( 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 - ) .

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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 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 .

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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 ) .

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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 ) .

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

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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 of control housing ( CH ) environmental enrichment ( EE ) cages . B Schematic diagram of 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 of DCX + in DG of CH EE ( C ) , of number of DCX + of WT Tau - / - 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 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 ) .

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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 B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-GENEPROD O O O O B-ORGANISM 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 B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR 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 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 ) .

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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 .

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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 )

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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 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 ) .

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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 )

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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

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


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

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 .

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

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

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

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 .

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

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 .

O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 I-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 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-TISSUE O O O B-ORGANISM O O O O O B-SMALL_MOLECULE 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 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 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-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 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 B-GENEPROD O B-GENEPROD O B-GENEPROD 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 of COASY mRNA from human fibroblasts treated with pPanSH . n = 3 for both genotypes .

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

B-EXP_ASSAY O O O B-ORGANISM B-CELL_TYPE O O O 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 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-GENEPROD O B-GENEPROD I-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 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 for 30 min , fixed , stained with anti - SKAP55 ( red ) , anti - LFA - 1 ( green ) 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 B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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-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 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 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 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 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 .

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

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 ) .

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 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

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 ) .

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 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 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 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 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 .

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 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 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 ) .

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

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 ) .

O O B-GENEPROD O B-CELL_TYPE 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-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 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 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 ) .

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 B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE 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 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 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 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 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 ) .

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 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 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 . 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 ) .

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 B-GENEPROD O O B-GENEPROD 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 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 ) .

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 B-CELL_TYPE O O O O B-CELL_LINE O O O O O O O B-ORGANISM O O O O O O O B-CELL_LINE 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 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 ) .

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 O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-GENEPROD 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 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 ) .

O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-TISSUE O B-TISSUE 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 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 ) .

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 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 ( 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 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-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 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 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 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE 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 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 ) .

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 B-SMALL_MOLECULE O O O O B-CELL_TYPE 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 O O O O B-ORGANISM 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 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 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 ) .

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 B-SMALL_MOLECULE O O O O B-CELL_TYPE 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 B-TISSUE 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 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 ) .

O O O B-ORGANISM O O O O O O O B-CELL_LINE 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 B-TISSUE 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 , 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 ) .

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 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 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 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 ) .

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 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 B-TISSUE 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 - / - 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

O O O B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD 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 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 B-GENEPROD 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 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 ) .

O O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD 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 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 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 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 .

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

O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O B-CELL_TYPE O O O O O B-GENEPROD 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 B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O B-CELL_TYPE 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 - / - 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 ) .

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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-TISSUE O B-GENEPROD 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 B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM 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 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 of DIV4 hippocampal neurons from wild type , Ttll1 - / - Ttll7 - / - mice were incubated with MitoTracker ® ( black dots ) mitochondria movements were recorded in longest neurite ( orange lines ) .

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


B ) of fold - change ( log2 ) of each lipid in HF / HS compared to chow diet . Only lipid species detected in mice are shown .

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B) Heatmap of the fold-change (log2) of each lipid in HF/HS compared to chow diet. Only lipid species detected in all mice are shown.


C ) Examples of different hepatic lipids within one class that are regulated in different directions in HF / HS fed as compared chow fed mice .

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C) Examples of different hepatic lipids within one class that are regulated in different directions in HF/HS fed as compared chow fed mice.


A ) relative genetic variation of hepatic lipidome composition ; lipids were quantified in proportion to total lipidome . Each lipid class is shown in a different color where differences can be observed across strains .

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

A) The relative genetic variation of hepatic lipidome composition; all lipids were quantified in proportion to the total lipidome. Each lipid class is shown in a different color where differences can be observed across the strains.


B ) showing correlations between different lipid species ( x - axis ) abundance of gut microbes ( y - axis ) . Microbes were summarized at of order ( o _ ) , genus ( g _ ) or family ( f _ ) . * = p < 0 . 05 , * * = p < 0 . 01 Pvalues were calculated on significance of regression ( students ) adjusted for multiple comparisons ( FDR = 0 . 05 ) .

O O O O O O B-SMALL_MOLECULE O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap showing correlations between different lipid species (x-axis) and the abundance of gut microbes (y-axis). Microbes were summarized at the levels of order (o_), genus (g_) or family (f_). *=p<0.05, **=p<0.01 Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05).


A ) showing correlations among hepatic lipids .

A) Heatmap showing correlations among hepatic lipids.


B ) showing concordance between different lipid species ( class listed on y - axis ) certain phenotypic traits on x - axis .

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B) Heatmap showing concordance between different lipid species (class listed on y-axis) and certain phenotypic traits on the x-axis.


A ) Manhattan plot of genome - wide association for of hepatic Pex16 transcript , where only significant locus appears directly surrounding genomic location ( red arrow ) . Significant cut - offs are shown for FDR ( blue ) Bonferroni ( red ) . peak SNP ( rs27364570 ) is highlighted with a red box . Y - axis shows - log10 ( pvalue ) vs x - axis showing each SNP measured . P - values for GWAS associations were calculated using FaST - LMM .

O O O O O O O O O O O B-TISSUE 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 O O O O O O 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) Manhattan plot of genome-wide association for the levels of hepatic Pex16 transcript, where the only significant locus appears directly surrounding the genomic location (red arrow). Significant cut-offs are shown for FDR (blue) and Bonferroni (red). The peak SNP (rs27364570) is highlighted with a dark red box. Y-axis shows the -log10(pvalue) vs x-axis showing each SNP measured. P-values for GWAS associations were calculated using FaST-LMM.


B ) Allelic distribution comparing GG vs TT ( x - axis ) for peak SNP of Pex16 association ( rs27364570 ) , where abundance of each LPC species ( y - axis ) showed significantly different depending on allele . P - values for GWAS associations were calculated using FaST - LMM . Boxplots show mean ( middle ) , 25 - 75 % quantiles ( colored box ) 5 - 95 % quantiles ( vertical lines )

O O B-EXP_ASSAY I-EXP_ASSAY 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 O 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) Allelic distribution comparing GG vs TT (x-axis) for the peak SNP of the Pex16 association (rs27364570), where the abundance of each LPC species (y-axis) showed significantly different levels depending on the allele. P-values for GWAS associations were calculated using FaST-LMM. Boxplots show mean (middle line), 25-75% quantiles (colored box) and 5-95% quantiles (vertical lines)


C ) Correlations between expression of hepatic Pex16 with LPC species phenotypic traits . Box color indicates bicor value , where relationships are positive number in each box shows p - value for each correlation . Pvalues were calculated on significance of regression ( students ) adjusted for multiple comparisons ( FDR = 0 . 05 )

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

C) Correlations between expression of hepatic Pex16 with LPC species and phenotypic traits. Box color indicates bicor value, where all relationships are positive and number in each box shows p-value for each correlation. Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)


A ) Manhattan plot of genome - wide association for TAG ( 48 : 2 ) . Red shows Bonferroni - corrected threshold of significance calculated on FaST - LMM pvalues .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-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

A) Manhattan plot of genome-wide association for TAG(48:2). Red line shows Bonferroni-corrected threshold of significance calculated based on FaST-LMM pvalues.


B ) Locuszoom plots showing focused genomic region ( x - axis ) plotted against - log10 ( p - value ) of association for liver mRNA expression of Map2k6 . P - values for GWAS associations were calculated using FaST -

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B) Locuszoom plots showing the focused genomic region (x-axis) plotted against the -log10 (p-value) of association for liver mRNA expression of Map2k6. P-values for GWAS associations were calculated using FaST-LMM


( C ) Correlations between hepatic expression of Map2k6 TAGs identified in study . Blue represents negative correlations .

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(C) Correlations between hepatic expression of Map2k6 and all TAGs identified in the study. Blue represents negative correlations.


E ) Western blots of liver homogenate using anti - Map2k6 anti - β - actin .

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E) Western blots of liver homogenate using anti-Map2k6 and anti-β-actin.


F ) Comparison of total hepatic TAG between Map2k6 - overexpressing mice ( black bar ) control mice ( empty bar ) .

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F) Comparison of total hepatic TAG between Map2k6-overexpressing mice (black bar) and control mice (empty bar).


G ) Differences in total phospholipid ( PC ) , total cholesterol ( TC ) unesterified cholesterol ( UC ) between Map2k6 - overexpressing mice ( black bars ) control mice ( empty bars ) .

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G) Differences in total phospholipid (PC), total cholesterol (TC) and unesterified cholesterol (UC) between Map2k6-overexpressing mice (black bars) and control mice (empty bars).


H ) fat % of both experimental cohorts prior to ( week 0 ) or 8 on a HF / HS diet .

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H) Body fat % of both experimental cohorts prior to (week 0) or 8 weeks on a HF/HS diet.


I , J ) Plasma of glucose ( I ) insulin ( J ) at end of 8 study .

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I,J) Plasma concentration of glucose (I) and insulin (J) at the end of the 8 weeks study.


A ) Manhattan plot of genome - wide association for expression of Ifi203 in liver . Red shows Bonferroni - corrected threshold blue shows an FDR = 0 . 01 pvalue of significance calculated on FaST - LMM pvalues .

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

A) Manhattan plot of genome-wide association for expression of Ifi203 in liver. Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.


B - D ) Allelic variation plots showing peak SNP for Ifi203 expression ( rs31614030 ) at CC or TT allele ( x - axis ) plotted against expression of Ifi203 ( B ) , of hepatic PC ( 38 : 3 ) ( C ) , plasma insulin ( D ) . Red shows Bonferroni - corrected threshold blue shows an FDR = 0 . 01 pvalue of significance calculated on FaST - LMM pvalues .

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

B-D) Allelic variation plots showing the peak SNP for Ifi203 expression (rs31614030) at the CC or TT allele (x-axis) plotted against expression of Ifi203 (B), levels of hepatic PC(38:3) (C), and plasma insulin levels (D). Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.


E - G ) Correlation between parameters listed above showing significant relationships between hepatic Ifi203 PC ( 38 : 3 ) ( E ) , hepatic Ifi203 plasma insulin ( F ) or PC ( 38 : 3 ) plasma insulin ( G ) . Pvalues were calculated on significance of regression ( students ) adjusted for multiple comparisons ( FDR = 0 . 05 )

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

E-G) Correlation between the parameters listed above showing significant relationships between hepatic Ifi203 and PC(38:3) (E), hepatic Ifi203 and plasma insulin levels (F) or PC (38:3) and plasma insulin (G). Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)


Mice receiving control ( open bars ) or shIfi203 ( black bars ) were analyzed for liver expression of Ifi203 ( I ) , total PC in liver ( J ) P - values calculated using a students t - between groups . Data represent means ± ( n = 4 - 5 per group ) .

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

Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Ifi203 (I), total PC levels in liver (J) P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).


Mice receiving control ( open bars ) or shIfi203 ( black bars ) were analyzed for liver expression of Pemt ( K ) , total liver TAG content ( L ) , or plasma insulin ( M ) . P - values calculated using a students t - between groups . Data represent means ± ( n = 4 - 5 per group ) .

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

Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Pemt (K), total liver TAG content (L), or plasma insulin levels (M). P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).


B . Bimolecular complementation between subunits of Dsl COPI coat . BiFC pair β ' - COPVN • Dsl3pVC yielded foci that showed dramatically reduced in COPI binding - deficient dsl1 - 5xWA mutant . Schematic yeast representations depict typical BiFC foci localization patterns . Scale bar 10 μm .

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B. Bimolecular fluorescence complementation between subunits of the Dsl complex and the COPI coat. The BiFC pair β'-COPVN•Dsl3pVC yielded fluorescent foci that showed dramatically reduced fluorescence intensity in the COPI binding-deficient dsl1-5xWA mutant. Schematic yeast cell representations depict the typical BiFC foci localization patterns. Scale bar 10 μm.


C . of β ' - COPVN • Dsl3pVC BiFC signals in wild type in COPI binding - deficient dsl1 - 5xWA dsl1 - Δlasso mutants ( as shown exemplarily in ( B ) ) . Mean values + of at least three independent experiments ( n = 3 - 8 ) are displayed . Statistical was carried out by comparing number of with BiFC signals over integrated thresholds to those without signals ( * = p < 0 , 05 ) . dsl1 mutant strains show a significant decrease in brightness , spot number spot in comparison with their corresponding DSL1 wild type strains ( two - sample t - ) . dsl1 - 5xWA mutation also led to comparable signal reduction in carrying VNI152L variant in β ' - COPVN I152L • Dsl3pVC combination .

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

C. Quantification of the β'-COPVN•Dsl3pVC BiFC signals in wild type cells and in COPI binding-deficient dsl1-5xWA and dsl1-Δlasso mutants (as shown exemplarily in (B)). Mean values + SEM of at least three independent experiments (n=3-8) are displayed. Statistical analysis was carried out by comparing the number of cells with BiFC signals over integrated thresholds to those without signals (*=p<0,05). dsl1 mutant strains show a significant decrease in brightness, spot number and spot size in comparison with their corresponding DSL1 wild type strains (two-sample t-test). The dsl1-5xWA mutation also led to comparable signal reduction in cells carrying the VNI152L variant in the β'-COPVN I152L•Dsl3pVC combination.


D . Subcellular localization of different BiFC - tagged pairs . Dsl1pVN • Dsl3pVC combination involving subunits of ER - resident Dsl1 exhibited typical ER localization . Sec16pVN • Sec16pVC signals in a diploid heterozygous displayed an pattern typical for ER exit sites , while diploid carrying α - COPVN • α - COPVC BiFC pair showed a Golgi - like pattern . Scale bar 10 μm .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O

D. Subcellular localization of different BiFC-tagged protein pairs. The Dsl1pVN•Dsl3pVC combination involving subunits of the ER-resident Dsl1 complex exhibited typical ER localization. Sec16pVN•Sec16pVC signals in a diploid heterozygous strain displayed an pattern typical for ER exit sites, while diploid cells carrying the α-COPVN•α-COPVC BiFC pair showed a Golgi-like fluorescence pattern. Scale bar 10 μm.


E . localization of COPI • Dsl BiFC foci compared to autophagy marker Ape1p . Double of BiFC pair β ' - COPVN • Dsl3pVC autophagy marker mRFPApe1p are superimposed with DIC image . BiFC - YFP signals are pseudocolored green . signals appeared distinct from each other . Also no co - localization was observed when we analyzed COPI • COPII ( Sec16p ) BiFC spots in expressing mRFP tagged APE1 . Scale bar 10 µm .

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

E. The localization of COPI•Dsl BiFC foci compared to the autophagy marker Ape1p. Double fluorescence micrographs of the BiFC pair β'-COPVN•Dsl3pVC and the autophagy marker mRFPApe1p are superimposed with the DIC image. BiFC-YFP signals are pseudocolored green. Fluorescent signals appeared distinct from each other. Also no co-localization was observed when we analyzed COPI•COPII(Sec16p) BiFC spots in cells expressing mRFP tagged APE1. Scale bar 10 µm.


F . Growth effects of Venus fragment tags in dsl1 mutant . Serial tenfold dilutions of liquid cultures were spotted on agar plates incubated at 30 ° C for two days . in second row show that β ' COPVN producing expressing dsl1 - 5xWA mutation could not grow . complementation of β ' COPVN by its cognate interaction partners Dsl1pVC Dsl3pVC , but not by non - cognate BiFC partners ( Sec24pVC , Sec16pVC or VCRer1p ) , suppressed these growth defects . Spot assays of exemplary BiFC partners are depicted . Asterisk : Dsl1pVC carrying dsl1 - 5xWA defect was expressed from a plasmid . See Fig EV1 , EV2 Appendix Fig S1 for full data display .

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

F. Growth effects of Venus fragment tags in dsl1 mutant cells. Serial tenfold dilutions of liquid cell cultures were spotted on agar plates and incubated at 30 °C for two days. The images in the second row show that β'COPVN producing cells expressing the dsl1-5xWA mutation could not grow. The complementation of β'COPVN by its cognate interaction partners Dsl1pVC and Dsl3pVC, but not by non-cognate BiFC partners (Sec24pVC, Sec16pVC or VCRer1p), suppressed these growth defects. Spot assays of exemplary BiFC partners are depicted. Asterisk: Dsl1pVC carrying the dsl1-5xWA defect was expressed from a plasmid. See Fig EV1, EV2 and Appendix Fig S1 for full data display.


G . BiFC signal quantifications of strains presented in ( F ) . β ' - COPVN yielded BiFC signals with tested complementation partners . This rules out possibility that lack of suppression observed in F simply reflects inability of BiFC partners to form a , shows that successful complementation of split - YFP fragments does not per se suppress synthetic lethal effect of VN tag in dsl1 - 5xWA . Mean values + of at least three independent experiments ( n = 3 - 8 ) are displayed .

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

G. BiFC signal quantifications of the strains presented in (F). β'-COPVN yielded BiFC fluorescence signals with all tested complementation partners. This rules out the possibility that the lack of suppression observed in F simply reflects the inability of the BiFC partners to form a complex, and shows that the successful complementation of the split-YFP fragments does not per se suppress the synthetic lethal effect of the VN tag in dsl1-5xWA cells. Mean values + SEM of at least three independent experiments (n=3-8) are displayed.


H . BiFC signal intensities greatly depend on which BiFC combination is analyzed . intensities of COPI • Dsl BiFC pairs ( left part of H ) were much higher than those of COPII • Dsl BiFC pairs ( right part of H ) .

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H. BiFC signal intensities greatly depend on which BiFC combination is analyzed. Fluorescence intensities of COPI•Dsl BiFC pairs (left part of H) were much higher than those of COPII•Dsl BiFC pairs (right part of H).


I . Localization of Dsl1pCFP in β ' - COPVN • Dsl3pVCcells . An overall ER localization of Dsl1pCFP was retained in β ' - COPVN • Dsl3pVCcells , while some of signal was found in foci . BiFC - YFP signals are pseudocolored red . Scale bar 10 µm .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

I. Localization of Dsl1pCFP in β'-COPVN•Dsl3pVCcells. An overall ER localization of Dsl1pCFP was retained in β'-COPVN•Dsl3pVCcells, while some of the signal was found in the foci. BiFC-YFP signals are pseudocolored red. Scale bar 10 µm.


A . Dsl1pVN • ε - COPVC BiFC signal localization pattern throughout budding cycle . Time - lapse of agarose - embedded were recorded at RT with continuous PM Glc + ura medium supply . Arrowheads mark positions of BiFC foci . signals exhibited a characteristic reproducible localization pattern , which favored sites of high material turnover during budding . Scale bar 10 µm .

O O B-GENEPROD 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 O 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. Dsl1pVN•ε-COPVC BiFC signal localization pattern throughout the budding cycle. Time-lapse images of agarose-embedded cells were recorded at RT with continuous PM Glc+ura medium supply. Arrowheads mark positions of BiFC foci. Fluorescence signals exhibited a characteristic and reproducible localization pattern, which favored sites of high material turnover during budding. Scale bar 10 µm.


B . Dsl1pVN • ε - COPVC BiFC foci showed different subcellular distribution depending on growth status of . were cultured in PM + ura medium with or without 2 % glucose at 30 ° C for 90 min , imaged . BiFC signals dispersed under glucose starvation , resulting in an ER exit site - like pattern . Scale bar 10 µm .

O O B-GENEPROD 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 B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O

B. Dsl1pVN•ε-COPVC BiFC foci showed different subcellular distribution depending on the growth status of cells. Cells were cultured in PM+ura medium with or without 2 % glucose at 30 °C for 90 min, and imaged. BiFC signals dispersed under glucose starvation, resulting in an ER exit site-like fluorescence pattern. Scale bar 10 µm.


C . effect of carbon source depletion on COPI • Dsl BiFC signal polarization . carrying Dsl1pVN • ε - COPVC were starved in glucose - free PM + ura medium for 90 min . Then medium was replaced by PM Glc + ura with 2 % glucose , while imaging continuously at 1 min intervals . Two exemplary are shown ( upper lower panel ) . Starvation - induced dispersed BiFC signals reversibly returned to polarized distribution within minutes after addition of glucose - containing medium . Arrowheads mark point of signal repolarization , arrows indicate resumed bud outgrowth . Scale bar 5 µm .

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

C. The effect of carbon source depletion on COPI•Dsl BiFC signal polarization. Cells carrying Dsl1pVN•ε-COPVC were starved in glucose-free PM+ura medium for 90 min. Then the medium was replaced by PM Glc+ura with 2 % glucose, while imaging the cells continuously at 1 min intervals. Two exemplary cells are shown (upper and lower panel). Starvation-induced dispersed BiFC signals reversibly returned to polarized distribution within minutes after addition of glucose-containing medium. Arrowheads mark the point of signal repolarization, arrows indicate resumed bud outgrowth. Scale bar 5 µm.


D . βʼ - COPVN • Dsl3pVC BiFC signals during mating . of BiFC pair β ' - COPVN • Dsl3pVC ( green ) are superimposed with DIC image ( grey ) . Two haploid strains carrying βʼ - COPVN • Dsl3pVC were allowed to mate in YEPD liquid culture for 2 h at 30 ° C . Then time - lapse recordings were made in agarose embedding at RT with continuous PM Glc + ura medium supply . Arrowheads mark positions of BiFC foci . signals exhibited a characteristic reproducible localization at sites of high material turnover during zygote formation . Scale bar 10 µm .

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

D. βʼ-COPVN•Dsl3pVC BiFC signals during mating. Fluorescence micrographs of the BiFC pair β'-COPVN•Dsl3pVC (green) are superimposed with the DIC image (grey). Two haploid strains carrying βʼ-COPVN•Dsl3pVC were allowed to mate in YEPD liquid culture for 2 h at 30 °C. Then time-lapse recordings were made in agarose embedding at RT with continuous PM Glc+ura medium supply. Arrowheads mark positions of BiFC foci. Fluorescence signals exhibited a characteristic and reproducible localization at sites of high material turnover during zygote formation. Scale bar 10 µm.


E . Comparison of BiFC localization pattern in COPI • Dsl COPI • COPI strains . Time - lapse of agarose - embedded carrying COPI • Dsl BiFC pair βʼ - COPVN • Dsl3pVC or COPI • COPI BiFC pair βʼ - COPVN • α - COPVC were recorded at RT with continuous PM Glc + ura medium supply at 5 min intervals . Single pseudokymographs were generated by concatenating central section of ( sliced along pole axis , frames at double width of bud neck ) of a time - lapse dataset . COPI • Dsl showed one single dominant spot at bud tip later bud neck . In contrast , COPI • COPI additionally exhibited multiple spots with no apparent polarization tendency , which were localized mainly in mother . Schematic yeast representations depict typical BiFC foci localization pattern of strains . Scale bar 10 µm .

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

E. Comparison of the BiFC localization pattern in COPI•Dsl and COPI•COPI strains. Time-lapse images of agarose-embedded cells carrying the COPI•Dsl BiFC pair βʼ-COPVN•Dsl3pVC or the COPI•COPI BiFC pair βʼ-COPVN•α-COPVC were recorded at RT with continuous PM Glc+ura medium supply at 5 min intervals. Single cell pseudokymographs were generated by concatenating the central section of the cell (sliced along the pole axis, frames at double the width of the bud neck) of a time-lapse dataset. The COPI•Dsl strain showed one single dominant spot at the bud tip and later the bud neck. In contrast, the COPI•COPI strain additionally exhibited multiple spots with no apparent polarization tendency, which were localized mainly in the mother cell. Schematic yeast cell representations depict the typical BiFC foci localization pattern of the strains. Scale bar 10 µm.