( C ) 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB / L inhibitor E64d or PBS as control and inoculated with pseudotypes bearing the indicated viral surface proteins . The average of three independent experiments is shown in panels A - C . Error bars indicate SEM . Statistical significance was tested by two - way ANOVA with Dunnett posttest .

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(C) 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB/L inhibitor E64d or PBS as control and inoculated with pseudotypes bearing the indicated viral surface proteins. The average of three independent experiments is shown in panels A-C. Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest.


entry . Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of serum from a convalescent SARS patient and subsequently inoculated onto 293T cells that transiently express ACE2 in order to evaluate cross - neutralization . The results from a representative experiment with triplicate samples are shown and were confirmed in a separate experiment . Error bars indicate SD . Statistical significance was tested by two - way ANOVA with Dunnett posttest .

O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

entry.Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of serum from a convalescent SARS patient and subsequently inoculated onto 293T cells that transiently express ACE2 in order to evaluate cross-neutralization. The results from a representative experiment with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate SD. Statistical significance was tested by two-way ANOVA with Dunnett posttest.


( B ) Structure of FRH from the large crystallographic cell with an accordingly colored domain map below .

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(B) Structure of FRH from the large crystallographic cell with an accordingly colored domain map below.


( A ) FRH large cell ( cyan ) , Mtr4 ( pdb 2XGJ , green , RNA backbone in orange ) , and FRH small alternate cell ( blue ) differ primarily in the orientation of the arch domain . The FRH RNA - bound structure also crystallizes in the small - cell configuration . The two different FRH structures have an overall RMSD of 3 . 9 Å and the RMSD between Mtr4 and FRH is 2 . 9 Å ( over 734 residues , for large cell FRH ) .

O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O

(A) FRH large cell (cyan), Mtr4 (pdb 2XGJ, green, RNA backbone in orange), and FRH small alternate cell (blue) differ primarily in the orientation of the arch domain. The FRH RNA-bound structure also crystallizes in the small-cell configuration. The two different FRH structures have an overall RMSD of 3.9 Å and the RMSD between Mtr4 and FRH is 2.9 Å (over 734 residues, for large cell FRH).


( B ) SAXS data indicates that FRH - Δ114 is monomeric and flexible . The crystal structure of the large cell structure predicts the solution scattering curve ( left , χ2 = 0 . 92 ) and fits well to the calculated molecular envelope ( right ) .

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(B) SAXS data indicates that FRH-Δ114 is monomeric and flexible. The crystal structure of the large cell structure predicts the solution scattering curve (left, χ2 = 0.92) and fits well to the calculated molecular envelope (right).


( A ) Comparison of the FRH N - terminus ( large cell structure , magenta ) to that of Mtr4 ( green ) aligned on their RecA domains . Only the additional elements of FRH are shown ( cyan ) . The FRH N - terminal extension has little ordered secondary structure , although regions 143 - 147 and 129 - 135 form small turns that interact with the helicase core . ( B ) The very N - terminus of FRH extends away from the RecA - 2 domain to interact with a symmetry related molecule in the crystal ( below ) , whereas the Mtr4 N - terminus turns back to form an outer β − strand . Center panel shows the crystal contacts , with close - ups for each region highlighted as insets on the left and right .

O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD 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-GENEPROD O O O O O O O O O O O O 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) Comparison of the FRH N-terminus (large cell structure, magenta) to that of Mtr4 (green) aligned on their RecA domains. Only the additional elements of FRH are shown (cyan). The FRH N-terminal extension has little ordered secondary structure, although regions 143-147 and 129-135 form small turns that interact with the helicase core. (B) The very N-terminus of FRH extends away from the RecA-2 domain to interact with a symmetry related molecule in the crystal (below), whereas the Mtr4 N-terminus turns back to form an outer β−strand. Center panel shows the crystal contacts, with close-ups for each region highlighted as insets on the left and right.


( A ) The two RecA domains are rotated relative to one another in FRH ( cyan , large cell ) compared to Mtr4 ( green , RNA backbone in orange ) . The change in positioning of RecA - 2 with respect to RecA - 1 results in an overall displacement from the Mtr4 domain juxtaposition that ranges from 1 . 1 - 4 . 0 Å throughout RecA - 2 with the largest differences at the external helices .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O 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-GENEPROD O O 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) The two RecA domains are rotated relative to one another in FRH (cyan, large cell) compared to Mtr4 (green, RNA backbone in orange). The change in positioning of RecA-2 with respect to RecA-1 results in an overall displacement from the Mtr4 domain juxtaposition that ranges from 1.1 - 4.0 Å throughout RecA-2 with the largest differences at the external helices.


( B ) Comparison of arch domains conformations from Mtr4 ( green ) and FRH ( large cell in cyan , small cell in marine ) after superposition of the most N - terminal helical region . The isolated KOW modules of FRH , Mtr4 ( with FRH superimposed ) , and Ski2 have considerably different loop configurations .

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(B) Comparison of arch domains conformations from Mtr4 (green) and FRH (large cell in cyan, small cell in marine) after superposition of the most N-terminal helical region. The isolated KOW modules of FRH, Mtr4 (with FRH superimposed), and Ski2 have considerably different loop configurations.


( A ) 3 . 8 Å resolution mFo - DFc omit map density ( green , calculated with sharpened B - factors and contoured at 2 . 0  ) reveals the presence of ADP and a short segment of RNA bound to FRH .

O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O

(A) 3.8 Å resolution mFo-DFc omit map density (green, calculated with sharpened B-factors and contoured at 2.0) reveals the presence of ADP and a short segment of RNA bound to FRH.


( B ) 3 . 8 Å resolution 2mFo - DFc calculated without ADP or RNA contributing to the model . Green electron density shown for ADP and RNA ( 0 . 6     orange density shown for surrounding protein ( 0 . 6   .

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(B) 3.8 Å resolution 2mFo-DFc calculated without ADP or RNA contributing to the model. Green electron density shown for ADP and RNA (0.6orange density shown for surrounding protein (0.6.


( C ) Comparison of Mtr4 and FRHADP binding ( FRH in cyan with ADP , RNA , and F179 in orange bonds ; Mtr4 in green with ADP , RNA , and F148 in yellow bonds ) . ( c ) Unwound DNA from Hel308 ( pdb 2PR6 ; magenta , DNA in orange bonds ) overlaid in FRH with RNA and ADP ( FRH in cyan with yellow RNA and cyan ADP in stick ) after superposition of the respective RecA domains .

O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O

(C) Comparison of Mtr4 and FRHADP binding (FRH in cyan with ADP, RNA, and F179 in orange bonds; Mtr4 in green with ADP, RNA, and F148 in yellow bonds). (c) Unwound DNA from Hel308 (pdb 2PR6; magenta, DNA in orange bonds) overlaid in FRH with RNA and ADP (FRH in cyan with yellow RNA and cyan ADP in stick) after superposition of the respective RecA domains.


( d ) Hel308 DNA helicase DNA ( pdb 2PR6 ; backbone in orange , bases in gray bonds ) after aligning the RecA domains of Hel308 with those of FRH ( cyan , RNA in yellow ) ; see also Appendix Fig S4 . The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains .

O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Hel308 DNA helicase DNA (pdb 2PR6; backbone in orange, bases in gray bonds) after aligning the RecA domains of Hel308 with those of FRH (cyan, RNA in yellow); see also Appendix Fig S4. The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains.


( A ) Key residues for FRH function shown on the large - cell structure : K208 and S324 compose the ATP binding site , E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis ; P871 resides in the elbow region of the arch domain . K811 , R806 , R712 and K766 contribute to a positively charged surface on the KOW domain . R806H disrupts the clock . Q131 , G132 and V142 in the N - terminus provide interactions to the helicase RecA domains ( FRH domains are colored according to Fig1 , ADP and RNA are in orange bonds ) . ( B ) Same as ( A ) after rotation about a horizontal axis .

O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

(A) Key residues for FRH function shown on the large-cell structure: K208 and S324 compose the ATP binding site, E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis; P871 resides in the elbow region of the arch domain. K811, R806, R712 and K766 contribute to a positively charged surface on the KOW domain. R806H disrupts the clock. Q131, G132 and V142 in the N-terminus provide interactions to the helicase RecA domains (FRH domains are colored according to Fig1, ADP and RNA are in orange bonds). (B) Same as (A) after rotation about a horizontal axis.


( C ) Close - up of the ADP - binding pocket and N - terminal interaction for the large - cell structure ( cyan ribbons ) and the small - cell structure ( blue ribbons ) .

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(C) Close-up of the ADP-binding pocket and N-terminal interaction for the large-cell structure (cyan ribbons) and the small-cell structure (blue ribbons).


( D ) Electrostatic potential surface of FRH ( blue positive , red negative ) shown with the same orientation as in ( B ) .

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(D) Electrostatic potential surface of FRH (blue positive, red negative) shown with the same orientation as in (B).


( E ) Close - up of the KOW domain showing the large number of basic ( aqua sticks ) and acidic side chains ( yellow sticks ) on the surface that also includes the mutational sites R806 , R712 , K811 and K766 ( grey sticks )

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

(E) Close-up of the KOW domain showing the large number of basic (aqua sticks) and acidic side chains (yellow sticks) on the surface that also includes the mutational sites R806, R712, K811 and K766 (grey sticks)


( A ) Race tube assays with FRH mutants . Every circadian cycle N . crassa lays down aerial hyphae that appear as fluffy yellow / orange conidial bands . The distance between the bands reflects the clock period . The strains were grown in constant darkness and growth fronts were labeled every 24 h ( black vertical lines ) , rhythmicity on right ( τ = period in hours , σ = standard deviation , n = number of race tubes , ARR , arrhythmic ) . The race tubes shown here are representative samples from three replicate tubes .

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(A) Race tube assays with FRH mutants. Every circadian cycle N. crassa lays down aerial hyphae that appear as fluffy yellow/orange conidial bands. The distance between the bands reflects the clock period. The strains were grown in constant darkness and growth fronts were labeled every 24 h (black vertical lines), rhythmicity on right (τ= period in hours, σ = standard deviation, n = number of race tubes, ARR, arrhythmic). The race tubes shown here are representative samples from three replicate tubes.


( B ) Pull - down assays evaluating interactions among FRH , FRQ , WC - 1 and WC - 2 . Ratio of FRQ , WC - 1 and WC - 2 binding to the V5 tagged FRH mutants as compared to the inter - strain variations in FRH levels , error bars represent standard deviation for the assay run in triplicate . * P < 0 . 05 ( unpaired two - tailed t - test with Gaussian distribution assumption ) . Representative immunoblots are shown in Appendix Fig S5 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O

(B) Pull-down assays evaluating interactions among FRH, FRQ, WC-1 and WC-2. Ratio of FRQ, WC-1 and WC-2 binding to the V5 tagged FRH mutants as compared to the inter-strain variations in FRH levels, error bars represent standard deviation for the assay run in triplicate. *P < 0.05 (unpaired two-tailed t-test with Gaussian distribution assumption). Representative immunoblots are shown in Appendix Fig S5.


( a ) Relative quantification ( RQ ) of CG7630 transcript after RNAi with a 120 - bp inverted - repeat sequence ( act5c - gal4 > UAS - CG7630RNAi ) compared to control ( act5c - gal4 > + ) .

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(a) Relative quantification (RQ) of CG7630 transcript after RNAi with a 120-bp inverted-repeat sequence (act5c-gal4>UAS-CG7630RNAi) compared to control (act5c-gal4>+).


( b ) Relative percentage of egg to adult viability of CG7630 RNAi cross ( act5c - gal4 / CyO . GFP x UAS - CG7630RNAi ) and control cross ( act5c - gal4 / CyO . GFP x w1118 ) , calculated at three developmental stages ( eggs , pupae and adults ) , ( n > 250 , Chi - square test 9 . 920 df ( 2 ) , * * p = 0 . 007 ) .

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(b) Relative percentage of egg to adult viability of CG7630 RNAi cross (act5c-gal4/CyO.GFP x UAS-CG7630RNAi) and control cross (act5c-gal4/CyO.GFP x w1118), calculated at three developmental stages (eggs, pupae and adults), (n > 250, Chi-square test 9.920 df(2), **p = 0.007).


( d ) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase ( CS ) activity .

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(d) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase (CS) activity.


( e ) In gel - activity assays for MRC complexes II and IV in DDM - solubilized mitochondria from RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control larvae ( act5c - gal4 > + ) .

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(e) In gel-activity assays for MRC complexes II and IV in DDM-solubilized mitochondria from RNAi (act5c-gal4>UAS-CG7630RNAi) and control larvae (act5c-gal4>+).


( f ) BN - PAGE , Western blot immunodetection of MRC complexes from CG7630 RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control ( act5c - gal4 > + ) larvae using antibodies against specific subunits : anti - UQCRC2 ( complex III ) , anti - NDUFS3 ( complex I ) , anti - COX4 ( complex IV ) and anti - SDHA ( complex II ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O

(f) BN-PAGE, Western blot immunodetection of MRC complexes from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II).


( g ) Western blot analysis of steady state levels ( SDS - PAGE ) of MRC subunits COX4 ( complex IV ) , SDHA ( complex II ) and UQCR - C2 ( complex III ) in whole lysates from CG7630 RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control ( act5c - gal4 > + ) larvae .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O

(g) Western blot analysis of steady state levels (SDS-PAGE) of MRC subunits COX4 (complex IV), SDHA (complex II) and UQCR-C2 (complex III) in whole lysates from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae.


( h ) Western blot analysis of anti - HA affinity purified samples from S2R + D . melanogaster cells . EV = samples from cells carrying the empty expression vector . CG7630 - HA = samples from cells expressing HA - tagged CG7630 . Tot = total mitochondrial - enriched fractions ; unb = unbound material ; IP : HA = immunoprecipitated material . Samples were probed with antibodies against MRC subunits of complexes I ( NDUFS3 ) , II ( SDHA ) , III ( UQCR - C2 ) , IV ( COX4 ) and V ( ATP5A ) .

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(h) Western blot analysis of anti-HA affinity purified samples from S2R+ D. melanogaster cells. EV = samples from cells carrying the empty expression vector. CG7630-HA = samples from cells expressing HA-tagged CG7630. Tot = total mitochondrial-enriched fractions; unb = unbound material; IP:HA = immunoprecipitated material. Samples were probed with antibodies against MRC subunits of complexes I (NDUFS3), II (SDHA), III (UQCR-C2), IV (COX4) and V (ATP5A).


( a ) Relative quantification ( RQ ) of CG7630 transcript in wild - type D . melanogaster S2R + cells treated with a dsRNA targeting CG7630 ( CG7630 KD ) and a mock control dsRNA ( mock ) .

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(a) Relative quantification (RQ) of CG7630 transcript in wild-type D. melanogaster S2R+ cells treated with a dsRNA targeting CG7630 (CG7630 KD) and a mock control dsRNA (mock).


( b ) Relative quantification ( RQ ) of CG7630 and COX7B transcripts in stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . Cells were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) and a mock control dsRNA ( solid bars , mock ) . N / D = not detected .

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(b) Relative quantification (RQ) of CG7630 and COX7B transcripts in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock). N/D = not detected.


( c ) Immunoblot analysis of stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . The CG7630 transcript was knocked - down ( KD ) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA ( mock ) . Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control .

O O O B-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-ORGANISM B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O

(c) Immunoblot analysis of stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). The CG7630 transcript was knocked-down (KD) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA (mock). Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control.


( d ) Kinetic enzyme activity of COX normalized by citrate synthase activity ( CS ) in stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . Cells were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) and a mock control dsRNA ( solid bars , mock ) .

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(d) Kinetic enzyme activity of COX normalized by citrate synthase activity (CS) in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock).


( e ) Steady - state levels of MRC subunits in samples from rescue experiment performed as in ( d ) .

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(e) Steady-state levels of MRC subunits in samples from rescue experiment performed as in (d).


A - B Representative image of a proliferative cell in the DG labeled with an anti - PH3 antibody ( green ) ( A ) and quantification of the number of PH3 + cells in the DG of WT and Tau - / - mice ( B ) ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - test ) .

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A-B Representative image of a proliferative cell in the DG labeled with an anti-PH3 antibody (green) (A) and quantification of the number of PH3+ cells in the DG of WT and Tau-/- mice (B) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).


C - D Representative image of an apoptotic cell in the DG labeled with an anti - fractin antibody ( red ) ( C ) and quantification of the number of fractin + cells in the DG of WT and Tau - / - mice ( D ) ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - test ) .

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C-D Representative image of an apoptotic cell in the DG labeled with an anti-fractin antibody (red) (C) and quantification of the number of fractin+ cells in the DG of WT and Tau-/- mice (D) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).


E - F Schematic diagram of the cell survival experimental design using thymidine analogs ( F ) and quantification of the percentage of surviving cells in Tau - / - mice as compared to WT mice at each cell age ( 1 - , 2 - , 4 - , 6 - and 8 - week - old cells ) ( F ) ( mean ± SEM ( normalized data ) ; Student ´ s t - test ) .

O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-F Schematic diagram of the cell survival experimental design using thymidine analogs (F) and quantification of the percentage of surviving cells in Tau-/- mice as compared to WT mice at each cell age (1-, 2-, 4-, 6- and 8-week-old cells) (F) (mean ± SEM (normalized data); Student´s t-test).


H Quantification of the percentage of 1 - , 4 - and 8 - week - old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau - / - mice ( χ2 test ) .

O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O

H Quantification of the percentage of 1-, 4- and 8-week-old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau-/- mice (χ2 test).


I - J Representative images of progenitor cells in the DG labeled with anti - Sox2 ( red ) and anti - BLBP ( green ) antibodies ( I ) , and neuroblasts and immature neurons labeled with anti - DCX ( red ) and anti - calretinin ( green ) antibodies , respectively ( J ) . K - N Quantification of the number of Sox2 + ( K ) , BLBP + ( L ) , DCX + ( M ) and calretinin + ( N ) cells in the DG of WT and Tau - / - mice ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - test ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O

I-J Representative images of progenitor cells in the DG labeled with anti-Sox2 (red) and anti-BLBP (green) antibodies (I), and neuroblasts and immature neurons labeled with anti-DCX (red) and anti-calretinin (green) antibodies, respectively (J).K-N Quantification of the number of Sox2+ (K), BLBP+ (L), DCX+ (M) and calretinin+ (N) cells in the DG of WT and Tau-/- mice (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).


A Representative images of 4 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus . A schematic representation of Sholl ´ s analysis is shown in the WT image . B - C Quantification of total dendritic length ( B ) and Sholl ´ s analysis ( C ) of 4 - week - old newborn granule neurons in WT and Tau - / - mice .

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A Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus. A schematic representation of Sholl´s analysis is shown in the WT image.B-C Quantification of total dendritic length (B) and Sholl´s analysis (C) of 4-week-old newborn granule neurons in WT and Tau-/- mice.


D Representative images of 8 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus . E - F Quantification of total dendritic length ( E ) and Sholl ´ s analysis ( F ) of 8 - week - old newborn granule neurons in WT and Tau - / - mice .

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D Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus.E-F Quantification of total dendritic length (E) and Sholl´s analysis (F) of 8-week-old newborn granule neurons in WT and Tau-/- mice.


G Representative images of 4 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus and their corresponding high - power magnifications showing PSDs ( green ) . H - IQuantification of the number of PSDs / µm ( H ) and PSD area ( I ) in each dendritic branching order of 4 - week - old newborn granule neurons in WT and Tau - / - mice .

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G Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).H-IQuantification of the number of PSDs/µm (H) and PSD area (I) in each dendritic branching order of 4-week-old newborn granule neurons in WT and Tau-/- mice.


J Representative images of 8 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus and their corresponding high - power magnifications showing PSDs ( green ) . K - L Quantification of the number of PSDs / µm ( K ) and PSD area ( L ) in each dendritic branching order of 8 - week - old newborn granule neurons in WT and Tau - / - mice .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O B-ORGANISM O

J Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).K-L Quantification of the number of PSDs/µm (K) and PSD area (L) in each dendritic branching order of 8-week-old newborn granule neurons in WT and Tau-/- mice.


A Representative traces of mEPSCs recorded at a holding potential of − 65 mV from WT ( black traces ) and Tau - / - ( red traces ) mice .

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A Representative traces of mEPSCs recorded at a holding potential of −65 mV from WT (black traces) and Tau-/- (red traces) mice.


B Exemplary traces of mEPSCs from one cell from WT ( black trace ) and Tau - / - ( red trace ) mice .

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B Exemplary traces of mEPSCs from one cell from WT (black trace) and Tau-/- (red trace) mice.


C Resting membrane potential of WT and Tau - / - mice ( mean ± SEM ; n = 13 cells WT , n = 10 cells Tau - / - ; * 0 . 05 > p ≥ 0 . 01 , Student ´ s t - test ) .

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C Resting membrane potential of WT and Tau-/- mice (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; *0.05 > p ≥ 0.01, Student´s t-test).


D - E mEPSC amplitude ( D ) and mEPSC frequency ( E ) of WT and Tau - / - animals ( mean ± SEM ; n = 13 cells WT , n = 10 cells Tau - / - ; Student ´ s t test or Mann - Whitney ´ s U test ) .

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D-E mEPSC amplitude (D) and mEPSC frequency (E) of WT and Tau-/- animals (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; Student´s t test or Mann-Whitney´s U test).


F Cumulative probability plot of the total mEPSC amplitudes , small mEPSCs ( i ) and big mEPSCs ( ii ) from both WT and Tau - / - mice ( black and red traces , respectively ) ( * * * p < 0 . 001 ; Kolmogorov - Smirnov Z test ) .

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F Cumulative probability plot of the total mEPSC amplitudes, small mEPSCs (i) and big mEPSCs (ii) from both WT and Tau-/- mice (black and red traces, respectively) (***p < 0.001; Kolmogorov-Smirnov Z test).


A Representative image of a mouse subjected to forced swimming in the Porsolt test .

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A Representative image of a mouse subjected to forced swimming in the Porsolt test.


C Representative images of 1 - week - old newborn granule neurons labeled with CldU ( green ) belonging to WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice . Cell nuclei were labeled with DAPI ( blue ) . Scale bar 50 µm . D - E Quantification of the number of 1 - week - old CldU + cells ( D ) and apoptotic fractin + cells ( E ) in each experimental group .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-ORGANISM O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O

C Representative images of 1-week-old newborn granule neurons labeled with CldU (green) belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice. Cell nuclei were labeled with DAPI (blue). Scale bar 50 µm.D-E Quantification of the number of 1-week-old CldU+ cells (D) and apoptotic fractin+ cells (E) in each experimental group.


B Representative images of 8 - week - old newborn granule neurons of WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice infected with a PSD95 - GFP - expressing retrovirus . In the WT CNP image a schematic representation of Sholl ´ s analysis is shown . C - D Quantification of total dendritic length ( C ) and Sholl ´ s analysis ( D ) of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O O

B Representative images of 8-week-old newborn granule neurons of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice infected with a PSD95-GFP-expressing retrovirus. In the WT CNP image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).


E - F Representative images of an 8 - week - old newborn granule neuron , in which proximal dendrites are highlighted ( E ) , and of PSDs ( green ) in proximal dendrites belonging to WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice ( F ) . G - H Quantification of the number of PSDs / µm ( G ) and PSD area ( H ) in each dendritic branching order of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) .

O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O O

E-F Representative images of an 8-week-old newborn granule neuron, in which proximal dendrites are highlighted (E), and of PSDs (green) in proximal dendrites belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).


I - J Representative Western Blot ( I ) and quantification ( J ) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau - / - mice subjected or not to the Porsolt test ( n = 6 mice per experimental condition ) .

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I-J Representative Western Blot (I) and quantification (J) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau-/- mice subjected or not to the Porsolt test (n= 6 mice per experimental condition).


K Representative image showing 8 - week - old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field ( indicated by a white square ) .

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K Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).


L - M Representative images ( L ) and quantification of the area ( M ) of MFTs of 8 - week - old newborn neurons in the CA3 region of WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice ( n = 3 mice per experimental condition ) . Arrows indicate MFTs .

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L-M Representative images (L) and quantification of the area (M) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (n= 3 mice per experimental condition). Arrows indicate MFTs.


A Representative images of control housing ( CH ) and environmental enrichment ( EE ) cages . B Schematic diagram of the experimental design .

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A Representative images of control housing (CH) and environmental enrichment (EE) cages.B Schematic diagram of the experimental design.


C - D Representative images of DCX + cells in the DG of CH and EE animals ( C ) , and quantification of the number of DCX + cells of WT and Tau - / - animals in CH or EE conditions ( D ) .

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C-D Representative images of DCX+ cells in the DG of CH and EE animals (C), and quantification of the number of DCX+ cells of WT and Tau-/- animals in CH or EE conditions (D).


E Representative images of 8 - week - old newborn granule neurons labeled with IdU ( red ) belonging to WT CH , WT EE , Tau - / - CH and Tau - / - EE mice . F - G Quantification of the number of 8 - week - old IdU + cells ( F ) and apoptotic fractin + cells ( G ) .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O

E Representative images of 8-week-old newborn granule neurons labeled with IdU (red) belonging to WT CH, WT EE, Tau-/- CH and Tau-/- EE mice.F-G Quantification of the number of 8-week-old IdU+ cells (F) and apoptotic fractin+ cells (G).


A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD - 65 antibody ( red ) , and high - power magnifications of the DG molecular layer showing GAD - 65 + terminals in WT CH , Tau - / - CH , WT EE and Tau - / - EE mice . Cell nuclei were labeled with DAPI ( blue ) . White scale bar 300 µm . Red scale bar 50 µm . Green scale bar 5 µm . B - C Quantification of the density ( B ) and area ( C ) of GAD - 65 + terminals in the molecular layer of mice in each experimental condition ( n = 10 mice WT CH , n = 8 miceTau - / - CH , n = 6 mice WT EE , n = 10 miceTau - / - EE ) .

O O O O O O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-TISSUE I-TISSUE O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O

A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD-65 antibody (red), and high-power magnifications of the DG molecular layer showing GAD-65+ terminals in WT CH, Tau-/- CH, WT EE and Tau-/- EE mice. Cell nuclei were labeled with DAPI (blue). White scale bar 300 µm. Red scale bar 50 µm. Green scale bar 5 µm.B-C Quantification of the density (B) and area (C) of GAD-65+ terminals in the molecular layer of mice in each experimental condition (n= 10 mice WT CH, n= 8 miceTau-/- CH, n= 6 mice WT EE, n= 10 miceTau-/- EE).


B Representative images of 8 - week - old newborn granule neurons of WT CH , WT EE , Tau - / - CH , and Tau - / - EE mice infected with a PSD95 - GFP - expressing retrovirus . In the WT CH image a schematic representation of Sholl ´ s analysis is shown . C - D Quantification of total dendritic length ( C ) and Sholl ´ s analysis ( D ) of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) .

B Representative images of 8-week-old newborn granule neurons of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice infected with a PSD95-GFP-expressing retrovirus. In the WT CH image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).


E - F Representative images of an 8 - week - old newborn granule neuron in which proximal and distal dendrites are highlighted ( E ) and of PSDs ( green ) in distal and proximal dendrites of WT and Tau - / - mice housed under either CH or EE conditions ( F ) . G - H Quantification of the number of PSDs / µm ( G ) and PSD area ( H ) in each dendritic branching order of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) .

O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O O

E-F Representative images of an 8-week-old newborn granule neuron in which proximal and distal dendrites are highlighted (E) and of PSDs (green) in distal and proximal dendrites of WT and Tau-/- mice housed under either CH or EE conditions (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).


I Representative image showing 8 - week - old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field ( indicated by a white square ) .

I Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).


J - K Representative images ( J ) and quantification of the area ( K ) of MFTs of 8 - week - old newborn neurons in the CA3 region of WT CH , WT EE , Tau - / - CH , and Tau - / - EE mice ( n = 3 mice per experimental condition ) . Arrows indicate MFTs .

O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O B-SUBCELLULAR O

J-K Representative images (J) and quantification of the area (K) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice (n= 3 mice per experimental condition). Arrows indicate MFTs.


Relative quantification of mRNA for Ppcs , Ppcdc , and Coasy by genotype from each of the three study regions . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively )

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Relative quantification of mRNA for Ppcs, Ppcdc, and Coasy by genotype from each of the three study regions. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively)


Western blot and quantification for Coasy from GP by genotype and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . Ponceau S was used as a loading control . n = 8 , 4 for WT and n = 8 , 6 for KO ( vehicle and pPanSH groups , respectively ) .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-TISSUE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O 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-SMALL_MOLECULE O O O O O

Western blot and quantification for Coasy from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as a loading control. n=8, 4 for WT and n=8, 6 for KO (vehicle and pPanSH groups, respectively).


Relative mRNA expression of Tfrc , Ireb2 , and Hamp by genotype and brain region . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively )

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O

Relative mRNA expression of Tfrc, Ireb2, and Hamp by genotype and brain region. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively)


Total iron quantity in cytosol and mitochondria from GP using ICP - MS . The data distribution is presented using a box plot , including minimum , first quartile , median , third quartile , and maximum . The magenta - colored line represent mean . n = 6 for both

O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Total iron quantity in cytosol and mitochondria from GP using ICP-MS. The data distribution is presented using a box plot, including minimum, first quartile, median, third quartile, and maximum. The magenta-colored line represent mean. n=6 for both genotypes


Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype . Quantification of aconitase activity in two tissues ( brain and liver ) from animals of each genotype by band density . n = 4 for WT , n = 5 for KO ( D - E ) Activity of pyruvate

O O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD

Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenase


Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype . Quantification of aconitase activity in two tissues ( brain and liver ) from animals of each genotype by band density . n = 4 for WT , n = 5 for KO ( D - E ) Activity of pyruvate dehydrogenase ( D ) and complex I ( E ) in GP by genotype and treatment status ( veh = vehicle ; pPanSH = 4 ' - phosphopantetheine ) . n = 5 for both genotypes and treatment groups .

O O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenase (D) and complex I (E) in GP by genotype and treatment status (veh = vehicle; pPanSH = 4'-phosphopantetheine). n=5 for both genotypes and treatment groups.


Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O

Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control.


Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype , brain region and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively ) QRT - PCR . n = 5 for Western blot for all

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O

Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype, brain region and treatment status (pPanSH = 4'-phosphopantetheine). n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively) QRT-PCR. n=5 for Western blot for all groups


Relative mRNA expression of Drd2 and GABA receptor Gabra3 expression by genotype and brain region . n = same as 3A .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O O O O O O

Relative mRNA expression of Drd2 and GABA receptor Gabra3 expression by genotype and brain region. n= same as 3A.


Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype . n = 5 for WT and n = 3 for KO .

B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype. n=5 for WT and n=3 for KO.


Relative quantification of COASY mRNA in human PKAN primary cell lines . n = 5 for both genotypes and cell

O O O B-GENEPROD O O B-ORGANISM B-DISEASE O O O O O O O O O O O O

Relative quantification of COASY mRNA in human PKAN primary cell lines. n= 5 for both genotypes and cell types


Relative quantification of COASY / Coasy in fresh , blood - derived lymphocytes from human and mouse compared by genotype . n = 51 , 35 for human ( control , PKAN , respectively ) and n = 5 for both mouse genotypes .

O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O B-CELL_TYPE O B-ORGANISM O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O B-DISEASE O O O O O O O O O B-ORGANISM O O

Relative quantification of COASY/Coasy in fresh, blood-derived lymphocytes from human and mouse compared by genotype. n=51,35 for human (control, PKAN, respectively) and n=5 for both mouse genotypes.


Extracellular flux analysis in human fibroblasts showing differences in OCR ( oxygen consumption rate ) and ECAR ( extracellular acidification rate ) by genotype . Oligom ; Oligomycin , FCCP ; Carbonyl cyanide - ptrifluoromethoxyphenylhydrazone , 2 - DG ; 2 - deoxy - glucose . Quantification of baseline OCR and ECAR is shown , as well . n = 3 per genotype with four technical replicates .

Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; Oligomycin, FCCP; Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, 2-DG; 2-deoxy-glucose. Quantification of baseline OCR and ECAR is shown, as well. n=3 per genotype with four technical replicates.


Complex I activity assay using fibroblast lysates . Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n = 4 for both genotypes and treatment groups .

B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n=4 for both genotypes and treatment groups.


Relative mRNA expression of mitochondrially - encoded genes important for oxidative phosphorylation from complex I ( MT - ND1 ) , complex III ( MT - CYB ) , cytochrome C ( MT - CO1 ) , and ATP synthase ( MT - ATP6 ) in human mutant fibroblasts versus control cells . n = 3 for both genotypes .

O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O B-CELL_TYPE O O O O O O O O O O O

Relative mRNA expression of mitochondrially-encoded genes important for oxidative phosphorylation from complex I (MT-ND1), complex III (MT-CYB), cytochrome C (MT-CO1), and ATP synthase (MT-ATP6) in human mutant fibroblasts versus control cells. n=3 for both genotypes.


Calculation of active mitochondria using the JC - 1 assay by genotype in control and PKAN lymphoblasts .

O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-DISEASE B-CELL_TYPE O

Calculation of active mitochondria using the JC-1 assay by genotype in control and PKAN lymphoblasts.


Relative expression of Pank1α , Pank1β , total Pank1 and Pank3 in mouse brain regions . n = 8 , 8 , 4 for both genotypes ( GP , SN , Cerebellum , respectively ) .

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O

Relative expression of Pank1α, Pank1β, total Pank1 and Pank3 in mouse brain regions. n=8,8,4 for both genotypes (GP, SN, Cerebellum, respectively).


Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate ( 0 . 8 mg / g body weight ) or gemfibrozil ( 1 . 2 mg / g body weight ) . n = 8 for both genotypes treated with vehicle , n = 5 for both KO groups treated with either bezafibrate or gemfibrozil .

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O

Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate (0.8 mg/g body weight) or gemfibrozil (1.2 mg/g body weight). n=8 for both genotypes treated with vehicle, n=5 for both KO groups treated with either bezafibrate or gemfibrozil.


Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds : 4 ' - phosphopantetheine ( pPanSH ) , pantetheine , vitamin B5 , and CoA . n = 8 ( WT - veh ) , 5 ( KO - veh ) , 5 ( KO - pPanSH ) , 4 ( KO - all other intermediates ) . Correction of Coasy , Tfrc , Ireb2 , and Drd1 expression in GP by 4 ' - phosphopantetheine over a range of doses . n = 8 ( WT - veh ) , 5 ( KO - veh ) , 5 ( KO - 0 . 82 ) , 4 ( WT - 5 ) , 5 ( KO - 5 ) , 5 ( KO - 8 . 2 ) , 5 ( KO - 10 ) . 5 ( WT - 20 ) , 5 ( KO - 20 )

O O O B-GENEPROD O O B-TISSUE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds: 4'-phosphopantetheine (pPanSH), pantetheine, vitamin B5, and CoA. n=8 (WT-veh), 5 (KO-veh), 5 (KO-pPanSH), 4 (KO-all other intermediates). Correction of Coasy, Tfrc, Ireb2, and Drd1 expression in GP by 4'-phosphopantetheine over a range of doses. n=8 (WT-veh), 5 (KO-veh), 5 (KO-0.82), 4 (WT-5), 5 (KO-5), 5 (KO-8.2), 5 (KO-10). 5 (WT-20), 5 (KO-20)


Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH . n = 3 for both genotypes .

O O O B-GENEPROD O O B-ORGANISM B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O

Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH. n=3 for both genotypes.


ECAR measured in cultured human fibroblasts by genotype and treatment status . Cells were treated for three days with 4 ' - phosphopantetheine ( pPanSH ) or vehicle ( veh ) . n = 3 for both genotypes .

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ECAR measured in cultured human fibroblasts by genotype and treatment status. Cells were treated for three days with 4'-phosphopantetheine (pPanSH) or vehicle (veh). n=3 for both genotypes.


Relative quantification of Coasy , Tfrc and Drd1 mRNA from GP by number of days post - cessation of treatment with 5μg of 4 ' - phosphopantetheine per g of body weight . n = 11 ( WT - veh - D0 ) , 5 ( KO - veh - D0 ) , 4 ( WT - pPanSH - D0 ) , 5 ( KO - pPanSH - D0 ) , 5 ( KO - pPanSH - D1 ) , 5 ( KO - pPanSH - D2 ) , 5 ( KO - pPanSH - D3 ) , 5 ( WT - pPanSH - D7 ) , 4 ( KO - pPanSH - D7 ) ,

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O

Relative quantification of Coasy, Tfrc and Drd1 mRNA from GP by number of days post-cessation of treatment with 5μg of 4'-phosphopantetheine per g of body weight. n= 11 (WT-veh-D0), 5 (KO-veh-D0), 4 (WT-pPanSH-D0), 5 (KO-pPanSH-D0), 5 (KO-pPanSH-D1), 5 (KO-pPanSH-D2), 5 (KO-pPanSH-D3), 5 (WT-pPanSH-D7), 4 (KO-pPanSH-D7),


A OT - I CD8 + CTLs were conjugated with 10 nM OVA257 - 264 - pulsed EL - 4 cells for 30 min , fixed , and stained with anti - SKAP55 ( red ) , anti - LFA - 1 ( green ) and Hoechst ( blue ) .

O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O

A OT-I CD8+ CTLs were conjugated with 10 nM OVA257-264-pulsed EL-4 cells for 30 min, fixed, and stained with anti-SKAP55 (red), anti-LFA-1 (green) and Hoechst (blue).


B , C WT and SKAP55 − / − OT - ICD8 + CTLs were generated from WT or SKAP55 − / − OT - I Tg mice , then incubated with 10 nM OVA257 - 264 - pulsed EL4 targets for 4 h to assess the in vitro cytotoxicity at different Effector : Target ratios ( B ; mean of triplicates ± SD ) ; surface expression and the mRNA levels of PD - 1 ( C ) . Graphs are representative of at least three independent experiments .

O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

B, C WT and SKAP55−/−OT-ICD8+CTLs were generated from WT or SKAP55−/−OT-I Tg mice, then incubated with 10 nM OVA257-264-pulsed EL4 targets for 4 h to assess the in vitro cytotoxicity at different Effector:Target ratios (B; mean of triplicates ± SD); surface expression and the mRNA levels of PD-1 (C). Graphs are representative of at least three independent experiments.


D WT and SKAP55 − / − OT - I CD8 + CTLs ( 3 × 106 ) were injected into C57BL / 6 mice , followed by injection of non - pulsed ( CFSEhi ) or 10 nM OVA257 - 264 - pulsed ( CFSElo ) splenocytes ( 5 × 106 ) to measure in vivo cytotoxicity ( mean ± SD , n = 3 mice per group ) . Representative data of three independent experiments .

O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

D WT and SKAP55−/− OT-I CD8+ CTLs (3 × 106) were injected into C57BL/6 mice, followed by injection of non-pulsed (CFSEhi) or 10 nM OVA257-264-pulsed (CFSElo) splenocytes (5 × 106) to measure in vivo cytotoxicity (mean ± SD, n = 3 mice per group). Representative data of three independent experiments.


E WT and SKAP55 − / − OT - I CD8 + CTLs were pretreated with anti - PD - 1 antibody or IgG control , followed by incubation with OVA257 - 264 - pulsed EL4 cells to examine in vitro killing ability ( mean of triplicates ± SD ) . Graphs are representative of three independent experiments .

O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O

E WT and SKAP55−/− OT-I CD8+ CTLs were pretreated with anti-PD-1 antibody or IgG control, followed by incubation with OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD). Graphs are representative of three independent experiments.


F CD8 + CTLs were transfected with plasmids expressing SKAP55 - GFP or EGFP , then treated with non - pulsed or 10 nM OVA257 - 264 - pulsed EL4 cells to examine surface PD - 1 expression ( left panel ) or the in vitro killing assay ( mean of triplicates ± SD ) ( right panel ) .

O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

F CD8+ CTLs were transfected with plasmids expressing SKAP55-GFP or EGFP, then treated with non-pulsed or 10 nM OVA257-264-pulsed EL4 cells to examine surface PD-1 expression (left panel) or the in vitro killing assay (mean of triplicates ± SD) (right panel).


A OT - I CD8 + CTLs were conjugated with CFSE - labeled OVA257 - 264 - pulsed or non - pulsed EL - 4 cells for 30 min , fixed , and stained with anti - ADAP ( red ) .

O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O

A OT-I CD8+ CTLs were conjugated with CFSE-labeled OVA257-264-pulsed or non-pulsed EL-4 cells for 30 min, fixed, and stained with anti-ADAP (red).


B OT - I CD8 + CTLs were transfected with plasmids expressing ADAP or GFP , then stimulated with 10 nM OVA257 - 264 - pulsed EL - 4 cells to detect surface PD - 1 expression . Representative of three independent experiments .

O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

B OT-I CD8+ CTLs were transfected with plasmids expressing ADAP or GFP, then stimulated with 10 nM OVA257-264-pulsed EL-4 cells to detect surface PD-1 expression. Representative of three independent experiments.


C , D WT and ADAP − / − OT - I CD8 + CTLs were stimulated with 10 nM OVA257 - 264 - pulsed or unpulsed EL - 4 cells for 4 h to examine surface expression and the mRNA levels of PD - 1 ( C ) , in vitro cytotoxicity at different Effector : Target ratios ( D ; mean of triplicates ± SD ) . Graphs are representative of at least three independent experiments .

O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O 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, D WT and ADAP−/− OT-I CD8+ CTLs were stimulated with 10 nM OVA257-264-pulsed or unpulsed EL-4 cells for 4 h to examine surface expression and the mRNA levels of PD-1 (C), in vitro cytotoxicity at different Effector:Target ratios (D; mean of triplicates ± SD). Graphs are representative of at least three independent experiments.


E WT and ADAP − / − OT - I CD8 + CTLs were pretreated with 10 μg / ml anti - PD - 1 antibody or IgG control , followed by incubation with 10 nM OVA257 - 264 - pulsed EL4 cells to examine in vitro killing ability ( mean of triplicates ± SD ) .

O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O O O O O O O O O O

E WT and ADAP−/− OT-I CD8+ CTLs were pretreated with 10 μg/ml anti-PD-1 antibody or IgG control, followed by incubation with 10 nM OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD).


F Naïve CD8 + T cells were isolated from WT , SKAP55 − / − , ADAP − / − , and SKAP55 − / − ADAP − / − splenocytes , then stimulated with plate - bound anti - CD3 / CD28 for 12 h to assess PD - 1 mRNA levels , and for 48 h to check surface PD - 1 expression ( mean of triplicates ± SD ) .

O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

F Naïve CD8+ T cells were isolated from WT, SKAP55−/−, ADAP−/−, and SKAP55−/−ADAP−/− splenocytes, then stimulated with plate-bound anti-CD3/CD28 for 12 h to assess PD-1 mRNA levels, and for 48 h to check surface PD-1 expression (mean of triplicates ± SD).


A - C WT , ADAP − / − , or SKAP55 − / − OT - I CD8 + CTLs were stimulated with OVA257 - 264 - pulsed or unpulsed EL4 cells to detect the levels of NFATc1 expression , activation and nuclear entry by immunoblotting ( A ) or immunostaining ( B ) . Alternatively , nuclear extracts from these CTLs were incubated with the N1 DNA probes containing NFAT - binding sites for an EMSA . The unlabeled competitor probe ( Comp . N1 ) and the mutant probe that contains the same sequence except for carrying a mutated NFAT - binding site ( Comp . mutN1 ) were included as controls ( C ) .

O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY 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-GENEPROD O O O O O O O O O O O O O O O O

A-C WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were stimulated with OVA257-264-pulsed or unpulsed EL4 cells to detect the levels of NFATc1 expression, activation and nuclear entry by immunoblotting (A) or immunostaining (B). Alternatively, nuclear extracts from these CTLs were incubated with the N1 DNA probes containing NFAT-binding sites for an EMSA. The unlabeled competitor probe (Comp. N1) and the mutant probe that contains the same sequence except for carrying a mutated NFAT-binding site (Comp. mutN1) were included as controls (C).


D WT , ADAP − / − , or SKAP55 − / − OT - I CD8 + CTLs were pretreated with DMSO or 5 μM CsA , then stimulated with OVA257 - 264 - pulsed EL - 4 cells for 4 h to examine surface PD - 1 expression ( mean of triplicates ± SD ) .

O O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

D WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were pretreated with DMSO or 5 μM CsA, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).


E WT , ADAP − / − , or SKAP55 − / − OT - I CTLs were pretreated with the DMSO control , 10 μM PP2 or 75 μM 2APB , then stimulated with OVA257 - 264 - pulsed EL - 4 cells for 4 h to examine surface PD - 1 expression ( mean of triplicates ± SD ) .

O O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

E WT, ADAP−/−, or SKAP55−/− OT-I CTLs were pretreated with the DMSO control, 10 μM PP2 or 75 μM 2APB, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).


F GFP , ADAP , or SKAP55 was overexpressed with pGL3 - NFAT Luciferase reporter plasmid into Jurkat cells . Cells were stimulated with anti - CD3 and anti - CD28 in the presence or absence of PP2 or 2APB for 6 h , followed by measuring luciferase readings ( mean of triplicates ± SD ) .

O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O

F GFP, ADAP, or SKAP55 was overexpressed with pGL3-NFAT Luciferase reporter plasmid into Jurkat cells. Cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of PP2 or 2APB for 6 h, followed by measuring luciferase readings (mean of triplicates ± SD).


A WT and SKAP55 KO C57BL / 6 mice were s . c . immunized on day − 14 and day − 7 by DCs that were prepulsed with B16F10 tumor lysates . On day 0 , the immunized mice were i . v . inoculated with B16F10 cells . Number of lung tumors were counted 26 days after B16F10 challenge ( n ≥ 5 mice ) .

O O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O B-CELL_LINE O O O O B-TISSUE I-TISSUE O O O O O B-CELL_LINE O O O O O O O O

A WT and SKAP55 KO C57BL/6 mice were s.c. immunized on day −14 and day −7 by DCs that were prepulsed with B16F10 tumor lysates. On day 0, the immunized mice were i.v. inoculated with B16F10 cells. Number of lung tumors were counted 26 days after B16F10 challenge (n ≥ 5 mice).


B , C At day 0 ( B , before tumor injection ) or at day 26 ( C , after tumor injection ) , surface PD - 1 expression in CD44hi CD8 + or CD44hi CD4 + T cells , and the percentages of CD44hi CD8 + T cells or Foxp3 + CD4 + cells were analyzed ( mean ± SD , n ≥ 5 mice ) .

O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

B, C At day 0 (B, before tumor injection) or at day 26 (C, after tumor injection), surface PD-1 expression in CD44hi CD8+ or CD44hi CD4+T cells, and the percentages of CD44hi CD8+T cells or Foxp3+ CD4+ cells were analyzed (mean ± SD, n ≥ 5 mice).


D , E The mRNA levels of PD - 1 , NFATc1 , or the mRNA and protein levels of granzyme B in lung infiltrating CD8 + T cells were examined at day 26 ( mean of triplicates ± SD , and the mRNA samples were prepared from at least 3 mice per group ) .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O B-TISSUE O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E The mRNA levels of PD-1, NFATc1, or the mRNA and protein levels of granzyme B in lung infiltrating CD8+ T cells were examined at day 26 (mean of triplicates ± SD, and the mRNA samples were prepared from at least 3 mice per group).


F CTLA - 4 levels in the lung - infiltrated CD44hiCD8 + T cells or the percentages of CD4 + Foxp3 + T cells in lungs , MLN , and spleen were measured at day 26 ( mean ± SD , n ≥ 5 mice ) .

O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O

F CTLA-4 levels in the lung-infiltrated CD44hiCD8+ T cells or the percentages of CD4+Foxp3+ T cells in lungs, MLN, and spleen were measured at day 26 (mean ± SD, n ≥ 5 mice).


A The same procedures as in Fig4A were performed with WT and ADAP − / − C57BL / 6 mice . HE staining of lung tissue was examined on day 26 ( representative graph of 5 mice ) .

O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O B-ORGANISM O O

A The same procedures as in Fig4A were performed with WT and ADAP−/− C57BL/6 mice. HE staining of lung tissue was examined on day 26 (representative graph of 5 mice).


B , C PD - 1 expression in CD44hiCD8 + or CD44hiCD4 + T cells ( mean ± SD , n ≥ 5 mice per group ) or granzyme B expression in CD44hiCD8 + T cells were examined from the lungs at day 26 ( representative data of 3 mice per group ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE O O O O O O O O B-ORGANISM O O O O

B, C PD-1 expression in CD44hiCD8+ or CD44hiCD4+ T cells (mean ± SD, n ≥ 5 mice per group) or granzyme B expression in CD44hiCD8+ T cells were examined from the lungs at day 26 (representative data of 3 mice per group).


D , E CTLA - 4 levels in lung infiltrating CD44hiCD8 + T cells and the percentages of CD4 + Foxp3 + T cells in the lungs or MLN were measured at day 26 ( mean ± SD , n ≥ 5 mice per group ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O O

D, E CTLA-4 levels in lung infiltrating CD44hiCD8+ T cells and the percentages of CD4+Foxp3+ T cells in the lungs or MLN were measured at day 26 (mean ± SD, n ≥ 5 mice per group).


A , B WT and SKAP55 − / − mice were s . c . injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257 - 264 - pulsed DCs or the PBS control respectively ( n ≥ 5 ) . The growth of tumors under skin was measured every three days according to tumor diameter ( mean ± SEM , n ≥ 5 mice per group ) ( A ) . Surface expression levels of PD - 1 were checked on OVA257 - 264 - specific Vα2high / Vβ5 + CD8 + effector cells at day 30 ( mean ± SD , n ≥ 5 mice ) ( B ) .

O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O

A, B WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257-264-pulsed DCs or the PBS control respectively (n ≥ 5). The growth of tumors under skin was measured every three days according to tumor diameter (mean ± SEM, n ≥ 5 mice per group) (A). Surface expression levels of PD-1 were checked on OVA257-264-specific Vα2high/Vβ5+ CD8+ effector cells at day 30 (mean ± SD, n ≥ 5 mice) (B).


C WT and SKAP55 − / − mice were s . c . injected with MO5 melanoma cells followed by s . c . two injection of OVA257 - 264 - pulsed DCs at day 7 and day 14 . anti - PD - 1 monoantibody or IgG controls were i . v . injected three times at day 7 , day 10 , and day 14 ( 50 μg / injection ) . The diameter and volume of tumors were measured every three days ( mean ± SEM , n ≥ 5 mice ) .

O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O 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-TISSUE O O O O O O O O O O O O O B-ORGANISM O O

C WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by s.c. two injection of OVA257-264-pulsed DCs at day 7 and day 14. anti-PD-1 monoantibody or IgG controls were i.v. injected three times at day 7, day 10, and day 14 (50 μg/injection). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 5 mice).


D The WT recipient mice were s . c . injected with MO5 melanoma cells followed by injection of 10 nM OVA257 - 264 - stimulated WT or SKAP55 − / − CTLs at day 8 ( n ≥ 8 ) . The diameter and volume of tumors were measured every three days ( mean ± SEM , n ≥ 8 mice ) .

O O O O B-ORGANISM O O O O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-ORGANISM O O

D The WT recipient mice were s.c. injected with MO5 melanoma cells followed by injection of 10 nM OVA257-264-stimulated WT or SKAP55−/− CTLs at day 8 (n ≥ 8). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 8 mice).


A , B During generation of OVA257 - 264 peptide - specific CTLs , naïve OT - I splenocytes were stimulated with 10 nM OVA257 - 264 with or without CsA ( 10 nM ) for 3 days , then washed , and further cultured in the RPMI growth medium until day 6 . The CsA - treated or untreated OVA257 - 264 peptide - specific CTLs were incubated with OVA257 - 264 - pulsed EL - 4 cells for 4 h to assess the in vitro cytotoxicity ( A , mean of triplicates ± SD ) or i . v . injected into WT mice to measure in vivo cytotoxicity ( B , mean ± SD , n = 4 mice ) .

O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O 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-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O

A, B During generation of OVA257-264 peptide-specific CTLs, naïve OT-I splenocytes were stimulated with 10 nM OVA257-264 with or without CsA (10 nM) for 3 days, then washed, and further cultured in the RPMI growth medium until day 6. The CsA-treated or untreated OVA257-264 peptide-specific CTLs were incubated with OVA257-264-pulsed EL-4 cells for 4 h to assess the in vitro cytotoxicity (A, mean of triplicates ± SD) or i.v. injected into WT mice to measure in vivo cytotoxicity (B, mean ± SD, n = 4 mice).


C WT mice were s . c . injected with B16 - MO5 melanoma cells followed by injection of the CsA - treated or untreated OVA257 - 264 peptide - specific CTLs at day 9 ( n ≥ 12 ) . The diameter and volume of tumors were measured every 3 days ( mean ± SEM , n ≥ 12 mice ) .

O O B-ORGANISM O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-ORGANISM O O

C WT mice were s.c. injected with B16-MO5 melanoma cells followed by injection of the CsA-treated or untreated OVA257-264 peptide-specific CTLs at day 9 (n ≥ 12). The diameter and volume of tumors were measured every 3 days (mean ± SEM, n ≥ 12 mice).


C ) Representative immunoblots of brain extracts of wild - type , Ttll1 - / - and Ttll7 - / - mice . Polyglutamylation of α - tubulin , detected with polyE and GT335 , is specifically lost in Ttll1 - / - brain . By contrast , β - tubulin glutamylation , which is specifically detected with β ‑ monoE , is absent from brains of Ttll7 - / - mice . D ) Representative immunoblots of extracts of wild - type , Ttll1 ‑ / - and Ttll7 - / - primary hippocampal neurons at DIV4 . As in brain ( C ) , α - tubulin polyglutamylation is specifically lost in Ttll1 - / - neurons , while β - tubulin glutamylation lacks in Ttll7 - / - neurons . E

O O O B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O B-CELL_TYPE O O

C) Representative immunoblots of brain extracts of wild-type, Ttll1-/- and Ttll7-/- mice. Polyglutamylation of α-tubulin, detected with polyE and GT335, is specifically lost in Ttll1-/- brain. By contrast, β-tubulin glutamylation, which is specifically detected with β‑monoE, is absent from brains of Ttll7-/- mice. D) Representative immunoblots of extracts of wild-type, Ttll1‑/- and Ttll7-/- primary hippocampal neurons at DIV4. As in brain (C), α-tubulin polyglutamylation is specifically lost in Ttll1-/- neurons, while β-tubulin glutamylation lacks in Ttll7-/- neurons. E


F , G ) Immunocytochemistry of DIV4 primary hippocampal neurons from wild type , Ttll1 - / - and Ttll7 ‑ / - mice . Microtubules in Ttll1 - / - cells have reduced levels of polyE signal , indicative of lower α - tubulin polyglutamylation ( F ) , Ttll7 ‑ / - neurons are specifically reduced in β ‑ monoE reactivity , indicating the lack of β ‑ tubulin glutamylation ( G ) .

O O O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O

F,G) Immunocytochemistry of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7‑/- mice. Microtubules in Ttll1-/- cells have reduced levels of polyE signal, indicative of lower α-tubulin polyglutamylation (F), Ttll7‑/- neurons are specifically reduced in β‑monoE reactivity, indicating the lack of β‑tubulin glutamylation (G).


A ) Representative histology images of cerebella from 3 - months - old mice stained with anti - calbindin antibody , which specifically labels Purkinje cells ( Sequier et al , 1988 ) . Note the presence of an intact Purkinje - cell layer in cerebella of wild - type , Ttll1 - / - , Ttll7 - / - and Ccp1 - / - Ttll1 - / - mice , in contrast to the complete absence of these cells in Ccp1 - / - and Ccp1 - / - Ttll7 - / - mice .

O O O B-EXP_ASSAY O O B-TISSUE O O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O

A) Representative histology images of cerebella from 3-months-old mice stained with anti-calbindin antibody, which specifically labels Purkinje cells (Sequier et al, 1988). Note the presence of an intact Purkinje-cell layer in cerebella of wild-type, Ttll1-/-, Ttll7-/- and Ccp1-/-Ttll1-/- mice, in contrast to the complete absence of these cells in Ccp1-/- and Ccp1-/-Ttll7-/- mice.


B ) Histology of 25 - day old mice with partial degeneration of Purkinje Cells . Note that both , Ccp1 - / - and Ccp1 - / - Ttll7 - / - brains , show highly similar pattern of partial Purkinje - cell loss ( red arrow heads : gaps in the Purkinje - cell layer , indicating degenerated Purkinje cells ; orange arrow heads : swellings of Purkinje - cell axons , indicative of ongoing degeneration ) .

O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O

B) Histology of 25-day old mice with partial degeneration of Purkinje Cells. Note that both, Ccp1-/- and Ccp1-/-Ttll7-/- brains, show highly similar pattern of partial Purkinje-cell loss (red arrow heads: gaps in the Purkinje-cell layer, indicating degenerated Purkinje cells; orange arrow heads: swellings of Purkinje-cell axons, indicative of ongoing degeneration).


C ) Representative immunoblots of cerebellum extracts of wild - type , Ccp1 - / - , Ttll7 - / - , Ccp1 - / - Ttll7 - / - , Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice . Loss of CCP1 leads to hyperglutamylation of α - and β - tubulin as revealed with polyE and GT335 antibodies . Concomitant deletion of TTLL7 lead to a loss of glutamylation from β - tubulin ( β ‑ monoE , GT335 ) , while hyperglutamylation of α - tubulin persists ( polyE , GT335 ) . Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both , α - and β - tubulin ( polyE , GT335 ) , while β - tubulin glutamylation is unaltered ( β - monoE ) .

O O O B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O

C) Representative immunoblots of cerebellum extracts of wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Loss of CCP1 leads to hyperglutamylation of α- and β-tubulin as revealed with polyE and GT335 antibodies. Concomitant deletion of TTLL7 lead to a loss of glutamylation from β-tubulin (β‑monoE, GT335), while hyperglutamylation of α-tubulin persists (polyE, GT335). Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both, α- and β-tubulin (polyE, GT335), while β-tubulin glutamylation is unaltered (β-monoE).


Semi - thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue . Note the smaller size of Ccp1 - / - and Ccp1 - / - Ttll7 - / - nerves ( A ) .

O O O O O O B-TISSUE I-TISSUE I-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE O O O O

Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. Note the smaller size of Ccp1-/- and Ccp1-/-Ttll7-/- nerves (A).


Semi - thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue . These sections were quantified for ( B ) number of myelinated axons , ( C ) nerve area , ( D ) nerve perimeter ( Fig . EV2 ) of femoral quadriceps nerves from wild - type , Ccp1 - / - , Ttll7 - / - , Ccp1 - / - Ttll7 - / - , Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice . Scatter dot plots in which each point represents one nerve of an individual mouse are shown . The black lines indicate means ± SEM . Significance was tested with one - way ANOVA and Holm - Šídák ' s multiple comparisons test . Note that all three parameters are decreased for Ccp1 - / - and Ccp1 - / - Ttll7 ‑ / - mice , while Ccp1 - / - Ttll1 - / - mice showed no difference to the Ttll1 - / - control .

O O O O O O B-TISSUE I-TISSUE I-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O O O O

Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. These sections were quantified for (B) number of myelinated axons, (C) nerve area, (D) nerve perimeter (Fig. EV2) of femoral quadriceps nerves from wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Scatter dot plots in which each point represents one nerve of an individual mouse are shown. The black lines indicate means ±SEM. Significance was tested with one-way ANOVA and Holm-Šídák's multiple comparisons test. Note that all three parameters are decreased for Ccp1-/- and Ccp1-/-Ttll7‑/- mice, while Ccp1-/-Ttll1-/- mice showed no difference to the Ttll1-/- control.


E , F ) Representative electron microscopy images of cross sections of femoral quadriceps nerves . ( E ) Nerves from Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice show no abnormalities of myelinated axons ( green asterisks ) , indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1 . ( F ) Ccp1 - / - and Ccp1 - / - Ttll7 - / - mice both show the presence of foamy macrophages ( magenta arrowheads ) , which are signs for myelin phagocytosis during axon degeneration . This indicates that loss of TTLL7 cannot prevent the CCP1 - induced degeneration of femoral quadriceps nerves .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-CELL_TYPE O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE O

E,F) Representative electron microscopy images of cross sections of femoral quadriceps nerves. (E) Nerves from Ttll1-/- and Ccp1-/-Ttll1-/- mice show no abnormalities of myelinated axons (green asterisks), indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1. (F) Ccp1-/- and Ccp1-/-Ttll7-/- mice both show the presence of foamy macrophages (magenta arrowheads), which are signs for myelin phagocytosis during axon degeneration. This indicates that loss of TTLL7 cannot prevent the CCP1-induced degeneration of femoral quadriceps nerves.


A ) Representative images of DIV4 primary hippocampal neurons from wild type , Ttll1 - / - and Ttll7 - / - mice Cells were incubated with MitoTracker ® ( black dots ) and mitochondria movements were recorded in the longest neurite ( orange lines ) .

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O

A) Representative images of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7-/- mice Cells were incubated with MitoTracker® (black dots) and mitochondria movements were recorded in the longest neurite (orange lines).


B ) Kymographs of one representative experiment representing mitochondrial movements C - F ) Analysis of one single experiment shown in ( B ) . Scatter dot plots of ( C ) run length and ( D ) speed distributions , where each point represents a single run extracted from the kymographs shown in ( B ) . The black lines indicate the median of the distribution , with whiskers at interquartile range . Numerical values of medians are indicated below each plot . ( E ) A bar graph showing the overall motility of mitochondria ( the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s ) . ( F ) A bar graph showing mitochondrial density in analysed axons . Values of the overall motilities and mitochondrial densities are given below the bar plots .

O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O

B) Kymographs of one representative experiment representing mitochondrial movements C - F) Analysis of one single experiment shown in (B). Scatter dot plots of (C) run length and (D) speed distributions, where each point represents a single run extracted from the kymographs shown in (B). The black lines indicate the median of the distribution, with whiskers at interquartile range. Numerical values of medians are indicated below each plot. (E) A bar graph showing the overall motility of mitochondria (the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s). (F) A bar graph showing mitochondrial density in analysed axons. Values of the overall motilities and mitochondrial densities are given below the bar plots.


A ) Volcano plot of the fold - change ( x - axis ) plotted against significance ( y - axis ) of lipids changing upon HF / HS feeding . Lipids are colored according to fold - change ( log2 , absolute ) > 1 ( orange ) , p - value < 0 . 05 ( red ) or both ( green ) . Pvalues calculated from differential expression using

O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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) Volcano plot of the fold-change (x-axis) plotted against significance (y-axis) of lipids changing upon HF/HS feeding. Lipids are colored according to fold-change (log2, absolute) > 1 (orange), p-value <0.05 (red) or both (green). Pvalues calculated from differential expression using limma