropy ( L ) in control ( green , nControl : 6 ) in βH - spectrin knock - down ( red , nβH - spectrin KD : 7 ) . Solid lines indicate mean values semi - transparent regions corresponding standard deviation .

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(K, L) Quantification of apical surface area (K) and anisotropy (L) in control embryos (green, nControl: 6 embryos) and in βH-spectrin knock-down embryos (red, nβH-spectrin KD: 7 embryos). Solid lines indicate the mean values and the semi-transparent regions the corresponding standard deviation.


( D , E ) Still frames of a movie showing endogenously tagged mVenus : : βH - spectrin in cross section of a gastrulating wild type ( D ) twist mutant ( E ) imaged using two - photon microscopy . top frame corresponds to time point where an initial bending of apical surface was observed , middle frame was taken 2 min lower frame 3 min afterwards . Scale bars , 30 µm .

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(D, E) Still frames of a movie showing endogenously tagged mVenus::βH-spectrin in the cross section of a gastrulating wild type (D) and twist mutant (E) embryo imaged using two-photon microscopy. The top frame corresponds to the time point where an initial bending of the apical surface was observed, the middle frame was taken 2 min and the lower frame 3 min afterwards. Scale bars, 30 µm.


( F , G ) Confocal showing apical βH - spectrin ( endogenously tagged mVenus : : βH - spectrin ) in a wild type ( F ) twist mutant ( G ) . Scale bars , 20 μm . Medio - apical βH - spectrin observed in wild type were absent in twist mutant .

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(F, G) Confocal images showing apical βH-spectrin (endogenously tagged mVenus::βH-spectrin) in a wild type (F) and twist mutant (G) embryo. Scale bars, 20 μm. Medio-apical βH-spectrin fibers observed in wild type embryos were absent in twist mutant embryos.


( H - Q ) Confocal or two - photon microscopy of apical surface of an mounted with dorsal side facing objective co - expressing endogenously tagged mVenus : : βH - spectrin ( green , H , K , M , P ) , myosin - II marker Sqh : : mCherry ( magenta , I , N ) , optogenetic modules as explained in ( C ) . apical surface is shown before photoactivation ( H - L ) 1 min after photoactivation ( M - Q ) . Panels ( J , O ) show a merge of βH - spectrin myosin - II dashed rectangle indicates regions shown in ( L Q ) at higher magnification . Panels ( K , L ) ( P , Q ) are zoom - ins into region indicated in ( J ) an ( O ) , respectively . Scale bars , 20 μm ( H - J , M - O ) . 2 . 5 μm ( K , L , P , Q ) .

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(H-Q) Confocal or two-photon microscopy images of the apical surface of an embryo mounted with the dorsal side facing the objective and co-expressing endogenously tagged mVenus::βH-spectrin (green, H, K, M, P), the myosin-II marker Sqh::mCherry (magenta, I, N), and the optogenetic modules as explained in (C). The apical surface is shown before photoactivation (H-L) and 1 min after photoactivation (M-Q). Panels (J, O) show a merge of βH-spectrin and myosin-II and the dashed rectangle indicates regions shown in (L and Q) at higher magnification. Panels (K, L) and (P, Q) are zoom-ins into the region indicated in (J) an (O), respectively. Scale bars, 20 μm (H-J, M-O). 2.5 μm (K, L, P, Q).


( R ) of apical βH - spectrin upon optogenetic stimulation of Rho signaling . graph shows mean of βH - spectrin ( green ) at apical surface over time as fold change relative to initial time point ( before photo - activation ) . semi - transparent region indicates standard deviation dots indicate data points ( n = 5 ) .

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(R) Quantification of apical βH-spectrin upon optogenetic stimulation of Rho signaling. The graph shows the mean intensity of βH-spectrin (green) at the apical surface over time as the fold change relative to the initial time point (before photo-activation). The semi-transparent region indicates the standard deviation and dots indicate individual data points (n = 5 embryos).


( S - T ) Two - photon microscopy of apical βH - spectrin ( green ) 1 . 5 min after optogenetic stimulation of Rho signaling in that were previously injected with water ( S ) or ROCK - inhibitor Y - 27634 ( T ) . Dashed boxes indicate photo - activated . Y - 27634 - injected did not undergo apical constriction did not accumulate βH - spectrin upon photo - activation , while water - injected constricted showed increased βH - spectrin . Scale bars : 10 µm .

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(S-T) Two-photon microscopy images of apical βH-spectrin (green) 1.5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water (S) or the ROCK-inhibitor Y-27634 (T). Dashed boxes indicate photo-activated cells. Y-27634-injected embryos did not undergo apical constriction and did not accumulate βH-spectrin upon photo-activation, while water-injected embryos constricted and showed increased βH-spectrin levels. Scale bars: 10 µm.


( A - C ) Confocal of apical surface of a βH - spectrin knock - down showing myosin - II marker Sqh : : mCherry ( green ) E - cadherin : : mNeonGreen ( magenta ) during ventral furrow formation . ( A ) myosin - II accumulates normally at apical surface undergo an initial phase of constriction . dashed rectangle indicates region shown in ( B C ) at higher magnification . Scale bar , 30 μm . Panels ( B , C ) show a sub - region within ventral tissue at different time points . Initially a supracellular myosin - II network formed ( 0 s ) , before intercellular connections broke ( 13 s ) resulting in tears ( dashed ) with tissue retractions ( small arrow heads ) along anterior - posterior axis . intercellular network re - established ( 39 s , 65 s ) as new actomyosin formed ( big arrow heads ) to reconnect neighboring . intercellular network broke again causing tears ( dashed lines ) at different positions ( 104 s ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Confocal images of the apical cell surface of a βH-spectrin knock-down embryo showing the myosin-II marker Sqh::mCherry (green) and E-cadherin::mNeonGreen (magenta) during ventral furrow formation. (A) myosin-II accumulates normally at the apical surface and cells undergo an initial phase of constriction. The dashed rectangle indicates the region shown in (B and C) at higher magnification. Scale bar, 30 μm. Panels (B,C) show a sub-region within the ventral tissue at different time points. Initially a supracellular myosin-II network formed (0 s), before intercellular connections broke (13 s) resulting in membrane tears (dashed line) with tissue retractions (small arrow heads) along the anterior-posterior axis. The intercellular network re-established (39 s, 65 s) as new actomyosin fibers formed (big arrow heads) to reconnect neighboring cells. The intercellular network broke again causing tears (dashed lines) at different positions (104 s). Scale bars, 5 μm.


( D , E ) Confocal showing myosin - II marker Sqh : : mCherry ( green ) , E - cadherin : : mNeonGreen ( magenta ) segmented ( red ) of a single at different time points in a control undergoing ratcheted constriction ( D ) in a βH - spectrin knock - down undergoing non - ratcheted pulsations ( E ) . Scale bars , 2 . 5 μm .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(D,E) Confocal images showing the myosin-II marker Sqh::mCherry (green), E-cadherin::mNeonGreen (magenta) and the segmented membrane (red) of a single cell at different time points in a control embryo undergoing ratcheted constriction (D) and in a βH-spectrin knock-down embryo undergoing non-ratcheted pulsations (E). Scale bars, 2.5 μm.


( F ) Heatmaps showing apical surface of over time of control ( left ) βH - spectrin knock - down ( right ) normalized to initial time point ( nControl = 233 ; nβH - spectrinKD = 368 ; of 5 different each ) . color - coded scale bar is depicted on left . blue indicates a bigger than initial value ( > 1 ) yellow indicates an constricted to less than 1 / 5 of initial ( < 0 . 2 ) . Three different behaviors in βH - spectrin knock - down were identified : that constricted , that showed impaired constriction that expanded .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(F) Heatmaps showing the apical surface of individual cells over time of control embryos (left) and βH-spectrin knock-down embryos (right) normalized to the initial time point (nControl=233; nβH-spectrinKD=368; of 5 different embryos each). The color-coded scale bar is depicted on the left. Dark blue indicates a cell area bigger than the initial value (>1) and yellow indicates an area constricted to less than 1/5 of the initial area (<0.2). Three different cell behaviors in βH-spectrin knock-down embryos were identified: cells that constricted, cells that showed impaired constriction and cells that expanded.


( G ) of of that constricted ( to an < 50 % , yellow ) , that showed impaired constriction ( to an > 50 < 100 % , green ) that expanded ( to an > 100 % , blue ) after 7 min in control in βH - spectrin knock - down .

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(G) Quantification of the percentage of cells that constricted (to an area < 50%, yellow), cells that showed impaired constriction (to an area >50 and <100%, green) and cells that expanded (to an area >100%, blue) after 7 min in control embryos and in βH-spectrin knock-down embryos.


( H , I ) Graph showing average over time of control ( H , n = 235 ) in βH - spectrin knock - down ( I ) that constricted ( yelow , n = 274 ) , of that showed impaired constriction ( green , n = 232 ) of that expanded ( blue , n = 32 ) . lines indicate mean value semi - transparent region standard deviation .

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(H,I) Graph showing the average area over time of control cells (H, n=235 cells) and cells in βH-spectrin knock-down embryos (I) that constricted (yelow, n=274), of cells that showed impaired constriction (green, n=232) and of cells that expanded (blue, n=32). The lines indicate the mean value and the semi-transparent region the standard deviation.


( J ) Boxplots showing distribution of apical values 7 . 5 min after onset of ventral furrow formation of ventral in control , of ventral in βH - spectrin knock - down that constricted , ventral in βH - spectrin knock - down that displayed impaired constriction expanded . Sample numbers are equal to I . values are presented as of initial . ANOVA result ( data points ) : F ( 3 , 717 ) = 763 . 9 , p = 9 . 6e - 223 ; Cohen ' s d compared to control : dconstricted = 0 . 1 , dimpaired = 1 . 5 , dexpanded = 3 . 7 . Due to a big effect ( high sample number ) , statistical significance was assessed on Cohen ' s d . A Cohen ' s d < 0 . 5 was considered not significant ( n . s . ) .

O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Boxplots showing the distribution of apical cell area values 7.5 min after the onset of ventral furrow formation of ventral cells in control embryos, of ventral cells in βH-spectrin knock-down embryos that constricted, and ventral cells in βH-spectrin knock-down embryos that displayed impaired constriction and expanded. Sample numbers are equal to I. The values are presented as percentage of initial cell area. ANOVA result (all data points): F(3,717)=763.9, p=9.6e-223; Cohen's d compared to the control: dconstricted=0.1, dimpaired=1.5, dexpanded=3.7. Due to a big effect size (high sample number), statistical significance was assessed based on Cohen's d. A Cohen's d < 0.5 was considered not significant (n. s.).


( K ) of peak constriction rate expansion rate relative to control . Control in βH - spectrin knock - down that showed impaired constriction or expanded were compared . mean value standard deviation are shown . Please note logarithmic scale . While in βH - spectrin knock - down constriction rate was ~ 24 % lower ( mean value of 0 . 8 ) compared to control , expansion rate increased to ~ 60 % ( mean value of 1 . 6 ) . absolute change of expansion rate was 2 . 5 times higher than change of constriction rate . ANOVA result ( data points ) : F ( 3 , 717 ) = 349 . 2 , p = 9 . 7e - 140 ; pairwise comparison of βH - spectrin knock - down to control using Cohen ' s d > 1 . 4 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

(K) Quantification of the peak constriction rate and expansion rate relative to control cells. Control cells and cells in βH-spectrin knock-down embryos that showed impaired constriction or expanded were compared. The mean value and standard deviation are shown. Please note the logarithmic scale. While in βH-spectrin knock-down cells the constriction rate was ~24% lower (mean value of 0.8) compared to control cells, the expansion rate increased to ~60% (mean value of 1.6). The absolute change of the expansion rate was 2.5 times higher than the change of the constriction rate. ANOVA result (all data points): F(3,717)=349.2, p=9.7e-140; pairwise comparison of βH-spectrin knock-down cells to control using Cohen's d>1.4.


( B , C ) Kinetics of binding between scFv of FD20 ( scFD20 ) RBD from SARS - CoV - 2 ( B ) or SARS - CoV ( C ) . Biolayer interferometry ( BLI ) was performed with RBD immobilized scFD20 as analyte at indicated concentrations ( nM ) . Raw data are shown in color fitted lines are shown in grey . KD values were calculated using 1 : 1 global fitting . Note , although fitting of binding curves in D gave a KD of 4 . 8 nM , accuracy of kinetic parameters may be compromised owing to mismatch between raw data fitted curves at initial dissociation phase . reason for abnormal BIL profile is unknown but it may be caused by possible denaturation of commercial SARS - CoV RBD during storage . It was also noted that BLI signal in D was ~ 5 times less than that with SARS - CoV - 2 RBD under similar conditions ( C ) .

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(B, C) Kinetics of the binding between the scFv of FD20 (scFD20) and RBD from SARS-CoV-2 (B) or SARS-CoV (C). Biolayer interferometry (BLI) assay was performed with RBD immobilized and scFD20 as analyte at indicated concentrations (nM). Raw data are shown in color and fitted lines are shown in grey. KD values were calculated using 1:1 global fitting. Note, although the fitting of the binding curves in D gave a KD of 4.8 nM, the accuracy of the kinetic parameters may be compromised owing to the mismatch between the raw data and the fitted curves at the initial dissociation phase. The reason for the abnormal BIL profile is unknown but it may be caused by possible denaturation of the commercial SARS-CoV RBD during storage. It was also noted that the BLI signal in D was ~5 times less than that with SARS-CoV-2 RBD under similar conditions (C).


( D ) Neutralizing of scFD20 against SARS - CoV - 2 ( black ) SARS - CoV ( red ) pseudoviruses .

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(D) Neutralizing assay of scFD20 against SARS-CoV-2 (black) and SARS-CoV (red) pseudoviruses.


( E ) Neutralization assays using FD20 against pseudoviruses harboring wildtype SARS - CoV - 2 S ( blue ) , S D614G mutant ( red ) , or SARS - CoV S ( black ) .

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(E) Neutralization assays using FD20 against pseudoviruses harboring the wildtype SARS-CoV-2 S (blue), S D614G mutant (red), or SARS-CoV S (black).


( A ) Neutralization of FD20 using SARS - CoV - 2 pp harboring spike derived from four Variants of Concern as indicated . Mean ± s . d . are plotted ( n = 3 biological replicates ) . Numbers in brackets indicate IC50 values in nM .

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(A) Neutralization assay of FD20 using SARS-CoV-2 pp harboring spike derived from four Variants of Concern as indicated. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate IC50 values in nM.


( A ) weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS - CoV - 2 . mean % of starting ± s . e . m . ( n = 5 biological replicates ) are plotted .

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(A) Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. The mean % of starting weight ± s.e.m. (n = 5 biological replicates) are plotted.


( B - E ) SARS - CoV - 2 viral ( B D ) or infectious ( C E ) was detected in lung ( B C ) nasal turbinates ( D E ) . mean copy number or mean infectious titer is shown , error bars represent s . e . m . ( n = 5 biological replicates ) .

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(B-E) SARS-CoV-2 viral RNA (B and D) or infectious virus (C and E) was detected in the lung (B and C) and nasal turbinates (D and E). The mean copy number or the mean infectious titer is shown, error bars represent s.e.m. (n = 5 biological replicates).


( D ) Modest perturbation of scFD20 for RBD - ACE2 interaction . A sensor with RBD immobilized was soaked in 200 nM of scFD20 before being further soaked in scFD20 - containing buffer with ( red ) or without ( blue ) 25 nM of ACE2 for BLI signal recording . As a control , ACE2 - RBD interaction was monitored in absence of scFD20 ( black ) .

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(D) Modest perturbation of scFD20 for RBD-ACE2 interaction. A sensor with RBD immobilized was soaked in 200 nM of scFD20 before being further soaked in scFD20-containing buffer with (red) or without (blue) 25 nM of ACE2 for BLI signal recording. As a control, the ACE2-RBD interaction was monitored in the absence of scFD20 (black).


( A ) Inhibition of - fusion by mAbs . fusion was quantified by measuring luciferase expression induced by full fusion mixing of cytoplasm , rate of inhibition by indicated antibodies compared to condition without antibody is reported . Data depict mean ± s . d . ( n = 4 biological replicates ) .

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(A) Inhibition of cell-cell fusion by mAbs. Cell fusion was quantified by measuring the luciferase expression induced by full cell membrane fusion and mixing of cytoplasm, and the rate of inhibition by indicated antibodies compared to the condition without antibody is reported. Data depict mean ± s.d. (n = 4 biological replicates).


( C ) FD20 pre - incubation with SARS - CoV - 2 pp in absence of strongly impairs entry . To direct action on particles , SARS - CoV - 2 pp FD20 were co - incubated for 1 h , then premixes were used to infect VeroE6 - hACE2 for 6 h at 37 ° C . Mean ± s . d . are plotted ( n = 3 biological replicates ) . Numbers in brackets indicate gained neutralization ( % ) by pre - incubation . As control , were incubated at each step with an equal volume of PBS or Y112R . Percentages of infection were calculated according to viral titers of PBS control conditions . p < 0 . 0001 ( * * * * ) unless specified otherwise ( 2 - , unpaired student ' s t - ) . ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) FD20 pre-incubation with SARS-CoV-2 pp in the absence of cells strongly impairs entry. To test direct action on particles, SARS-CoV-2 pp and FD20 were co-incubated for 1 h, then the premixes were used to infect VeroE6-hACE2 cells for 6 h at 37 °C. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate the gained neutralization (%) by pre-incubation. As control, cells were incubated at each step with an equal volume of PBS or Y112R. Percentages of primary infection were calculated according to viral titers of PBS control conditions. p < 0.0001 (****) unless specified otherwise (2-tailed, unpaired student's t-test). ns, not significant.


( D ) Inhibition of surface binding of SARS - CoV - 2 VLP by FD20 . VLP - GFP were preincubated , or not , with FD20 RBM - targeting mAbs ( CB6 , REGN10933 , REGN10987 ) for 30 min at 37 ° C . Then VLP premixes were incubated with VeroE6 - hACE2 for 1 h at 37 ° C binding detected by flow cytometry . Control binding assays were performed using soluble ACE2 as a competitor . Data are representatives from three experiments .

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(D) Inhibition of cell surface binding of SARS-CoV-2 VLP by FD20. VLP-GFP were preincubated, or not, with FD20 and RBM-targeting mAbs (CB6, REGN10933, REGN10987) for 30 min at 37 °C. Then the VLP premixes were incubated with VeroE6-hACE2 for 1 h at 37 °C and the binding detected by flow cytometry. Control binding assays were performed using soluble ACE2 as a competitor. Data are representatives from three experiments.


( B ) FD20 acts irreversibly on particles in presence or absence of other mAbs . As described in ( A ) , 5 different conditions were used to evaluate effect of SARS - CoV - 2 pp pre - incubation with mAbs . In some experiments , FD20 was mixed with other mAbs synergic effects were observed . Inhibition rate was calculated relatively to infection without antibodies at similar SARS - CoV - 2 pp input ( diluted 20 times or not , Group 2 / 1 in A ) . Mean ± s . d . are plotted ( n = 3 biological replicates ) . p < 0 . 0001 ( * * * * ) unless specified otherwise ( one - way ANOVA with Tukey ' s ) ; ns , not significant . Source data is available online .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FD20 acts irreversibly on particles in the presence or absence of other mAbs. As described in (A), 5 different conditions were used to evaluate the effect of SARS-CoV-2 pp pre-incubation with mAbs. In some experiments, FD20 was mixed with other mAbs and synergic effects were observed. Inhibition rate was calculated relatively to infection without antibodies at the similar SARS-CoV-2 pp input (diluted 20 times or not, Group 2/1 in A). Mean ± s.d. are plotted (n = 3 biological replicates). p < 0.0001 (****) unless specified otherwise (one-way ANOVA with Tukey's test); ns, not significant. Source data is available online.


( C - F ) destruction of SARS - CoV - 2 S by FD20 . ( C ) Negative - stain of FD20 . ( D - F ) Negative - stain image ( i ) 2D class averages ( ii ) of S alone ( D ) , S incubated with FD20 ( E ) or 5E1 ( F ) which is an unrelated mAb targeting a called Hedgehog ( Maun et al , 2010 ) . Bar = 100 nm . In D F , red circles indicate typical side views of S . Results are representative of two independent experiments with two different S purification batches .

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(C-F) The destruction of SARS-CoV-2 S by FD20. (C) Negative-stain of FD20. (D-F) Negative-stain image (i) and 2D class averages (ii) of S alone (D), and S incubated with FD20 (E) or 5E1 (F) which is an unrelated mAb targeting a protein called Hedgehog (Maun et al, 2010). Bar = 100 nm. In D and F, red circles indicate typical side views of S. Results are representative of two independent experiments with two different S purification batches.


Graph represents brain of TKIs over a 24 hr period after IP injection with Imatinib , Nilotinib , Bosutinib DMSO ( N = 7 ) . [ Correction added after publication on 4 July 2013 : y axis of graph shown in Fig . 1A has been corrected from " ( mM ) " to " ( nM ) " ]

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Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib, Nilotinib, Bosutinib and DMSO (N = 7). [Correction added after publication on 4 July 2013: The y axis of the graph shown in Fig. 1A has been corrected from "concentration (mM)" to "concentration (nM)"]


WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) show total Abl ( top blot ) T412 Abl ( 2nd blot ) , soluble parkin level ( 3rd blot ) , LC3 ( 4th blot ) relative to MAP - 2 ( N = 9 ) .

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WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Abl (top blot) T412 Abl (2nd blot), soluble parkin level (3rd blot), and LC3 (4th blot) relative to MAP-2 (N = 9).


Quantitative ELISA showing soluble ( STEN extract ) insoluble ( 4 M urea extract ) mouseparkin in Tg - APP ( N = 9 ) . Parkin − / − brains were used as a specificity control .

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Quantitative ELISA showing soluble (STEN extract) and insoluble (4 M urea extract) mouseparkin in Tg-APP (N = 9). Parkin−/−brains were used as a specificity control.


( D ) Blots represent immunoprecipitatedBeclin - 1 in mice probed with parkin

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR

(D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody


( D ) Blots represent immunoprecipitatedBeclin - 1 in mice probed with parkin antibody , ( E ) control IgG in parallel with immunoprecipitates .

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(D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody, and (E) control IgG in parallel with immunoprecipitates.


( F ) immunoprecipitatedBeclin - 1 probed with parkin reverse experiment in post - mortem human AD cortex analyzed on 4 - 12 % SDS - NuPAGE gel .

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(F) immunoprecipitatedBeclin-1 probed with parkin and the reverse experiment in post-mortem human AD cortex analyzed on 4-12% SDS-NuPAGE gel.


In situ proximity ligation ( PLA ) shows endogenous parkin - Beclin - 1 complexes in ( G ) WT C57BL / 6mice ( N = 5 ) ( H ) parkin − / − as control . PLA in Tg - APPmice IP injected once daily for 3 with ( I ) DMSO ( J ) 5 mg / kg Bosutinib ( K ) 10 mg / kg Nilotinib ( N = 5 ) .

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In situ proximity ligation assay (PLA) shows endogenous parkin-Beclin-1 complexes in (G) WT C57BL/6mice (N = 5) and (H) parkin−/− as control.PLA in Tg-APPmice IP injected once daily for 3 weeks with (I) DMSO (J) 5 mg/kg Bosutinib and (K) 10 mg/kg Nilotinib (N = 5).


PLA in human post - mortem brains in ( L ) cortex of a normal subject ( M ) cortex of an AD ; hippocampus of ( N ) a normal subject ( O ) an AD ; caudate of ( P ) a normal subject ( Q ) an AD .

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PLA in human post-mortem brains in the (L) cortex of a normal subject and (M) cortex of an AD patient; the hippocampus of (N) a normal subject and (O) an AD patient; the caudate of (P) a normal subject and (Q) an AD patient.


Graph represents human Aβ1 - 42ELISA in rat B35 neuroblastoma transfected with human cDNA Aβ1 - 42 ( or LacZ ) or Beclin - 1 shRNA for 24 hr , then treated with 1 μM Bosutinib for an additional 24 hr ( N = 12 ) . * Significantly different to control or as indicated , Mean ± , ANOVA with NeumannKeuls multiple comparison .

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Graph represents human Aβ1-42ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1-42 (or LacZ) or Beclin-1 shRNA for 24 hr, and then treated with 1 μM Bosutinib for an additional 24 hr (N = 12). *Significantly different to control or as indicated, Mean ± SEM, ANOVA with NeumannKeuls multiple comparison.


of 20 μm brain sections shows plaque formation with 6E10 antibody DAB in brain in different ( A , B ) Tg - APP + DMSO ( C ) thioflavin - S in Tg - APP treated with DMSO ( N = 7 ) . ( D , E ) Tg - APP treated with 5 mg / kg Bosutinib for 3 - ( F ) thioflavin - s .

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Staining of 20 μm brain sections shows plaque formation with 6E10 antibody and DAB in the brain in different (A,B) Tg-APP + DMSO and (C) thioflavin-S staining in Tg-APP treated with DMSO (N = 7). (D,E) Tg-APP treated with 5 mg/kg Bosutinib for 3-weeks and (F) thioflavin-s staining.


of 20 μm thick brain sections shows ( G ) parkin , ( H ) Aβ1 - 42 ( I ) merged figure in cortex of Tg - APPmice after 3 of DMSO treatment , ( J ) parkin , ( K ) Aβ1 - 42 ( L ) merged figure in cortex of Tg - APPmice after 3 of 5 mg / kg Bosutinib treatment . ( M ) Parkin , ( N ) Aβ1 - 42 ( O ) merged figure in hippocampus of Tg - APPmice after 3 of DMSO treatment , ( P ) parkin , ( Q ) Aβ1 - 42 ( R ) merged figure in cortex of Tg - APPmice after 3 of Bosutinib treatment ( N = 7 ) .

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Staining of 20 μm thick brain sections shows (G) parkin, (H) Aβ1-42 and (I) merged figure in cortex of Tg-APPmice after 3 weeks of DMSO treatment, and (J) parkin, (K) Aβ1-42 and (L) merged figure in cortex of Tg-APPmice after 3 weeks of 5 mg/kg Bosutinib treatment. (M) Parkin, (N) Aβ1-42 and (O) merged figure in hippocampus of Tg-APPmice after 3 weeks of DMSO treatment, and (P) parkin, (Q) Aβ1-42 and (R) merged figure in cortex of Tg-APPmice after 3 weeks of Bosutinib treatment (N = 7).


( S ) Graphs represent of amyloidplaques in Tg - APP with without Bosutinib .

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(S) Graphs represent quantification of amyloidplaques in Tg-APP with and without Bosutinib.


WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing ( T ) of BACE1 ( 1st blot ) , ADAM - 10 ( 2nd blot ) presinilin - 1 ( 3rd blot ) relative to

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WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) showing (T) levels of BACE1 (1st blot), ADAM-10 (2nd blot) and presinilin-1 (3rd blot) relative to actin


WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing ( U ) total APP ( top blot ) , CTFs ( 2nd blot ) phospho - tyrosine ( 3rd blot ) relative to actin ( N = 9 ) .

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WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) showing (U) total APP (top blot), CTFs (2nd blot) and phospho-tyrosine (3rd blot) relative to actin (N = 9).


Graphs represent ELISA of human Aβ1 - 42 in ( A ) brain ( B ) bloodAβ1 - 42 in 8 - month old Tg - APPmice injected I . P . every other day for 6 with Bosutinib or Nilotinib ( N = 10 ) .

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Graphs represent ELISA of human Aβ1-42 in (A) brain levels and (B) bloodAβ1-42 levels in 8-month old Tg-APPmice injected I.P. every other day for 6 weeks with Bosutinib or Nilotinib (N = 10).


ELISA of human soluble insoluble brain ( C ) Aβ1 - 42 ( D ) Aβ1 - 40 in 8 - month old Tg - APPmice injected IP daily for 3 with 5 mg / kg Bosutinib ( N = 9 ) .

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ELISA of human soluble and insoluble brain (C) Aβ1-42 and (D) Aβ1-40 levels in 8-month old Tg-APPmice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).


ELISA of ( E ) mouse p - Tau in 8 - month old Tg - APP mice injected IP daily for 3 with 5 mg / kg Bosutinib ( N = 9 ) .

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ELISA of (E) mouse p-Tau levels in 8-month old Tg-APP mice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).


WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) show total Tau ( 1st blot ) , serine 396 p - Tau ( 2nd blot ) , threonine 231 ( AT180 , 3rd blot ) serine 199 p - Tau ( AT8 , 4th blot ) relative to actin ( N = 7 ) .

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WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Tau (1st blot), serine 396 p-Tau (2nd blot), threonine 231 (AT180, 3rd blot) and serine 199 p-Tau (AT8, 4th blot) relative to actin (N = 7).


of 20μm brain sections shows intracellular Aβ1 - 42 within ( A ) hippocampus of lentiviralAβ1 - 42 injected WT mice , ( B ) Bosutinib clearance of intracellular Aβ1 - 42 . of 20μm brain sections shows intracellular Aβ1 - 42 within ( C ) cortex of WT mice with lentiviralAβ1 - 42 , ( D ) Bosutinib clearance of intracellular Aβ1 - 42 ( N = 7 ) .

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Staining of 20μm brain sections shows intracellular Aβ1-42 within the (A) hippocampus of lentiviralAβ1-42 injected WT mice, and (B) Bosutinib clearance of intracellular Aβ1-42. Staining of 20μm brain sections shows intracellular Aβ1-42 within the (C) cortex of WT mice with the lentiviralAβ1-42, and (D) Bosutinib clearance of intracellular Aβ1-42 (N = 7).


ELISA of human soluble insoluble Aβ1 - 42 at 6 post - injection of lentiviralAβ1 - 42 daily treatment with 5 mg / kg Bosutinib ( N = 9 ) for 3 . * Significantly different to control or as indicated , Mean ± , ANOVA with Neumann Keuls multiple comparison .

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ELISA of human soluble and insoluble Aβ1-42 at 6 weeks post-injection of lentiviralAβ1-42 and daily treatment with 5 mg/kg Bosutinib (N = 9) for 3 weeks. * Significantly different to control or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.


WB on 4 - 12 % NuPAGE SDS gel of total brain lysates in 1 year old wild type parkin − / − mice treated with 5 mg / kg Bosutinib for 3 on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing parkin ( 1st blot ) total Abl ( 2nd blot ) , T412 Abl ( 3rd blot ) , Beclin - 1 ( 4th blot ) LC3 ( 5th blot ) relative to actin ( N = 7 ) .

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WB on 4-12% NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin−/−mice treated with 5 mg/kg Bosutinib for 3 weeks on 4-12% NuPAGE SDS gel (Invitrogen) showing parkin (1st blot)total Abl (2nd blot), T412 Abl (3rd blot), Beclin-1 (4th blot) and LC3 (5th blot) relative to actin (N = 7).


of 20μm brain sections shows plaque formation with 6E10 antibody DAB 6 post - injection with ( G ) lentiviralAβ1 - 42 + 3 DMSO treatment ( H ) IP injection with 5 mg / kg Bosutinib 3 post - lentiviral expression ( 3 treatment ) clears plaques in WT mice ( N = 7 ) . ( I ) Lentiviral Aβ1 - 42 + DMSO ( J ) IP injection with 5 mg / kg Bosutinib 3 post - lentiviral expression ( 3 treatment ) in parkin − / − mice ( N = 7 ) .

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Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post-injection with (G) lentiviralAβ1-42 + 3 weeks DMSO treatment and (H) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) clears plaques in WT mice (N = 7).(I) Lentiviral Aβ1-42 + DMSO and (J) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) in parkin−/−mice (N = 7).


Graphs represent ELISA ( N = 5 ) in autophagic vacuoles ( AVs ) in brain of 4 8 months old Tg - APPmice treated with 10 mg / kg Nilotinib or 5 mg / kg Bosutinib ( N = 5 ) for 3 showing ( A ) humanAβ1 - 42 ( Insert is WB of AVs showing LC3 - B in AV10 AV20 ( 1st blot ) LAMP2a in Lys ( 2nd blot ) fraction ) ( B ) Aβ1 - 40 , ( C ) p - Tau ( D ) parkin .

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Graphs represent ELISA (N = 5) in autophagic vacuoles (AVs) in the brain of 4 and 8 months old Tg-APPmice treated with 10 mg/kg Nilotinib or 5 mg/kg Bosutinib (N = 5) for 3 weeks showing (A)humanAβ1-42 (Insert is WB analysis of AVs showing LC3-B in AV10 and AV20 (1st blot) and LAMP2a in Lys (2nd blot) fraction) and (B)Aβ1-40,(C) p-Tau and (D) parkin.


ELISA in autophagic vacuoles of 1 year old WT parkin − / − mice ( N = 5 ) injected with lentiviralAβ1 - 42 for 3 treated with 10 mg / kg Nilotinib 5 mg / kg Bosutinib for 3 additional showing ( E ) humanAβ1 - 42 ( F ) p - Tau .

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ELISA in autophagic vacuoles of 1 year old WT and parkin−/−mice (N = 5) injected with lentiviralAβ1-42 for 3 weeks and treated with 10 mg/kg Nilotinib and 5 mg/kg Bosutinib for 3 additional weeks showing (E) humanAβ1-42 and (F) p-Tau levels.


( A ) Represents results of Morris water maze in lentiviralAβ1 - 42 - injected ± Bosutinib WT ( N = 12 ) parkin − / − ( N = 10 ) mice , ( B ) heat maps for each group . Graphs represent total number of entry into platform travelled .

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(A) Represents results of Morris water maze test in lentiviralAβ1-42-injected ± Bosutinib WT (N = 12) and parkin−/− (N = 10) mice, and (B) heat maps for each group.Graphs represent total number of entry into platform area and distance travelled.


Represents results of Morris water maze in Tg - APP ± Bosutinib ( N = 12 ) mice . Graphs represent total number of entry into platform travelled .

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Represents results of Morris water maze test in Tg-APP ± Bosutinib (N = 12) mice.Graphs represent total number of entry into platform area and distance travelled.


Histopathological ( A - D ) virological ( E ) examinations of lung tissues from an ill Malayan pangolin naturally infected with Pangolin - CoV ( A B , 200 × 400 × , respectively ) an uninfected Malayan pangolin ( C D , 200 × 400 × , respectively ) . Interstitial pneumonia with extensive infiltration of inflammatory is seen in lung tissue from infected pangolin . Viral particles are seen in double - vesicles in transmission electron microscopy image ( small scale = 50 nm ) taken from Verno E6 culture inoculated with supernatant of homogenized lung ( E ) , with indicative of coronavirus ( inserts at upper right corner of E ) .

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Histopathological (A-D) and virological (E) examinations of the lung tissues from an ill Malayan pangolin naturally infected with Pangolin-CoV (A and B, 200× and 400×, respectively) and an uninfected Malayan pangolin (C and D, 200× and 400×, respectively). Interstitial pneumonia with extensive infiltration of inflammatory cells is seen in the lung tissue from the infected pangolin. Viral particles are seen in double-membrane vesicles in the transmission electron microscopy image (small scale = 50 nm) taken from the Verno E6 cell culture inoculated with supernatant of homogenized lung (E), with morphology indicative of coronavirus (inserts at the upper right corner of E).


Similarity plot of near full - genome ( A ) S gene ( B ) of Pangolin - CoV to sequences of 2019 - nCoV _ WIV02 , Bat - CoV RaTG13 , Bat - CoV ZC45 Bat - CoV ZXC21 . While Pangolin - CoV has a high identity to 2019 - nCoV Bat - CoV - RaTG13 in most regions of S gene , it is more similar to Bat SARSr - CoV ZXC21 Bat SARSr - CoV ZC45 at 5 ′ end ( B ) . More importantly , Pangolin - CoV is highly similar to 2019 - nCoV in functional RBD region of S , with only one difference ( insert in B ) . Because of presence of genetic recombination , there is a discrepancy in cluster formation among outcomes of phylogenetic analyses of different regions of S gene ( C ) .

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Similarity plot of the near full-length genome sequence (A) and S gene sequence (B) of Pangolin-CoV to sequences of 2019-nCoV_WIV02, Bat-CoV RaTG13, Bat-CoV ZC45 and Bat-CoV ZXC21. While Pangolin-CoV has a high sequence identity to 2019-nCoV and Bat-CoV-RaTG13 in most regions of the S gene, it is more similar to Bat SARSr-CoV ZXC21 and Bat SARSr-CoV ZC45 at the 5′ end (B). More importantly, Pangolin-CoV is highly similar to 2019-nCoV in the functional RBD region of the S protein, with only one amino difference (insert in B). Because of the presence of genetic recombination, there is a discrepancy in cluster formation among the outcomes of phylogenetic analyses of different regions of the S gene (C).


Free energy ( kcal / mol ) for binding between S of four SARSr - CoV ACE2 of four likely hosts .

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Free energy (kcal/mol) for the binding between S proteins of four SARSr-CoV viruses and ACE2 of four likely hosts.


Human peripheral blood mononuclear ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T stimulation was performed through CD3 / CD28 Dynabeads at a bead : of 1 : 8 . Human pan T was isolated culture supernatant was sampled . A mRNA expression of CD4 + cytokines Il2 , IL4 , IL8 IL22 relative to endogenous controls , n = 13 / 11 / 10 / 8 biological replicates . Data information : Data depicted as mean ± ( data ) box plots with median , twenty - fifth seventy - fifth percentiles range ( other ) . Dots indicating values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - / Wilcoxon matched - pairs signed rank , as appropriate .

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Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. A mRNA expression of CD4+ cytokines Il2, IL4, IL8 and IL22 relative to endogenous controls, n=13/11/10/8 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human peripheral blood mononuclear ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T stimulation was performed through CD3 / CD28 Dynabeads at a bead : of 1 : 8 . Human pan T was isolated culture supernatant was sampled . D mRNA expression of CD8 + cytokines IFNγ , PRF1 , TNFα , GZMB CTLA4 in stimulated human T relative to internal controls , n = 15 / 14 / 10 / 14 / 9 biological replicates . Data information : Data depicted as mean ± ( data ) box plots with median , twenty - fifth seventy - fifth percentiles range ( other ) . Dots indicating values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - / Wilcoxon matched - pairs signed rank , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. D mRNA expression of CD8+ cytokines IFNγ, PRF1, TNFα, GZMB and CTLA4 in stimulated human T cells relative to internal controls, n=15/14/10/14/9 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human peripheral blood mononuclear ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T stimulation was performed through CD3 / CD28 Dynabeads at a bead : of 1 : 8 . Human pan T was isolated culture supernatant was sampled . F Foxp3 mRNA relative to internal control in human PBMCs of CD4 + CD25 + Foxp3 + regulatory T ( Treg ) following 5 days of Treg differentiation with a representative Foxp3 histogram plot ( right side ) , n = 7 / 11 biological replicates . Data information : Data depicted as mean ± ( data ) box plots with median , twenty - fifth seventy - fifth percentiles range ( other ) . Dots indicating values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - / Wilcoxon matched - pairs signed rank , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. F Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg) following 5 days of Treg differentiation with a representative Foxp3 histogram plot (right side), n=7/11 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human peripheral blood mononuclear ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T stimulation was performed through CD3 / CD28 Dynabeads at a bead : of 1 : 8 . Human pan T was isolated culture supernatant was sampled . G IL10 TGFβ1 mRNA expression of human Treg , n = 8 / 9 ( IL10 ) , 5 / 9 ( TGFβ1 ) biological replicates . Data information : Data depicted as mean ± ( data ) box plots with median , twenty - fifth seventy - fifth percentiles range ( other ) . Dots indicating values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - / Wilcoxon matched - pairs signed rank , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. G IL10 and TGFβ1 mRNA and protein expression of human Treg cells, n=8/9 (IL10), 5/9 (TGFβ1) biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.