( C ) Detection of endosomal PI ( 4 , 5 ) P2 in pre - treated with OSW1 for 1 h in serum - free medium using confocal microscopy PLCδ1PH - GFP PI ( 4 , 5 ) P2 reporter . Angiotensin II ( AngII , 100 nM ) ( a Gq PLC - activating agonist ) was used to liberate PLCδ1PH - GFP PI ( 4 , 5 ) P2 reporter from PM , which was necessary to detect endosomal PI ( 4 , 5 ) P2 . signal is transient as AngII receptors show desensitization . Note reduced signal in two PI4K2A K / O clones . Scale bars : 20 µm .

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(C) Detection of endosomal PI(4,5)P2 in cells pre-treated with OSW1 for 1 h in serum-free medium using confocal microscopy and the PLCδ1PH-GFP PI(4,5)P2 reporter. Angiotensin II (AngII, 100 nM) (a Gq and PLC-activating agonist) was used to liberate the PLCδ1PH-GFP PI(4,5)P2 reporter from the PM, which was necessary to detect the endosomal PI(4,5)P2. The signal is transient as the AngII receptors show desensitization. Note the reduced signal in the two PI4K2A K/O clones. Scale bars: 20 µm.


( D ) of same changes by BRET in wild - type using PI ( 4 , 5 ) P2 sensor with Rab7 - Q67L - targeting . Note transient signal increase after AngII stimulation only in treated with OSW1 . ( E ) This increase is barely detectable in PI4K2A K / O . D Data information : Means ± S . E . M . are shown from three experiments performed in triplicates . values in ( D ) ( E ) were measured in same 96 - well plate at same time .

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(D) Quantification of the same changes by BRET analysis in wild-type cells using the PI(4,5)P2 sensor with Rab7-Q67L-targeting. Note the transient signal increase after AngII stimulation only in cells treated with OSW1. (E) This increase is barely detectable in the PI4K2A K/O cells. D Data information: Means ± S.E.M. are shown from three experiments performed in triplicates. The values in (D) and (E) were measured in the same 96-well plate at the same time.


( A ) BRET experiment in parental HEK293 - AT1 in OSW1 - pretreated after RNAi mediated knock - down of PIP5Kβ or PIP5Kγ . ( means ± S . E . M . from three experiments , each performed in triplicates ) . Note reduced response in PIP5Kγ knock - down slight reduction in case of PIP5Kβ knock - down . ( B ) Areas below curves calculated from time of rapamycin addition for each of three separate experiments shown in panel A ( means ± S . E . M . , n = 3 ) . One - way ANOVA with Dunnett ' s multiple comparisons was used for statistical ( * P = 0 . 0241 ; * * P = 0 . 0088 ) . (

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(A) BRET experiment in parental HEK293-AT1 cells in OSW1-pretreated cells after RNAi mediated knock-down of PIP5Kβ or PIP5Kγ. (means ± S.E.M. from three experiments, each performed in triplicates). Note the reduced response in the PIP5Kγ knock-down cells and the slight reduction in the case of PIP5Kβ knock-down. (B) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P=0.0241; **P=0.0088). (


( C ) These reductions are proportional to reduction in PIP5Kγ as assessed by Western blot of lysates obtained from treated with siRNAs at same time .

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(C) These reductions are proportional to the reduction in PIP5Kγ levels as assessed by Western blot analysis of cell lysates obtained from cells treated with siRNAs at the same time.


( D ) Increased appearance of GFP - LC3 positive vesicles in PI4K2A K / O reduced acidification of LC3 - positive vesicles using GFP - mCherry - LC3 reporter . were transfected with indicated constructs for one day observed by confocal microscopy . Scale bars : 20 µm . ( E ) of LC3 positive vesicles . ( n = 67 , 34 , 26 , 13 , 30 , 27 for various groups , respectively from left to right ) . * * * designates P < 0 . 0001 using unpaired t - . same data set was also analyzed with non - parametric Mann - Whitney showing a P = 0 . 0004 0 . 0012 for # 20 # 26 clones relative to control , respectively . ( F ) Co - localization using Pearson coefficient ( n = 31 , 21 14 analyzed for different groups from left to right ) . * * * designates P < 0 . 0001 using unpaired t - . same data set was also analyzed with non - parametric Mann - Whitney showing a P value < 0 . 0001 for both groups .

O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Increased appearance of GFP-LC3 positive vesicles in PI4K2A K/O cells and reduced acidification of the LC3-positive vesicles using the GFP-mCherry-LC3 reporter. Cells were transfected with the indicated constructs for one day and observed by confocal microscopy. Scale bars: 20 µm. (E) Quantification of the LC3 positive vesicles. (n = 67, 34, 26, 13, 30, 27 for the various groups, respectively from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P = 0.0004 and 0.0012 for #20 and #26 clones relative to control, respectively. (F) Co-localization analysis using the Pearson coefficient (n = 31, 21 and 14 cells analyzed for the different groups from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P value < 0.0001 for both groups.


( A , B ) Distribution of mCherry - GFP - LC3 in HEK293 - AT1 ( without starvation ) after treatment with control siRNA ( A ) or siRNA for PIP5Kγ ( PIP5K1C ) . Scale bars : 20 µm . Note massive tubulation of LC3 compartment in some of knocked - down ( B , lower , scale bar : 10 µm ) . ( C ) Comparison of Pearson coefficients from treated with control or PIP5Kγ RNAi . For statistical unpaired t - was used ( n = 50 45 for control a PIP5Kγ knock down groups , respectively ; * * * P < 0 . 0001 ) . ( D ) Representative confocal of live HEK293 - AT1 expressing GFP - Rab7 treated with control or PIP5Kγ RNAi . Note tubulation in PIP5Kγ RNAi - treated . Scale bars : 10 µm .

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(A, B) Distribution of mCherry-GFP-LC3 in HEK293-AT1 cells (without starvation) after treatment with control siRNA (A) or siRNA for PIP5Kγ (PIP5K1C). Scale bars: 20 µm. Note the massive tubulation of the LC3 compartment in some of the knocked-down cells (B, lower images, scale bar: 10 µm). (C) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi. For statistical analysis the unpaired t-test was used (n = 50 and 45 cells for control a PIP5Kγ knock down groups, respectively; *** P< 0.0001). (D) Representative confocal images of live HEK293-AT1 cells expressing GFP-Rab7 treated with control or PIP5Kγ RNAi. Note the tubulation in the PIP5Kγ RNAi-treated cells. Scale bars: 10 µm.


( A ) HEK293 - AT1 were transfected with PLEKHM1 - GFP - Rab7 along with CFP - FKBP - PIP5Kγ iRFP - FRB - Rab7 constructs for one day examined live by confocal microscopy . Representative show reduced PLEKHM1 localization when PIP5Kγ was recruited to Rab7 endosomes ( lower row ) . ( B ) This response is reduced in PI4K2A K / O . ( Data information : Scale bars : 20 µm .

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(A) HEK293-AT1 cells were transfected with PLEKHM1-GFP-Rab7 along with CFP-FKBP-PIP5Kγ and iRFP-FRB-Rab7 constructs for one day and examined live by confocal microscopy. Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes (lower row images). (B) This response is reduced in PI4K2A K/O cells. ( Data information: Scale bars: 20 µm.


( C ) of these changes by BRET where full - PLEKHM1 was fused to Sluc Venus was fused to Rab7 in BRET construct . For PIP5Kγ recruitment a mutant CFP ( W66A ) - FKBP - PIP5Kγ was used with an iRFP - FRB - Rab7 . BRET values were normalized to those of DMSO treated . Comparisons were made for parental ( blue ) PI4K2A K / O ( green orange ) . ( D ) Similar BRET where PLEKHM1 was replaced by either RILP or Vps35 in BRET construct . D Data information Means ± S . E . M . are shown from three experiments performed in triplicates .

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(C) Quantification of these changes by BRET analysis where the full-length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct. For PIP5Kγ recruitment a mutant dark CFP(W66A)-FKBP-PIP5Kγ was used with an iRFP-FRB-Rab7. BRET values were normalized to those of DMSO treated cells. Comparisons were made for parental (blue) and PI4K2A K/O (green and orange) cells. (D) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct. D Data information Means ± S.E.M. are shown from three experiments performed in triplicates.


lines . of 2019 - nCoV - S expression ( A ) by Western blot . Representative blots from three experiments are shown . ß - Actin ( lysates )

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lines.Analysis of 2019-nCoV-S expression (A) by Western blot. Representative blots from three experiments are shown. ß-Actin (cell lysates)


ines . of 2019 - nCoV - S pseudotype incorporation ( B ) by Western blot . Representative blots from three experiments are shown . VSV - M ( particles ) served as loading controls .

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ines.Analysis of 2019-nCoV-S pseudotype incorporation (B) by Western blot. Representative blots from three experiments are shown. VSV-M (particles) served as loading controls.


( C ) lines of human origin were inoculated with pseudotyped VSV harboring VSV - G , SARS - S or 2019 - nCoV - S . At 16 h postinoculation , pseudotype entry was analyzed . Shown are combined data of three experiments . Error bars indicate .

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(C) Cell lines of human and animal origin were inoculated with pseudotyped VSV harboring VSV-G, SARS-S or 2019-nCoV-S. At 16 h postinoculation, pseudotype entry was analyzed. Shown are the combined data of three experiments. Error bars indicate SEM.


( C ) 293T transiently expressing ACE2 of human ( blue ) or bat ( blue ) origin , human APN ( purple ) or hDPP4 ( green ) were inoculated with pseudotyped VSV harboring VSV - G , SARS - S , 2019 - nCoV - S , MERS - S or 229E - S . At 16 h postinoculation , pseudotype entry was analyzed . average of three independent experiments is shown . Error bars indicate .

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(C) 293T cells transiently expressing ACE2 of human (dark blue) or bat (light blue) origin, human APN (purple) or hDPP4 (green) were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, 2019-nCoV-S, MERS-S or 229E-S. At 16 h postinoculation, pseudotype entry was analyzed. The average of three independent experiments is shown. Error bars indicate SEM.


priming . Ammonium chloride ( A ) , were added to indicated target before transduction with pseudotypes bearing indicated glycoproteins .

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priming.Ammonium chloride (A), were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.


E64d ( CatB / L inhibitor ) ( B ) / or camostat ( TMPRSS2 inhibitor ) ( B ) were added to indicated target before transduction with pseudotypes bearing indicated glycoproteins .

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E64d (CatB/L inhibitor) (B) and/or camostat (TMPRSS2 inhibitor) (B) were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.


( C ) 293T transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB / L inhibitor E64d or PBS as control inoculated with pseudotypes bearing indicated viral surface . average of three independent experiments is shown in panels A - C . Error bars indicate . 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 indicated viral surface were incubated with different dilutions of serum from a convalescent SARS subsequently inoculated onto 293T that transiently express ACE2 in order to evaluate cross - neutralization . results from a representative experiment with triplicate samples are shown were confirmed in a separate experiment . Error bars indicate SD . Statistical significance was tested by two - way ANOVA with Dunnett posttest .

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

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(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 flexible . crystal structure of large structure predicts solution scattering curve ( left , χ2 = 0 . 92 ) fits well to 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 FRH N - terminus ( large structure , magenta ) to that of Mtr4 ( green ) aligned on their RecA domains . Only additional elements of FRH are shown ( cyan ) . FRH N - terminal extension has little ordered secondary structure , although regions 143 - 147 129 - 135 form small turns that interact with helicase . ( B ) very N - terminus of FRH extends away from RecA - 2 domain to interact with a symmetry related molecule in crystal ( below ) , whereas Mtr4 N - terminus turns back to form an outer β − strand . Center panel shows crystal contacts , with close - ups for each region highlighted as insets on left right .

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

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(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 ) FRH ( large in cyan , small in marine ) after superposition of most N - terminal helical region . isolated KOW modules of FRH , Mtr4 ( with FRH superimposed ) , 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 contoured at 2 . 0  ) reveals presence of ADP a short segment of bound to FRH .

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(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 contributing to model . Green electron density shown for ADP ( 0 . 6     orange density shown for surrounding ( 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 FRHADP binding ( FRH in cyan with ADP , , F179 in orange bonds ; Mtr4 in green with ADP , , F148 in yellow bonds ) . ( c ) Unwound from Hel308 ( pdb 2PR6 ; magenta , in orange bonds ) overlaid in FRH with ADP ( FRH in cyan with yellow cyan ADP in stick ) after superposition of respective RecA domains .

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

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

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(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 ADP - binding pocket N - terminal interaction for large - structure ( cyan ribbons ) small - 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 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 KOW domain showing large number of basic ( aqua sticks ) acidic side chains ( yellow sticks ) on surface that also includes mutational sites R806 , R712 , K811 K766 ( grey sticks )

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(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 . between bands reflects clock period . strains were grown in constant darkness 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 ) . 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 WC - 2 . of FRQ , WC - 1 WC - 2 binding to V5 tagged FRH mutants as compared to inter - variations in FRH , error bars represent standard deviation for run in triplicate . * P < 0 . 05 ( unpaired two - t - with Gaussian distribution assumption ) . Representative immunoblots are shown in Appendix Fig S5 .

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(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 ( RQ ) of CG7630 transcript after RNAi with a 120 - bp inverted - repeat ( 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 of egg to adult viability of CG7630 RNAi cross ( act5c - gal4 / CyO . GFP x UAS - CG7630RNAi ) control cross ( act5c - gal4 / CyO . GFP x w1118 ) , calculated at three developmental stages ( eggs , pupae adults ) , ( n > 250 , Chi - square 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 MRC complexes in CG7630 RNAi 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 IV in DDM - solubilized mitochondria from RNAi ( act5c - gal4 > UAS - CG7630RNAi ) 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 ) control ( act5c - gal4 > + ) larvae using antibodies against specific subunits : anti - UQCRC2 ( III ) , anti - NDUFS3 ( I ) , anti - COX4 ( IV ) anti - SDHA ( II ) .

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

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(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 of anti - HA affinity purified samples from S2R + D . melanogaster . EV = samples from carrying empty expression vector . CG7630 - HA = samples from 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 ) 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 ( RQ ) of CG7630 transcript in wild - type D . melanogaster S2R + treated with a dsRNA targeting CG7630 ( CG7630 KD ) 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 ( RQ ) of CG7630 COX7B transcripts in stable S2R + lines carrying empty expression vector ( EV : GFP ; neo ) , expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) CG7630 ( CG7630 - HA ; neo ) . were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) 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 of stable S2R + lines carrying empty expression vector ( EV : GFP ; neo ) , expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) CG7630 ( CG7630 - HA ; neo ) . CG7630 transcript was knocked - down ( KD ) in by a dsRNA targeting CG7630 compared with treated with a mock control dsRNA ( mock ) . Samples were probed with an antibody anti HA tag with an antibody against Hsp70 as an endogenous control .

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(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 + lines carrying empty expression vector ( EV : GFP ; neo ) , expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) CG7630 ( CG7630 - HA ; neo ) . were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) 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 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 in DG labeled with an anti - PH3 antibody ( green ) ( A ) of number of PH3 + in DG of WT Tau - / - mice ( B ) ( mean ± ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - ) .

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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 in DG labeled with an anti - fractin antibody ( red ) ( C ) of number of fractin + in DG of WT Tau - / - mice ( D ) ( mean ± ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - ) .

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 survival experimental design using thymidine analogs ( F ) of of surviving in Tau - / - mice as compared to WT mice at each ( 1 - , 2 - , 4 - , 6 - 8 - week - old ) ( F ) ( mean ± ( normalized data ) ; Student ´ s t - ) .

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

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

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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 of 4 - week - old newborn granule neurons of WT Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus . A schematic representation of Sholl ´ s is shown in WT image . B - C of total dendritic ( B ) Sholl ´ s ( C ) of 4 - week - old newborn granule neurons in WT 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 of 8 - week - old newborn granule neurons of WT Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus . E - F of total dendritic ( E ) Sholl ´ s ( F ) of 8 - week - old newborn granule neurons in WT 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 of 4 - week - old newborn granule neurons of WT Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus their corresponding high - power magnifications showing PSDs ( green ) . H - IQuantification of number of PSDs / µm ( H ) PSD ( I ) in each dendritic branching order of 4 - week - old newborn granule neurons in WT 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 of 8 - week - old newborn granule neurons of WT Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus their corresponding high - power magnifications showing PSDs ( green ) . K - L of number of PSDs / µm ( K ) PSD ( L ) in each dendritic branching order of 8 - week - old newborn granule neurons in WT 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.


A Representative traces of mEPSCs recorded at a holding potential of − 65 mV from WT ( black traces ) 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 from WT ( black trace ) 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 potential of WT Tau - / - mice ( mean ± ; n = 13 WT , n = 10 Tau - / - ; * 0 . 05 > p ≥ 0 . 01 , Student ´ s t - ) .

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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 ) mEPSC frequency ( E ) of WT Tau - / - ( mean ± ; n = 13 WT , n = 10 Tau - / - ; Student ´ s t or Mann - Whitney ´ s U ) .

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

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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 Porsolt .

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


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

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

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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 of an 8 - week - old newborn granule neuron , in which proximal dendrites are highlighted ( E ) , of PSDs ( green ) in proximal dendrites belonging to WT CNP , WT P , Tau - / - CNP , Tau - / - P mice ( F ) . G - H of number of PSDs / µm ( G ) PSD ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) ( J ) of of GluR1 subunit of AMPA receptors in hippocampi of WT Tau - / - mice subjected or not to Porsolt ( n = 6 mice per experimental condition ) .

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

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 DG projecting axons through mossy fiber tract into CA3 field ( indicated by a square ) .

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

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 ( L ) of ( M ) of MFTs of 8 - week - old newborn neurons in CA3 region of WT CNP , WT P , Tau - / - CNP , Tau - / - P 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

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 of control housing ( CH ) environmental enrichment ( EE ) cages . B Schematic diagram of experimental design .

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

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


C - D Representative of DCX + in DG of CH EE ( C ) , of number of DCX + of WT Tau - / - in CH or EE conditions ( D ) .

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

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

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

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 of whole hippocampus showing GABAergic innervation labeled with GAD - 65 antibody ( red ) , high - power magnifications of DG molecular layer showing GAD - 65 + terminals in WT CH , Tau - / - CH , WT EE Tau - / - EE mice . nuclei were labeled with DAPI ( blue ) . scale bar 300 µm . Red scale bar 50 µm . Green scale bar 5 µm . B - C of density ( B ) ( C ) of GAD - 65 + terminals in 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 O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of 8 - week - old newborn granule neurons of WT CH , WT EE , Tau - / - CH , Tau - / - EE mice infected with a PSD95 - GFP - expressing retrovirus . In WT CH image a schematic representation of Sholl ´ s is shown . C - D of total dendritic ( C ) Sholl ´ s ( 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 of an 8 - week - old newborn granule neuron in which proximal distal dendrites are highlighted ( E ) of PSDs ( green ) in distal proximal dendrites of WT Tau - / - mice housed under either CH or EE conditions ( F ) . G - H of number of PSDs / µm ( G ) PSD ( 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 O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 DG projecting axons through mossy fiber tract into CA3 field ( indicated by a 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 ( J ) of ( K ) of MFTs of 8 - week - old newborn neurons in CA3 region of WT CH , WT EE , Tau - / - CH , Tau - / - EE mice ( n = 3 mice per experimental condition ) . Arrows indicate MFTs .

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

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

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

O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 , Hamp by genotype brain region . n = 11 , 4 , 4 for WT n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively )

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mitochondria from GP using ICP - MS . data distribution is presented using a box plot , including minimum , first quartile , median , third quartile , maximum . magenta - colored represent mean . n = 6 for both

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

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


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

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

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


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

O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 of TfR1 PDHE1α1 from GP by genotype treatment status ( pPanSH = 4 ' - phosphopantetheine ) . Ponceau S was used as total loading control VDAC for mitochondrial loading control .

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

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

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

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 GABA receptor Gabra3 expression by genotype brain region . n = same as 3A .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O 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 from whole brain of tyrosine hydroxylase compared by genotype . n = 5 for WT n = 3 for KO .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 of COASY mRNA in human PKAN lines . n = 5 for both genotypes

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

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

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

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

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.


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

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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 I ( MT - ND1 ) , III ( MT - CYB ) , cytochrome C ( MT - CO1 ) , ATP synthase ( MT - ATP6 ) in human mutant fibroblasts versus control . n = 3 for both genotypes .

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

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 JC - 1 by genotype in control PKAN lymphoblasts .

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


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

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

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

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 of Coasy mRNA from GP compared by treatment status using four compounds : 4 ' - phosphopantetheine ( pPanSH ) , pantetheine , vitamin B5 , CoA . n = 8 ( WT - veh ) , 5 ( KO - veh ) , 5 ( KO - pPanSH ) , 4 ( KO - other intermediates ) . Correction of Coasy , Tfrc , Ireb2 , 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 B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR 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 treatment status . were treated for three days with 4 ' - phosphopantetheine ( pPanSH ) or vehicle ( veh ) . n = 3 for both genotypes .

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

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 of Coasy , Tfrc Drd1 mRNA from GP by number of days post - cessation of treatment with 5μg of 4 ' - phosphopantetheine per g of . 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR 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 for 30 min , fixed , stained with anti - SKAP55 ( red ) , anti - LFA - 1 ( green ) Hoechst ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O 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 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 in vitro cytotoxicity at different Effector : Target ratios ( B ; mean of triplicates ± SD ) ; surface expression mRNA of PD - 1 ( C ) . Graphs are representative of at least three independent experiments .

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

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

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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 to examine surface PD - 1 expression ( left panel ) or in vitro killing ( mean of triplicates ± SD ) ( right panel ) .

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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 for 30 min , fixed , stained with anti - ADAP ( red ) .

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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 to detect surface PD - 1 expression . Representative of three independent experiments .

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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 ADAP − / − OT - I CD8 + CTLs were stimulated with 10 nM OVA257 - 264 - pulsed or unpulsed EL - 4 for 4 h to examine surface expression mRNA 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 .

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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 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 to examine in vitro killing ability ( mean of triplicates ± SD ) .

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

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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 to detect of NFATc1 expression , activation entry by immunoblotting ( A ) or immunostaining ( B ) . Alternatively , extracts from these CTLs were incubated with N1 probes containing NFAT - binding sites for an EMSA . unlabeled competitor probe ( Comp . N1 ) mutant probe that contains same except for carrying a mutated NFAT - binding site ( Comp . mutN1 ) were included as controls ( C ) .

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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 for 4 h to examine surface PD - 1 expression ( mean of triplicates ± SD ) .

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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 DMSO control , 10 μM PP2 or 75 μM 2APB , then stimulated with OVA257 - 264 - pulsed EL - 4 for 4 h to examine surface PD - 1 expression ( mean of triplicates ± SD ) .

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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 . were stimulated with anti - CD3 anti - CD28 in presence or absence of PP2 or 2APB for 6 h , followed by measuring luciferase readings ( mean of triplicates ± SD ) .

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

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

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

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

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

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

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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 in lung infiltrating CD44hiCD8 + T percentages of CD4 + Foxp3 + T in lungs or MLN were measured at day 26 ( mean ± SD , n ≥ 5 mice per group ) .

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

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

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

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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 , further cultured in RPMI growth medium until day 6 . CsA - treated or untreated OVA257 - 264 peptide - specific CTLs were incubated with OVA257 - 264 - pulsed EL - 4 for 4 h to assess 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 ) .

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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 followed by injection of CsA - treated or untreated OVA257 - 264 peptide - specific CTLs at day 9 ( n ≥ 12 ) . diameter volume of tumors were measured every 3 days ( mean ± , n ≥ 12 mice ) .

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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 - / - Ttll7 - / - mice . Polyglutamylation of α - tubulin , detected with polyE 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 ‑ / - Ttll7 - / - 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

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

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

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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 . Note that both , Ccp1 - / - Ccp1 - / - Ttll7 - / - brains , show highly similar pattern of partial Purkinje - loss ( red arrow heads : gaps in Purkinje - layer , indicating degenerated Purkinje ; orange arrow heads : swellings of Purkinje - axons , indicative of ongoing degeneration ) .

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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 - / - Ccp1 - / - Ttll1 - / - mice . Loss of CCP1 leads to hyperglutamylation of α - β - tubulin as revealed with polyE 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 TTLL1 eliminates hyperglutamylation from both , α - β - tubulin ( polyE , GT335 ) , while β - tubulin glutamylation is unaltered ( β - monoE ) .

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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 smaller of Ccp1 - / - Ccp1 - / - Ttll7 - / - nerves ( A ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O 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 , ( D ) nerve perimeter ( Fig . EV2 ) of femoral quadriceps nerves from wild - type , Ccp1 - / - , Ttll7 - / - , Ccp1 - / - Ttll7 - / - , Ttll1 - / - Ccp1 - / - Ttll1 - / - mice . Scatter dot plots in which each point represents one nerve of an mouse are shown . black lines indicate means ± . Significance was tested with one - way ANOVA Holm - Šídák ' s multiple comparisons . Note that three parameters are decreased for Ccp1 - / - Ccp1 - / - Ttll7 ‑ / - mice , while Ccp1 - / - Ttll1 - / - mice showed no difference to Ttll1 - / - control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR 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.