Figure 3 ( A ) Representative maximum intensity Z - projections from confocal fluorescence imaging ( thickness of ~ 10 µm , in steps of 0 . 21 µm ) of HEK293 cells after mock or 1 , 6 - hexanediol ( 1 , 6 - HD ) treatment for 15 min stained for endogenous TDP - 43 ( endTDP - 43 ) . Scale bar : 5 µm . ( B ) Quantification of three - dimensional ( 3D ) reconstructions from the images of the conditions shown in A depicting the number of nuclear condensates per cell . N = 14 - 23 cells . Unpaired two - tailed t - test . ( C ) Fluorescence microscopy images of 10 μM purified full - length TDP - 43 and its oligomerization - deficient counterpart showing different abilities to undergo LLPS and its disruption by 1 , 6 - HD treatment for 10 min . Scale bar : 10 μm . ( D ) Quantification of the number of condensates in the conditions shown in C per 4000 µm2 field . N = 10 independent experiments . Kruskal - Wallis test with Dunn ' s multiple comparisons post hoc test . ( H ) GFP - TDP - 43 expression was induced with DOX for 4 h before crosslinking protein - protein interactions with DSG and subsequent analysis by western blot . * , * * and * * * indicate GFP - TDP - 43 monomers , dimers and trimers , respectively . o , oo , ooo and oooo indicate endTDP - 43 monomers , dimers , trimers and tetramers . ( I ) Quantification of GFP - TDP - 43 dimer / monomer ratio based on the GFP signal from H . N = 3 independent experiments . Repeated measures one - way ANOVA with Greenhouse - Geisser correction and Tukey ' s multiple comparisons post hoc test . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . Graph bars represent mean ± SD .

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

Figure 3(A) Representative maximum intensity Z-projections from confocal fluorescence imaging (thickness of ~10 µm, in steps of 0.21 µm) of HEK293 cells after mock or 1,6-hexanediol (1,6-HD) treatment for 15 min stained for endogenous TDP-43 (endTDP-43). Scale bar: 5 µm. (B) Quantification of three-dimensional (3D) reconstructions from the images of the conditions shown in A depicting the number of nuclear condensates per cell. N=14-23 cells. Unpaired two-tailed t-test.(C) Fluorescence microscopy images of 10 μM purified full-length TDP-43 and its oligomerization-deficient counterpart showing different abilities to undergo LLPS and its disruption by 1,6-HD treatment for 10 min. Scale bar: 10 μm. (D) Quantification of the number of condensates in the conditions shown in C per 4000 µm2 field. N=10 independent experiments. Kruskal-Wallis test with Dunn's multiple comparisons post hoc test.(H) GFP-TDP-43 expression was induced with DOX for 4 h before crosslinking protein-protein interactions with DSG and subsequent analysis by western blot. *, ** and *** indicate GFP-TDP-43 monomers, dimers and trimers, respectively. o, oo, ooo and oooo indicate endTDP-43 monomers, dimers, trimers and tetramers. (I) Quantification of GFP-TDP-43 dimer/monomer ratio based on the GFP signal from H. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test. * p<0.05, ** p<0.01, **** p<0.0001. Graph bars represent mean ± SD.


Figure 4 ( A ) HEK293 cells were treated with actinomycin D ( ActD ) for 4 h to inhibit transcription before treatment with the protein - protein cross - linker DSG and western blot analysis . o , oo and oooo indicate endogenous TDP - 43 ( endTDP - 43 ) monomers , dimers and trimers , respectively . ( B ) Quantification of the endTDP - 43 signal from A . N = 3 independent experiments . Paired two - tailed t - test . ( C ) Representative image of confocal fluorescence microscopy of HEK293 cells and neurons treated with 5 μg / ml ActD for 4h . Scale bar : 5 µm . ( D ) Single - plane quantification of the number of nuclear condensates per neuron in the conditions described in C . N = 25 - 26 cells . Mann - Whitney U test . ( F ) PLA using a monoclonal anti - GFP antibody reveals the localization of GFP - TDP - 43 dimers in the isogenic cell lines upon protein expression with doxycycline ( DOX ) for 48 h . Note the absence of dimers in the oligomerization - deficient variants ( 6M and 6M & RRMm ) . Scale bar : 5 µm . ( G ) Quantification of the nuclear PLA signal shown in F correlated to the protein expression levels of the different TDP - 43 variants , measured as the mean GFP fluorescence . N = 11 - 13 cells .

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

Figure 4(A) HEK293 cells were treated with actinomycin D (ActD) for 4 h to inhibit transcription before treatment with the protein-protein cross-linker DSG and western blot analysis. o, oo and oooo indicate endogenous TDP-43 (endTDP-43) monomers, dimers and trimers, respectively. (B) Quantification of the endTDP-43 signal from A. N=3 independent experiments. Paired two-tailed t-test.(C) Representative image of confocal fluorescence microscopy of HEK293 cells and neurons treated with 5 μg/ml ActD for 4h. Scale bar: 5 µm. (D) Single-plane quantification of the number of nuclear condensates per neuron in the conditions described in C. N=25-26 cells. Mann-Whitney U test.(F) PLA using a monoclonal anti-GFP antibody reveals the localization of GFP-TDP-43 dimers in the isogenic cell lines upon protein expression with doxycycline (DOX) for 48 h. Note the absence of dimers in the oligomerization-deficient variants (6M and 6M&RRMm). Scale bar: 5 µm.(G) Quantification of the nuclear PLA signal shown in F correlated to the protein expression levels of the different TDP-43 variants, measured as the mean GFP fluorescence. N=11-13 cells.


Figure 5 ( A ) Representative confocal microscopy images of HEK293 cells probed for endogenous TDP - 43 ( endTDP - 43 ) and different subnuclear compartment markers . Lower panel shows the three - dimensional ( 3D ) reconstruction of the endTDP - 43 condensates and the indicated nuclear body obtained from confocal Z - stacks . Scale bar : 5 µm . ( B ) Quantification of the 3D reconstructions shown in A depicting the percentage of nuclear TDP - 43 condensates colocalizing with markers of subnuclear compartments . N = 14 - 38 cells . ( C ) Quantification of the 3D reconstructions shown in A depicting the percentage of each of the analyzed subnuclear compartments that colocalize with endTDP - 43 . N = 14 - 38 cells . ( D ) Representative confocal microscopy images of human neurons showing the presence of endTDP - 43 in Cajal bodies . The neuron - specific marker MAP2 is shown in cyan . Scale bar : 20 µm ( inset : 5 µm ) . ( E ) Representative confocal microscopy images of the isogenic HEK293 lines expressing the different GFP - TDP - 43 variants for 24 h and stained for the Cajal body marker coilin . Scale bar : 5 µm . ( F ) Representative confocal microscopy images of the isogenic HEK293 lines expressing the different GFP - TDP - 43 variants for 24 h and hybridized with a fluorescent NEAT1 probe to mark the paraspeckles . Scale bar : 5 µm .

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

Figure 5(A) Representative confocal microscopy images of HEK293 cells probed for endogenous TDP-43 (endTDP-43) and different subnuclear compartment markers. Lower panel shows the three-dimensional (3D) reconstruction of the endTDP-43 condensates and the indicated nuclear body obtained from confocal Z-stacks. Scale bar: 5 µm. (B) Quantification of the 3D reconstructions shown in A depicting the percentage of nuclear TDP-43 condensates colocalizing with markers of subnuclear compartments. N=14-38 cells. (C) Quantification of the 3D reconstructions shown in A depicting the percentage of each of the analyzed subnuclear compartments that colocalize with endTDP-43. N=14-38 cells.(D) Representative confocal microscopy images of human neurons showing the presence of endTDP-43 in Cajal bodies. The neuron-specific marker MAP2 is shown in cyan. Scale bar: 20 µm (inset: 5 µm).(E) Representative confocal microscopy images of the isogenic HEK293 lines expressing the different GFP-TDP-43 variants for 24 h and stained for the Cajal body marker coilin. Scale bar: 5 µm. (F) Representative confocal microscopy images of the isogenic HEK293 lines expressing the different GFP-TDP-43 variants for 24 h and hybridized with a fluorescent NEAT1 probe to mark the paraspeckles. Scale bar: 5 µm.


Figure 6 ( A ) Volcano plots showing alternative splicing ( AS ) events upon expression of GFP - TDP - 43 variants for 48 h . ( E ) Western blot analysis of the isogenic HEK293 after GFP - TDP - 43 expression for 48 h showing that only the WT variant regulates endTDP - 43 levels . ( F ) Quantification of the endTDP - 43 signal from E . N = 4 independent experiments . Two - way ANOVA with Tukey ' s multiple comparisons post hoc test . ( G ) Tripartite GFP complementation assay involving the co - transfection of a pair of N - terminally T10 - and T11 - tagged TDP - 43 constructs in HeLa cells subjected to arsenite stress for 30 min and incubated with recombinant GFP1 - 9 after fixation to label T10 - and T11 - TDP - 43 dimers . TriFC : trimolecular fluorescence complementation . Scale bar : 10 µm . ( H ) Quantification of the trimolecular fluorescence complementation ( triFC ) signal of GFP in the TIA - 1 - marked SGs from the images shown in G . N = 3 independent experiments . Repeated measures one - way ANOVA with Greenhouse - Geisser correction and Tukey ' s multiple comparisons post hoc test . ( I ) Expression of GFP - TDP - 43 mutNLS variants was induced with doxycycline ( DOX ) for 4 h before crosslinking protein - protein interactions with DSG and subsequent analysis by western blot . * and * * indicate GFP - TDP - 43 monomers and dimers , respectively . ( J ) Quantification of the GFP signal from I . N = 3 independent experiments . Repeated measures one - way ANOVA with Greenhouse - Geisser correction and Tukey ' s multiple comparisons post hoc test . ( K ) After expression of GFP - TDP - 43 mutNLS variants for 48 h , the isogenic lines were treated with DSG to cross - link protein - protein interactions before performing nucleocytoplasmic fractionation and western blot analysis . * and * * indicate GFP - TDP - 43 monomers and dimers , respectively .

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

Figure 6(A) Volcano plots showing alternative splicing (AS) events upon expression of GFP-TDP-43 variants for 48 h.(E) Western blot analysis of the isogenic HEK293 after GFP-TDP-43 expression for 48 h showing that only the WT variant regulates endTDP-43 levels. (F) Quantification of the endTDP-43 signal from E. N=4 independent experiments. Two-way ANOVA with Tukey's multiple comparisons post hoc test.(G) Tripartite GFP complementation assay involving the co-transfection of a pair of N-terminally T10- and T11-tagged TDP-43 constructs in HeLa cells subjected to arsenite stress for 30 min and incubated with recombinant GFP1-9 after fixation to label T10- and T11-TDP-43 dimers. TriFC: trimolecular fluorescence complementation. Scale bar: 10 µm. (H) Quantification of the trimolecular fluorescence complementation (triFC) signal of GFP in the TIA-1-marked SGs from the images shown in G. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test.(I) Expression of GFP-TDP-43 mutNLS variants was induced with doxycycline (DOX) for 4 h before crosslinking protein-protein interactions with DSG and subsequent analysis by western blot. * and ** indicate GFP-TDP-43 monomers and dimers, respectively. (J) Quantification of the GFP signal from I. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test.(K) After expression of GFP-TDP-43 mutNLS variants for 48 h, the isogenic lines were treated with DSG to cross-link protein-protein interactions before performing nucleocytoplasmic fractionation and western blot analysis. * and ** indicate GFP-TDP-43 monomers and dimers, respectively.


Figure 7 ( A ) Representative confocal microscopy images of the isogenic GFP - TDP - 43 lines after 48 h of expression , treated with the proteasome inhibitor MG132 for the last 24 h and stained for lamin B to mark the nuclear envelope . Note the different localization of TDP - 43 inclusions in the oligomerization - ( 6M and 6M & RRMm ) versus RNA binding - deficient ( RRMm ) variants . Scale bar : 20 µm ( 5 µm for inset ) . ( B ) Quantification of the localization of GFP - TDP - 43 inclusions after MG132 treatment for the different variants in the isogenic HEK293 lines as shown in A . Represented values are averages from N = 3 independent experiments , with N = 189 - 497 cells quantified per condition and replicate . ( C ) Representative maximum intensity Z - projections from confocal fluorescence imaging ( thickness of 4 µm , in steps of 1 µm ) of human neurons transduced with TDP - 43 - HA variants and treated overnight with the proteasome inhibitor MG132 . Scale bar : 10 µm . ( D ) Quantification of the differentially localized TDP - 43 - HA inclusions in human neurons as described in C . Represented values correspond to the quantification of N = 85 - 97 neurons from two independent experiments . Nuclei are stained with DAPI The neuron - specific marker MAP2 is shown in magenta or cyan ( E ) Representative maximum intensity Z - projections from confocal fluorescence imaging ( thickness of 4 µm , in steps of 1 µm ) of the same experimental conditions as shown in A , with the addition of a DMSO control for MG132 treatment . GFP - TDP - 43 - expressing cells were double immunolabeled for TDP - 43 phosphorylated at the S403 / 404 and S409 / 410 epitope . Note the absence of phospho - S409 / 410 immunopositivity in all conditions and positivity for phospho - S403 / 404 in cytoplasmic inclusions and a subset of nuclear GFP - TDP - 43 RRMm inclusions ( appointed by arrowheads ) . Scale bar : 10 µm . Nuclei are stained with DAPI ( F - G ) Representative maximum intensity Z - projections from confocal fluorescence imaging ( thickness of 4 µm , in steps of 1 µm ) of the same experimental conditions as shown in C , with the addition of a DMSO control . TDP - 43 - HA expressing human neurons were stained for either phospho - S403 / 404 ( F ) or phospho - S409 / 410 ( G ) . Scale bar : 10 µm . Note the re - localization of the phospho - S403 / 404 signal from neuronal nuclei to inclusions ( F ) and the presence of phospho - S409 / 410 signal in only a subset of aggregate - bearing neurons ( G ) . Nuclei are stained with DAPI The neuron - specific marker MAP2 is shown in magenta or

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

Figure 7(A) Representative confocal microscopy images of the isogenic GFP-TDP-43 lines after 48 h of expression, treated with the proteasome inhibitor MG132 for the last 24 h and stained for lamin B to mark the nuclear envelope. Note the different localization of TDP-43 inclusions in the oligomerization- (6M and 6M&RRMm) versus RNA binding-deficient (RRMm) variants. Scale bar: 20 µm (5 µm for inset). (B) Quantification of the localization of GFP-TDP-43 inclusions after MG132 treatment for the different variants in the isogenic HEK293 lines as shown in A. Represented values are averages from N=3 independent experiments, with N=189-497 cells quantified per condition and replicate.(C) Representative maximum intensity Z-projections from confocal fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of human neurons transduced with TDP-43-HA variants and treated overnight with the proteasome inhibitor MG132. Scale bar: 10 µm. (D) Quantification of the differentially localized TDP-43-HA inclusions in human neurons as described in C. Represented values correspond to the quantification of N=85-97 neurons from two independent experiments. Nuclei are stained with DAPI The neuron-specific marker MAP2 is shown in magenta or cyan(E) Representative maximum intensity Z-projections from confocal fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of the same experimental conditions as shown in A, with the addition of a DMSO control for MG132 treatment. GFP-TDP-43-expressing cells were double immunolabeled for TDP-43 phosphorylated at the S403/404 and S409/410 epitope. Note the absence of phospho-S409/410 immunopositivity in all conditions and positivity for phospho-S403/404 in cytoplasmic inclusions and a subset of nuclear GFP-TDP-43 RRMm inclusions (appointed by arrowheads). Scale bar: 10 µm. Nuclei are stained with DAPI(F-G) Representative maximum intensity Z-projections from confocal fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of the same experimental conditions as shown in C, with the addition of a DMSO control. TDP-43-HA expressing human neurons were stained for either phospho-S403/404 (F) or phospho-S409/410 (G). Scale bar: 10 µm. Note the re-localization of the phospho-S403/404 signal from neuronal nuclei to inclusions (F) and the presence of phospho-S409/410 signal in only a subset of aggregate-bearing neurons (G). Nuclei are stained with DAPI The neuron-specific marker MAP2 is shown in magenta or cyan


Figure 8 ( B ) Representative fluorescent confocal microscopy images of FRAP experiments of the GFP - TDP - 43 aggregates formed upon MG132 treatment as described in Figure 7A . FRAP was performed in the areas highlighted in magenta . Numbers in images indicate the experimental time point in seconds ( ' ' ) of MG132 treatment . Scale bar : 5 µm . Bottom panel : Measured GFP intensity values are expressed as a fraction of the average pre - bleach fluorescence levels . ( D ) Representative confocal microscopy images of the isogenic GFP - TDP - 43 lines at the endpoint ( 24 h ) of MG132 treatment of the experimental conditions described in Figure 7A and stained for vimentin . Scale bar : 5 µm . Cell nuclei are visualized with DAPI ( E ) Representative confocal microscopy images of the isogenic GFP - TDP - 43 lines at the endpoint ( 24 h ) of MG132 treatment of the experimental conditions described in Figure 7A and stained for p62 . Cell nuclei are visualized with

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

Figure 8(B) Representative fluorescent confocal microscopy images of FRAP experiments of the GFP-TDP-43 aggregates formed upon MG132 treatment as described in Figure 7A. FRAP was performed in the areas highlighted in magenta. Numbers in images indicate the experimental time point in seconds ('') of MG132 treatment. Scale bar: 5 µm. Bottom panel: Measured GFP intensity values are expressed as a fraction of the average pre-bleach fluorescence levels.(D) Representative confocal microscopy images of the isogenic GFP-TDP-43 lines at the endpoint (24 h) of MG132 treatment of the experimental conditions described in Figure 7A and stained for vimentin. Scale bar: 5 µm. Cell nuclei are visualized with DAPI(E) Representative confocal microscopy images of the isogenic GFP-TDP-43 lines at the endpoint (24 h) of MG132 treatment of the experimental conditions described in Figure 7A and stained for p62. Cell nuclei are visualized with DAPI


Figure 1 ( B ) Immunoblot analysis of SLC25A33 and SLC25A36 depletion in the indicated knockout ( KO ) and double knockout ( DKO ) HeLa cells ( left ) alongside the relative mtDNA levels of two monoclonal SLC25A33 / SLC25A36 DKO cell lines calculated by qPCR ( CYTB / ACTB ; right ) ( n = 3 independent cultures ) . ( D ) Representative images taken from the arrayed CRISPR - SpCas9 screen of SLC25A33 / SLC25A36 DKO cells showing one field of view from wells transfected with non - targeting control ( NTC ) sgRNA or TFAM sgRNA ( scale bar = 20 µm ) ( G ) Relative mtDNA levels in NME4 KO and NME4 / SLC25A33 / SLC25A36 triple KO HeLa ( left ) or NME6 KO and two clones of NME6 / SLC25A33 / SLC25A36 triple KO HeLa ( right ) . MtDNA calculated by qPCR ( CYTB / ACTB ) and presented as log2 fold change compared to levels in WT HeLa cells ( n = 4 - 5 independent cultures ) . ( H ) Representative immunoblot analysis of the indicated HeLa cell lines ( UQCRC2 , Ubiquinol - cytochrome C reductase core protein 2 ; MT - CO2 , mitochondrial encoded cytochrome c oxidase II ; ATP5A , ATP synthase F1 subunit alpha ) .

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

Figure 1(B) Immunoblot analysis of SLC25A33 and SLC25A36 depletion in the indicated knockout (KO) and double knockout (DKO) HeLa cells (left) alongside the relative mtDNA levels of two monoclonal SLC25A33/SLC25A36 DKO cell lines calculated by qPCR (CYTB / ACTB ;right) (n=3 independent cultures).(D) Representative images taken from the arrayed CRISPR-SpCas9 screen of SLC25A33/SLC25A36 DKO cells showing one field of view from wells transfected with non-targeting control (NTC) sgRNA or TFAM sgRNA (scale bar = 20 µm)(G) Relative mtDNA levels in NME4 KO and NME4/SLC25A33/SLC25A36 triple KO HeLa (left) or NME6 KO and two clones of NME6/SLC25A33/SLC25A36 triple KO HeLa (right). MtDNA calculated by qPCR (CYTB / ACTB) and presented as log2 fold change compared to levels in WT HeLa cells (n = 4-5 independent cultures).(H) Representative immunoblot analysis of the indicated HeLa cell lines (UQCRC2, Ubiquinol-cytochrome C reductase core protein 2; MT-CO2, mitochondrial encoded cytochrome c oxidase II; ATP5A, ATP synthase F1 subunit alpha).


Figure 2 ( A ) The growth rate of NME6 KO and NME6 KO + NME6 - MycFlag HeLa relative to WT HeLa cells incubated in DMEM containing 25 mM glucose ( log2 ; n = 13 independent cultures ) . ( B ) Violin plot of gene effects of NME3 , NME4 or NME6 depletion in 1086 cell lines from the Cancer Cell Line Encyclopaedia ( CCLE ) determined by CRISPR screening ( DepMap 22Q2 Public + Score , Chronos ; solid line denotes median , dotted line denotes 25 % quartile ) . ( D ) Representative live - cell images of the indicated cell lines grown in Human Plasma Like Medium ( HPLM ) ( left ) and calculated cell death after 96 h ( right ) . Cell confluency is depicted with the yellow mask and dead cells are identified by SYTOX green staining in purple ( n = 3 independent cultures ) .

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

Figure 2(A) The growth rate of NME6 KO and NME6 KO + NME6-MycFlag HeLa relative to WT HeLa cells incubated in DMEM containing 25 mM glucose (log2; n=13 independent cultures).(B) Violin plot of gene effects of NME3, NME4 or NME6 depletion in 1086 cell lines from the Cancer Cell Line Encyclopaedia (CCLE) determined by CRISPR screening (DepMap 22Q2 Public+Score, Chronos; solid line denotes median, dotted line denotes 25 % quartile).(D) Representative live-cell images of the indicated cell lines grown in Human Plasma Like Medium (HPLM) (left) and calculated cell death after 96 h (right). Cell confluency is depicted with the yellow mask and dead cells are identified by SYTOX green staining in purple (n=3 independent cultures).


Figure 3 ( A ) Oxygen consumption rates ( OCR ) and extracellular acidification rates ( ECAR ) of the indicated HeLa cell lines during a mitochondrial stress test with inhibitor treatments at the indicated timepoints . P - values for OCR : two - way ANOVA P - value ( time ) = < 0 . 0001 , P - value ( genotype ) = < 0 . 0001 , P - value ( interaction ) = < 0 . 0001 ; P - values for ECAR : two - way ANOVA P - value ( time ) = < 0 . 0001 , P - value ( genotype ) = < 0 . 001 , P - value ( interaction ) = 0 . 0517 ; P - values for genotype are shown ( Oligo , oligomycin ; FCCP , carbonyl cyanide - p - trifluoromethoxyphenylhydrazone ; Rot + AA , rotenone and antimycin a ; n = 3 independent experiments ) . ( B ) Heat map representation of z - scores of log2 - transformed protein intensities determined by quantitative mass spectrometry and filtered for mitochondrial proteins according to MitoCarta 3 . 0 The proteins shown clustered together due to their significant ( permutation - based FDR < 0 . 05 , s0 = 0 . 1 ) depletion in NME6 KO HeLa cells compared to WT and NME6 KO + NME6 - MycFlag HeLa cells . OXPHOS subunits are indicated in the right column ( n = 4 independent cultures ) . ( E ) Immunoblot analysis of WT HeLa cells , NME6 KO cells and NME6 KO cells expressing NME6 - MycFlag ( WT ) or NME6H137N - MycFlag ( H137N ) . ( F ) Immunoblot analysis of WT cells and two NME6 KO clones ( # 1 , # 2 ) generated in three different liver cancer cell lines : HLE , Huh6 and HepG2 . ( H ) The relative growth of WT and NME6 KO liver cancer cell lines monitored on each day ( d ) using an ATP luminescence assay . P - values for HLE cells : two - way ANOVA P - value ( time ) = < 0 . 0001 , P - value ( genotype ) = < 0 . 0001 , P - value ( interaction ) = < 0 . 0001 ; P - values for Huh6 cells : two - way ANOVA P - value ( time ) = < 0 . 0001 , P - value ( genotype ) = 0 . 0005 , P - value ( interaction ) = < 0 . 0001 ; p - values for genotype are shown ( n = 4 independent cultures ) .

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

Figure 3(A) Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of the indicated HeLa cell lines during a mitochondrial stress test with inhibitor treatments at the indicated timepoints. P-values for OCR: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = <0.0001; P-values for ECAR:two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.001, P-value (interaction) = 0.0517; P-values for genotype are shown (Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot+AA, rotenone and antimycin a; n=3 independent experiments).(B) Heat map representation of z-scores of log2-transformed protein intensities determined by quantitative mass spectrometry and filtered for mitochondrial proteins according to MitoCarta 3.0 The proteins shown clustered together due to their significant (permutation-based FDR < 0.05, s0 = 0.1) depletion in NME6 KO HeLa cells compared to WT and NME6 KO + NME6-MycFlag HeLa cells. OXPHOS subunits are indicated in the right column (n=4 independent cultures).(E) Immunoblot analysis of WT HeLa cells, NME6 KO cells and NME6 KO cells expressing NME6-MycFlag (WT) or NME6H137N-MycFlag (H137N).(F) Immunoblot analysis of WT cells and two NME6 KO clones (#1, #2) generated in three different liver cancer cell lines: HLE, Huh6 and HepG2.(H) The relative growth of WT and NME6 KO liver cancer cell lines monitored on each day (d) using an ATP luminescence assay. P-values for HLE cells: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = <0.0001; P-values for Huh6 cells: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = 0.0005, P-value (interaction) = <0.0001;p-values for genotype are shown (n=4 independent cultures).


Figure 4 ( A ) Representative mitochondrial translation assay monitored by the incorporation of 35S methionine and cysteine into the indicated mtDNA - encoded proteins followed by autoradiography ( top panel ) . MtDNA encoded proteins are labelled according to expected size and NME6 and SDHA immunoblots are shown below . ( B ) Quantification of 35S methionine and cysteine incorporation into all mtDNA - encoded proteins labelled in ( B ) relative to WT HeLa cells at 15 min ( log2 ; mitochondrial preparations from n = 3 independent cultures ) . ( C ) Immunofluorescence of NME6 with mtDNA ( top ) or bromouridine ( BrU ) labelled nascent mtRNA ( bottom ) in HeLa cells imaged by confocal microscopy . Relative fluorescence intensities were calculated from linescans generated within the mitochondrial regions indicated by dotted lines . ( E ) MtDNA - encoded transcript levels analysed by qRT - PCR in NME6 KO and NME6 KO + NME6 - MycFlag cells relative to WT HeLa cells . P - values for mtDNA - encoded transcripts : two - way ANOVA P - value ( transcript ) = < 0 . 0001 , P - value ( genotype ) = < 0 . 0001 , P - value ( interaction ) = < 0 . 0001 ( log2 ; n = 3 independent cultures ) . ( F ) Nuclear DNA - encoded transcript levels analysed by qRT - PCR in NME6 KO and NME6 KO + NME6 - MycFlag HeLa cells relative to WT ( log2 ; n = 3 independent cultures ) .

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

Figure 4(A) Representative mitochondrial translation assay monitored by the incorporation of 35S methionine and cysteine into the indicated mtDNA-encoded proteins followed by autoradiography (top panel). MtDNA encoded proteins are labelled according to expected size and NME6 and SDHA immunoblots are shown below. (B) Quantification of 35S methionine and cysteine incorporation into all mtDNA-encoded proteins labelled in (B) relative to WT HeLa cells at 15 min (log2; mitochondrial preparations from n=3 independent cultures).(C) Immunofluorescence of NME6 with mtDNA (top) or bromouridine (BrU) labelled nascent mtRNA (bottom) in HeLa cells imaged by confocal microscopy. Relative fluorescence intensities were calculated from linescans generated within the mitochondrial regions indicated by dotted lines.(E) MtDNA-encoded transcript levels analysed by qRT-PCR in NME6 KO and NME6 KO + NME6-MycFlag cells relative to WT HeLa cells. P-values for mtDNA-encoded transcripts: two-way ANOVA P-value (transcript) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = <0.0001 (log2; n=3 independent cultures).(F) Nuclear DNA-encoded transcript levels analysed by qRT-PCR in NME6 KO and NME6 KO + NME6-MycFlag HeLa cells relative to WT (log2; n=3 independent cultures).


Figure 5 ( A ) Heat map of log2 transformed mean mitochondrial transcript levels of WT and NME6 KO HeLa cells incubated with 100 µM rNTPs or dNTPs for 48 h relative to untreated WT cells analysed by qRT - PCR ( n = 3 independent cultures ) . ( B ) Volcano plot representation of log2 fold change in proteins between NME6 KO cells treated with 100 µM rNTPs for 120 h and untreated NME6 KO HeLa cells determined by quantitative mass spectrometry . OXPHOS subunits are highlighted in teal ( n = 3 independent cultures ) ( D ) Oxygen consumption rates ( OCR ) and extracellular acidification rates ( ECAR ) of WT and NME6 KO HeLa cells incubated with or without nucleosides ( 100 µM ) for a minimum of 120 h . Mitochondrial stress test was performed as in Fig 3A ( n = 3 independent experiments ) . P - values for OCR : two - way ANOVA P - value ( time ) = < 0 . 0001 , P - value ( genotype ) = 0 . 0011 , P - value ( interaction ) = < 0 . 0001 ; P - values for ECAR : two - way ANOVA P - value ( time ) = < 0 . 0001 , P - value ( genotype ) = 0 . 8994 , P - value ( interaction ) = < 0 . 0001 ( G ) CYTB ( top ) and ND5 ( bottom ) transcript levels analysed by qRT - PCR in WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for 48 h . P - values were calculated using a one - way ANOVA ( A , adenosine ; G , guanosine ; C , cytidine ; U , uridine ; log2 ; 100 µM ; n = 4 independent cultures ) . ( H ) CTP ( top ) and dCTP ( bottom ) levels in the mitochondria enriched fraction of WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for 120 h as determined by quantitative mass spectrometry . P - values for CTP : two - way ANOVA P - value ( supplementation ) = < 0 . 0001 , P - value ( genotype ) = < 0 . 0001 , P - value ( interaction ) = 0 . 3179 ; P - values for dCTP : two - way ANOVA P - value ( supplementation ) = < 0 . 0001 , P - value ( genotype ) = < 0 . 0001 , P - value ( interaction ) = 0 . 0569 ; P - values for supplementation are shown ( C , cytidine ; U , uridine ; log2 ; 100 µM ; n = 4 independent cultures ) . ( I ) Basal ( top ) and maximal ( bottom ) oxygen consumption rates of WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for a minimum of 120 h relative to untreated WT cells ( A , adenosine ; G , guanosine ; C , cytidine ; U , uridine ; log2 ; 100 µM ; n = 4 independent experiments ) . P - values were calculated using a one - way ANOVA .

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

Figure 5(A) Heat map of log2 transformed mean mitochondrial transcript levels of WT and NME6 KO HeLa cells incubated with 100 µM rNTPs or dNTPs for 48 h relative to untreated WT cells analysed by qRT-PCR (n=3 independent cultures).(B) Volcano plot representation of log2 fold change in proteins between NME6 KO cells treated with 100 µM rNTPs for 120 h and untreated NME6 KO HeLa cells determined by quantitative mass spectrometry. OXPHOS subunits are highlighted in teal (n=3 independent cultures)(D) Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of WT and NME6 KO HeLa cells incubated with or without nucleosides (100 µM) for a minimum of 120 h. Mitochondrial stress test was performed as in Fig 3A (n=3 independent experiments). P-values for OCR: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = 0.0011, P-value (interaction) = <0.0001; P-values for ECAR: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = 0.8994, P-value (interaction) = <0.0001(G) CYTB (top) and ND5 (bottom) transcript levels analysed by qRT-PCR in WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for 48 h. P-values were calculated using a one-way ANOVA (A, adenosine; G, guanosine; C, cytidine; U, uridine; log2; 100 µM; n=4 independent cultures).(H) CTP (top) and dCTP (bottom) levels in the mitochondria enriched fraction of WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for 120 h as determined by quantitative mass spectrometry. P-values for CTP: two-way ANOVA P-value (supplementation) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = 0.3179; P-values for dCTP: two-way ANOVA P-value (supplementation) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = 0.0569; P-values for supplementation are shown (C, cytidine; U, uridine; log2; 100 µM; n=4 independent cultures).(I) Basal (top) and maximal (bottom) oxygen consumption rates of WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for a minimum of 120 h relative to untreated WT cells (A, adenosine; G, guanosine; C, cytidine; U, uridine; log2; 100 µM; n=4 independent experiments). P-values were calculated using a one-way ANOVA.


Figure 1 WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation . Cilia were stained with Arl13B ( green ) while the basal bodies were marked with C - Nap1 ( red ) . DNA was stained with DAPI . Cilia in the magenta box are shown enlarged on the right picture WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation . Cilia were stained with Arl13B ( green ) while the basal bodies were marked with C - Nap1 ( red ) . DNA was stained with DAPI Percent of ciliated cells , as well as the length of cilia , was quantified from A . Cilia length was measured by a semi - automated ImageJ macro as described in methods . Data points are the average of three independent experiments with N = 150 cilia for each experiment and condition . Error bars represent the mean ± SD . * * * * P ≤ 0 . 0001 . High means 80 - 90 % and low 40 - 50 % confluency of RPE1 cells Elongated cilia phenotype was confirmed by siRNA - mediated knockdown of hCDC14A . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SD . * * * * P ≤ 0 . 0001 hCDC14A - GFP and hCDC14AC278S - GFP under control of the TetON promoter were stably integrated into RPE1 cells . hCDC14A - GFP and hCDC14AC278S - GFP localized to the C - Nap1 marked basal body upon Dox induction . In addition , expression of hCDC14A - GFP but not the phosphatase dead hCDC14AC278S - GFP mutant inhibited cilia formation . Ciliated cells were quantified 48 h after serum starvation with and without Dox addition . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SD . * * * P ≤ 0 . 001 ; ns : not significant Expression of hCDC14A - GFP and hCDC14AC278S - GFP was validated by immunoblot analysis with anti - GFP antibodies . GAPDH was the loading control RPE1 cells over - expressing hCDC14AC278S have longer cilia than those over - expressing hCDC14A . Three independent experiments , N = 100 cilia for hCDC14A expressing cells and 200 for hCDC14AC278S expressing cells . Mean ± SD . * * * P ≤ 0 . 001 The dynamics of ciliogenesis was determined by serum starving WT and hCDC14APD cells for different time - points . The cilia from hCDC14APD cells continue to elongate even after 48 h of starvation whereas those from WT cells reached constant length within 12 to 24 h . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SEM . * P ≤ 0 . 05 , * * ; P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 In a time course experiment , the cilia disassembly kinetics was measured by first inducing ciliation through 48 h of serum starvation prior to incubation with serum containing media for different time points . Measurement of cilia length indicated the comparable rates of cilia disassembly in RPE1 WT and hCDC14APD cells . Cilia disassembly rates are shown in Fig . EV1D . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SEM . * * * * P < 0 . 0001 Images of cilia of WT RPE1 and hCDC14APD RPE1 cells from I , 0 , 6 and 24 h after serum addition . Fixed cells were analyzed for cilia length

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

Figure 1 WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI. Cilia in the magenta box are shown enlarged on the right picture  WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI Percent of ciliated cells, as well as the length of cilia, was quantified from A. Cilia length was measured by a semi-automated ImageJ macro as described in methods. Data points are the average of three independent experiments with N = 150 cilia for each experiment and condition. Error bars represent the mean ± SD. **** P≤ 0.0001. High means 80-90% and low 40-50% confluency of RPE1 cells Elongated cilia phenotype was confirmed by siRNA-mediated knockdown of hCDC14A. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. **** P≤ 0.0001  hCDC14A-GFP and hCDC14AC278S-GFP under control of the TetON promoter were stably integrated into RPE1 cells. hCDC14A-GFP and hCDC14AC278S-GFP localized to the C-Nap1 marked basal body upon Dox induction. In addition, expression of hCDC14A-GFP but not the phosphatase dead hCDC14AC278S-GFP mutant inhibited cilia formation. Ciliated cells were quantified 48 h after serum starvation with and without Dox addition. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. *** P≤ 0.001; ns: not significant  Expression of hCDC14A-GFP and hCDC14AC278S-GFP was validated by immunoblot analysis with anti-GFP antibodies. GAPDH was the loading control  RPE1 cells over-expressing hCDC14AC278S have longer cilia than those over-expressing hCDC14A. Three independent experiments, N = 100 cilia for hCDC14A expressing cells and 200 for hCDC14AC278S expressing cells. Mean ± SD. *** P≤ 0.001  The dynamics of ciliogenesis was determined by serum starving WT and hCDC14APD cells for different time-points. The cilia from hCDC14APD cells continue to elongate even after 48 h of starvation whereas those from WT cells reached constant length within 12 to 24 h. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SEM. * P≤ 0.05, **; P≤ 0.01; **** P≤ 0.0001 In a time course experiment, the cilia disassembly kinetics was measured by first inducing ciliation through 48 h of serum starvation prior to incubation with serum containing media for different time points. Measurement of cilia length indicated the comparable rates of cilia disassembly in RPE1 WT and hCDC14APD cells. Cilia disassembly rates are shown in Fig. EV1D. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SEM. **** P<0.0001  Images of cilia of WT RPE1 and hCDC14APD RPE1 cells from I, 0, 6 and 24 h after serum addition. Fixed cells were analyzed for cilia length


Figure 2TetON - hCDC14A - GFP RPE1 cells were serum starved for 48 h prior to fixation and immunofluorescence microscopy . hCDC14A - GFP colocalizes with Phalloidin 555 conjugated dye that marks F - actin fibers . The boxed area was 10 - fold enlarged and displayed below the corresponding images to indicate co - localization of hCDC14A - GFP and F - actin . Line scan inside the magenta colored box showed in ( A ) Sub - centrosomal localization of hCDC14AC278S - GFP was determined by correlative positioning of the GFP signal with the proteins Arl13B and IFT88 ( cilia ) , C - Nap1 / ninein ( proximal end ) , CEP164 ( distal appendage ) and ODF2 / ninein ( subdistal appendage ) . Results are fitted in the cartoons adjacent to the respective panels for better

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

Figure 2TetON-hCDC14A-GFP RPE1 cells were serum starved for 48 h prior to fixation and immunofluorescence microscopy. hCDC14A-GFP colocalizes with Phalloidin 555 conjugated dye that marks F-actin fibers. The boxed area was 10-fold enlarged and displayed below the corresponding images to indicate co-localization of hCDC14A-GFP and F-actin. Line scan inside the magenta colored box showed in (A)Sub-centrosomal localization of hCDC14AC278S-GFP was determined by correlative positioning of the GFP signal with the proteins Arl13B and IFT88 (cilia), C-Nap1/ninein (proximal end), CEP164 (distal appendage) and ODF2/ ninein (subdistal appendage). Results are fitted in the cartoons adjacent to the respective panels for better visualization


Figure 4hCDC14A , not the inactive hCDC14AC278S , dephosphorylates the serine residue 142 of DBN1 in vivo . RPE1 cells expressing GFP , hCDC14A - GFP , and hCDC14AC278S - GFP under TetON promoter were serum starved in presence or absence of Dox for 48 h prior to fixation for immunofluorescence . DBN1pS142 was detected with a phospho - specific antibody . DNA was stained with DAPI . Scale Bar = 10 μmImmunoblot analysis of samples from ( A ) . The DBN1pS142 / DBN1 ratio was densitometrically measured from the Dox treated samples and represented in the graph next to the immunoblot . In this experiment two GAPDH blots were used to normalize protein levels because DBN1 and DBN1pS142 were analyzed in separated blots . Three independent experiments . Mean ± SD . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001DBN1S142 was hyper - phosphorylated in hCDC14APD cell lines during ciliogenesis compared to the WT control . In this experiment we sequentially analyzed DBN1pS142 and DBN1 on the same blot . The specificity of the DBN1pS142 antibody was validated by siRNA mediated knock - down of DBN1 in both hCDC14APD cells and GFP / hCDC14A - GFP / hCDC14AC278S - GFP stable cell lines . Three independent experiments . Mean ± SD . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001In vitro dephosphorylation of DBN1 by purified hCDC14A . DBN1 - GFP was transfected into HEK293T cells and expressed for 48 h . DBN1 - GFP was immunoprecipitated from cell lysates with GFP binder . The immunoprecipitated phospho - DBN1 was then incubated with and without recombinant hCDC14A in vitro . The DBN1pS142 / DBN1 ratio was densitometrically measured . Three independent experiments . Mean ± SD . * * * * P ≤ 0 .

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

Figure 4hCDC14A, not the inactive hCDC14AC278S, dephosphorylates the serine residue 142 of DBN1 in vivo. RPE1 cells expressing GFP, hCDC14A-GFP, and hCDC14AC278S-GFP under TetON promoter were serum starved in presence or absence of Dox for 48 h prior to fixation for immunofluorescence. DBN1pS142 was detected with a phospho-specific antibody. DNA was stained with DAPI. Scale Bar = 10 μmImmunoblot analysis of samples from (A). The DBN1pS142/DBN1 ratio was densitometrically measured from the Dox treated samples and represented in the graph next to the immunoblot. In this experiment two GAPDH blots were used to normalize protein levels because DBN1 and DBN1pS142 were analyzed in separated blots. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001DBN1S142 was hyper-phosphorylated in hCDC14APD cell lines during ciliogenesis compared to the WT control. In this experiment we sequentially analyzed DBN1pS142 and DBN1 on the same blot. The specificity of the DBN1pS142 antibody was validated by siRNA mediated knock-down of DBN1 in both hCDC14APD cells and GFP/hCDC14A-GFP/hCDC14AC278S-GFP stable cell lines. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001In vitro dephosphorylation of DBN1 by purified hCDC14A. DBN1-GFP was transfected into HEK293T cells and expressed for 48 h. DBN1-GFP was immunoprecipitated from cell lysates with GFP binder. The immunoprecipitated phospho-DBN1 was then incubated with and without recombinant hCDC14A in vitro. The DBN1pS142/DBN1 ratio was densitometrically measured. Three independent experiments. Mean ± SD. **** P≤ 0.0001


Figure 5The absence of DBN1 protein in the targeted clones was further verified by immunoblotting of the whole cell lysate . GAPDH is the loading control . One targeted clone from each gRNA ( 1 . 16 , 2 . 18 , 3 . 3 ) including the non - targeted clone 2 . 19 and WT control were serum starved for 48 h to induce ciliogenesis . Subsequently , cilia length was measured . The cilia from DBN1 KO cells were significantly longer than those from WT or non - targeted controls . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SD . * * * * P ≤ 0 . 0001 . Overexpression of DBN1 , DBN1S142A and DBN1S142D in RPE1 WT cells . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SD . * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; ns : not specific . The phospho - inhibitory ( S142A ) version of DBN1 rescued the elongated cilia of DBN1 KO cells . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SD . * P ≤ 0 . 05 ; * * * P ≤ 0 . 001 ; ns : not significant . Pairwise comparison was done with unpaired student ' s t - test and the p - value was written in the figure . Global comparison was done with one - way ANOVA and the p - values were marked with stars . The overexpressed WT DBN1 was hyper - phosphorylated in cells as detected by phospho - specific DBN1pS142 antibodies . The DBN1pS142 antibodies did not detect the overexpressed DBN1S142A or DBN1S142D . Immunoblot analysis confirming the successful siRNA depletion of CDK5 and indicating hypo - phosphorylation of DBN1S142 in response to CDK5 depletion The DBN1pS142 / GAPDH ratio from ( H ) was densitometrically measured . Three independent experiments . Mean ± SD . * P ≤ 0 . 05 Depletion of CDK5 decreased the cilia length in WT cells . It restored cilia length in hCDC14APD cells . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SD . * * ; P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 ; ns : not significant . CDK5 mediated cilia length decrease can be completely blocked by expression of the phospho - mimetic DBN1S142D in hCDC14APD cells . Three independent experiments ; 150 cells per experiment and condition . Mean ± SD ; ns = not significant ; * * * * P ≤ 0 . 0001 .

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

Figure 5The absence of DBN1 protein in the targeted clones was further verified by immunoblotting of the whole cell lysate. GAPDH is the loading control.One targeted clone from each gRNA (1.16, 2.18, 3.3) including the non-targeted clone 2.19 and WT control were serum starved for 48 h to induce ciliogenesis. Subsequently, cilia length was measured. The cilia from DBN1 KO cells were significantly longer than those from WT or non-targeted controls. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **** P≤ 0.0001.Overexpression of DBN1, DBN1S142A and DBN1S142D in RPE1 WT cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. ** P≤ 0.01; *** P≤ 0.001; ns: not specific. The phospho-inhibitory (S142A) version of DBN1 rescued the elongated cilia of DBN1 KO cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. * P≤ 0.05; *** P≤ 0.001; ns: not significant. Pairwise comparison was done with unpaired student's t-test and the p-value was written in the figure. Global comparison was done with one-way ANOVA and the p-values were marked with stars.  The overexpressed WT DBN1 was hyper-phosphorylated in cells as detected by phospho-specific DBN1pS142 antibodies. The DBN1pS142 antibodies did not detect the overexpressed DBN1S142A or DBN1S142D.  Immunoblot analysis confirming the successful siRNA depletion of CDK5 and indicating hypo-phosphorylation of DBN1S142 in response to CDK5 depletion The DBN1pS142/GAPDH ratio from (H) was densitometrically measured. Three independent experiments. Mean ± SD. * P≤ 0.05 Depletion of CDK5 decreased the cilia length in WT cells. It restored cilia length in hCDC14APD cells. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **; P≤ 0.01; **** P≤ 0.0001; ns: not significant.CDK5 mediated cilia length decrease can be completely blocked by expression of the phospho-mimetic DBN1S142D in hCDC14APD cells. Three independent experiments; 150 cells per experiment and condition. Mean ± SD; ns = not significant; **** P≤ 0.0001.


Figure 6 Transferrin uptake was measured with serum starvation or 4 h after serum starvation as described [ 17 ] . Fluorescent transferrin was added for 45 min to the cells . Cells were analyzed after washing . The red circle around the mother centriole marker CEP164 indicates the fluorescence intensity that was determined for ( B ) . The encircled area in MERGE is shown 2 - fold enlarged on the right hand side of the figure Quantification of ( A ) . The fluorescence intensities of transferrin within a 2 μm - radius circle surrounding the CEP164 signal were quantified . An ImageJ macro was used to measure the intensity as described in methods . Data were calculated from a total of 120 cells pooled from two independent experiments . Mean ± SD . * * * * P ≤ 0 . 0001 hCDC14APD cells showed an increased localization of myosin Va containing vesicles within the 1 μm - radius of the basal body ( red cicle ) . The encircled area in MERGE is shown 4 - fold enlarged on the right hand side of the figure Quantification of C . Data was calculated from a total of 180 cells pooled from three independent experiments . Mean ± SD . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 Arp2 localization in REP1 WT , hCDC14APD and DBN1 KO cells . Signal intensity was measured 30 min after serum starvation . The encircled area in MERGE is shown 4 - fold enlarged on the right hand side of the figure Quantification of E . Sub - centrosomal ( 1 μm - radius ; red circle in E ) Arp2 was measured by quantifying the intensity of Arp2 in WT , hCDC14APD and DBN1 KO cells . Both hCDC14APD and DBN1 KO cells contain significantly higher Arp2 in the pericentrosomal area ( 1 μm - radius ) compared to WT cells after 30 min of serum starvation . Data were calculated from a total of 120 cells pooled from three independent experiments . Mean ± SD . * * * * P ≤ 0 . 0001 Pericentrosomal ARP2 enrichment was comparable in the RPE1 DBN1 KO cells ( Tet Control ) with DBN1 KO cells expressing different DBN1 versions ( WT , S142A , S142D ) . Data were calculated from a total of 200 cells each pooled from two independent experiments . Mean ± SD . ns : not specific

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

Figure 6 Transferrin uptake was measured with serum starvation or 4 h after serum starvation as described [17]. Fluorescent transferrin was added for 45 min to the cells. Cells were analyzed after washing. The red circle around the mother centriole marker CEP164 indicates the fluorescence intensity that was determined for (B). The encircled area in MERGE is shown 2-fold enlarged on the right hand side of the figure Quantification of (A). The fluorescence intensities of transferrin within a 2 μm-radius circle surrounding the CEP164 signal were quantified. An ImageJ macro was used to measure the intensity as described in methods. Data were calculated from a total of 120 cells pooled from two independent experiments. Mean ± SD. **** P≤ 0.0001  hCDC14APD cells showed an increased localization of myosin Va containing vesicles within the 1 μm-radius of the basal body (red cicle). The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure Quantification of C. Data was calculated from a total of 180 cells pooled from three independent experiments. Mean ± SD. * P≤ 0.05, *** P≤ 0.001, **** P≤ 0.0001  Arp2 localization in REP1 WT, hCDC14APD and DBN1 KO cells. Signal intensity was measured 30 min after serum starvation. The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure  Quantification of E. Sub-centrosomal (1 μm-radius; red circle in E) Arp2 was measured by quantifying the intensity of Arp2 in WT, hCDC14APD and DBN1 KO cells. Both hCDC14APD and DBN1 KO cells contain significantly higher Arp2 in the pericentrosomal area (1 μm-radius) compared to WT cells after 30 min of serum starvation. Data were calculated from a total of 120 cells pooled from three independent experiments. Mean ± SD. **** P≤ 0.0001  Pericentrosomal ARP2 enrichment was comparable in the RPE1 DBN1 KO cells (Tet Control) with DBN1 KO cells expressing different DBN1 versions (WT, S142A, S142D). Data were calculated from a total of 200 cells each pooled from two independent experiments. Mean ± SD. ns: not specific


Figure 1 ( B ) Representative leak - subtracted ClC - 3 , ClC - 4 and ClC - 5 whole - cell currents measured with ( upper row ) or without ( lower row ) 5 mM ATP added to the pipette solution . Currents were elicited every 10 s by voltage steps from 0 mV to + 140 mV , followed by a step to - 100 mV to maximize the off - gating currents visible as sharp peaks at the end of the pulses . The recording started shortly ( 10s - 30s ) after obtaining a whole - cell configuration . The first and last recorded currents in a series are depicted in green and red , respectively . NMDG - based solutions were used to characterize ClC - 4 . ( C ) Effects of various adenine nucleotides or their washout on the ionic transport of ClC - 3 , ClC - 4 and ClC - 5 . The current amplitudes were measured at the end of the test pulses from experiments as shown in ( B ) and normalized to the initial current amplitudes obtained after establishing the whole - cell configuration . Some of the experimental data points have been omitted for clarity . Monoexponential fits to the data are depicted as red lines . Asterisks indicate statistical significances evaluated by two - sample t - test analysis of the residual currents determined at the end of each 10 - minute measurement ( * , p < 0 . 05 ; * * * , p < 0 . 001 , n . s . , not significant ; n = 5 - 11 ) . ( D ) , Left panel : residual currents determined at the end of 10 - minute measurements as shown in ( C ) . ( D ) , Right panel : time constants ( τ ) of the current rundown obtained by fitting monoexponential functions to individual measurements as depicted in ( C ) , ( n = 5 - 11 ) . Asterisks indicate statistical significances evaluated using two - sample t - test analysis ( * , p < 0 . 05 ; * * * , p < 0 . 001 , n . s . , not significant ) .

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Figure 1(B) Representative leak-subtracted ClC-3, ClC-4 and ClC-5 whole-cell currents measured with (upper row) or without (lower row) 5 mM ATP added to the pipette solution. Currents were elicited every 10 s by voltage steps from 0 mV to +140 mV, followed by a step to -100 mV to maximize the off-gating currents visible as sharp peaks at the end of the pulses. The recording started shortly (10s - 30s) after obtaining a whole-cell configuration. The first and last recorded currents in a series are depicted in green and red, respectively. NMDG-based solutions were used to characterize ClC-4.(C) Effects of various adenine nucleotides or their washout on the ionic transport of ClC-3, ClC-4 and ClC-5. The current amplitudes were measured at the end of the test pulses from experiments as shown in (B) and normalized to the initial current amplitudes obtained after establishing the whole-cell configuration. Some of the experimental data points have been omitted for clarity. Monoexponential fits to the data are depicted as red lines. Asterisks indicate statistical significances evaluated by two-sample t-test analysis of the residual currents determined at the end of each 10-minute measurement (*, p < 0.05; ***, p < 0.001, n.s., not significant; n= 5-11).(D), Left panel: residual currents determined at the end of 10-minute measurements as shown in (C). (D), Right panel: time constants (τ) of the current rundown obtained by fitting monoexponential functions to individual measurements as depicted in (C), (n= 5-11). Asterisks indicate statistical significances evaluated using two-sample t-test analysis (*, p < 0.05; ***, p < 0.001, n.s., not significant).


Figure 2 ( B ) Voltage dependence of the WT ClC - 5 off - gating charge normalized to the ionic current at + 165 mV in the absence or presence of adenine nucleotides ( n = 7 - 12 ) . Error bars represent SEM . Red lines depict Boltzmann fits to the data ; fit parameters are provided in Appendix Table S1 . ( C ) Voltage dependence of the WT ClC - 3 off - gating charge normalized to the ionic current at + 165 mV in the absence or presence of adenine nucleotides ( n = 4 - 5 , Error bars represent SEM . ) . Red lines represent Boltzmann fits to the

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

Figure 2(B) Voltage dependence of the WT ClC-5 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=7-12). Error bars represent SEM. Red lines depict Boltzmann fits to the data; fit parameters are provided in Appendix Table S1.(C) Voltage dependence of the WT ClC-3 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=4-5, Error bars represent SEM.). Red lines represent Boltzmann fits to the data


Figure 3 ( A ) Representative whole - cell recordings of cells expressing E211G ClC - 5 upon voltage pulses to + 140 mV from a - 60 - mV holding potential applied in 10 - s intervals starting shortly after obtaining the whole - cell configuration with ( left ) or without ( right ) 5 mM ATP in the pipette solution . Leak subtraction was not applied . Asymmetric [ Cl - ] was used ( 110 mM internal NaCl substituted by TrisSO4 ) to achieve a stable negative reversal potential of < - 50 mV allowing to recognize recordings with high unspecific leak current contamination . ( B ) Normalized time course of the steady - state E211G ClC - 5 current amplitudes measured with ( n = 5 ) or without ( n = 4 ) internal ATP . Error bars show SEM . Red line represents a monoexponential fit to the data with a time constant of 108 ± 8 s ( SEM ) ; currents measured with ATP - free pipette solution declined to 57 . 9 ± 2 . 6 % ( SEM ) of the initial amplitude .

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

Figure 3(A) Representative whole-cell recordings of cells expressing E211G ClC-5 upon voltage pulses to +140 mV from a -60-mV holding potential applied in 10-s intervals starting shortly after obtaining the whole-cell configuration with (left) or without (right) 5 mM ATP in the pipette solution. Leak subtraction was not applied. Asymmetric [Cl-] was used (110 mM internal NaCl substituted by TrisSO4) to achieve a stable negative reversal potential of <-50 mV allowing to recognize recordings with high unspecific leak current contamination.(B) Normalized time course of the steady-state E211G ClC-5 current amplitudes measured with (n=5) or without (n=4) internal ATP. Error bars show SEM. Red line represents a monoexponential fit to the data with a time constant of 108±8 s (SEM); currents measured with ATP-free pipette solution declined to 57.9±2.6% (SEM) of the initial amplitude.


Figure 4 ( A ) Effects of different adenine nucleotides or their washout on the ClC - 5 current amplitudes ( n = 4 - 7 ) . The data were normalized to the initial current amplitude determined shortly after establishing whole - cell configuration . Some of the data points are omitted for clarity . For comparison , the ADP data set from Figure 1 is also depicted . Red lines indicate monoexponential fits to the averaged data . ( B ) Averaged residual currents determined after 600 - s cell dialysis as shown in ( A ) , and normalized to the current at the start of the measurements ( n = 4 - 7 ) . Asterisks indicate statistically significant differences from a two - sample t - test analysis with p < 0 . 001 ; n . d . - not defined . Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in ( A ) .

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

Figure 4(A) Effects of different adenine nucleotides or their washout on the ClC-5 current amplitudes (n=4-7). The data were normalized to the initial current amplitude determined shortly after establishing whole-cell configuration. Some of the data points are omitted for clarity. For comparison, the ADP data set from Figure 1 is also depicted. Red lines indicate monoexponential fits to the averaged data.(B) Averaged residual currents determined after 600-s cell dialysis as shown in (A), and normalized to the current at the start of the measurements (n=4-7). Asterisks indicate statistically significant differences from a two-sample t-test analysis with p < 0.001; n.d. - not defined.Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in (A).


Figure 1A . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom left inset . Scale bars = 5 μm . B . Quantification of the number of CETN3 foci per spermatocyte ( n = 30 ) at each meiotic prophase substage . C . Representative images of unseparated ( < 1 μm apart ) , initial separation ( 1 - 5 μm apart ) , intermediate separation ( 5 - 11 μm apart ) , and pole separation ( > 11 μm apart ) of centrosomes . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom and top left insets . Scale bars = 5 μm . . Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using the four centrosome separation classifications P value obtained from a Kruskal - Wallis one - way ANOVA . * * P < 0 . 01 . E . Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT ( purple ) , CETN3 ( green ) , and SYCP3 ( red ) , and stained with DAPI ( blue ) . Zoomed images of centrosomes below each panel . Scale bars = 5 μm .

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Figure 1A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.C. Representative images of unseparated (< 1 μm apart), initial separation (1-5 μm apart), intermediate separation (5-11 μm apart), and pole separation (>11 μm apart) of centrosomes. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.. Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using the four centrosome separation classifications P value obtained from a Kruskal-Wallis one-way ANOVA. **P<0.01.E. Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT (purple), CETN3 (green), and SYCP3 (red), and stained with DAPI (blue). Zoomed images of centrosomes below each panel. Scale bars = 5 μm.


Figure 2A . Hematoxylin and eosin staining of 5 - micron thick testis sections of adult control , Plk1 cHet , and Plk1 cKO mice . Stars indicate examples of spermatocytes with condensed chromosomes . B . Percent testis to body weight ratios from control ( n = 8 ) , Plk1 cHet ( n = 4 ) , and Plk1 cKO ( n = 7 ) adult mice . C . TUNEL staining of paraffin embedded testis sections of adult control , Plk1 cHet , and Plk1 cKO mice . D . The number of TUNEL positive cells per seminiferous tubule cross - section was quantified in control Plk1 cHet , and Plk1 cKO mice . Two technical replicates were performed per genotype , and 100 seminiferous tubule cross sections were analyzed per replicate . E . Hematoxylin and eosin staining of 5 - micron thick testis sections of adult control , Aurka cHet , and Aurka cKO mice . Stars indicate examples of spermatocytes with condensed chromosomes . F . Percent testis to body weight ratios from control ( n = 5 ) , Aurka cHet ( n = 3 ) , and Aurka cKO ( n = 5 ) adult mice . G . TUNEL staining of paraffin embedded testis sections of adult control , Aurka cHet , and Aurka cKO mice . H . The number of TUNEL positive cells per seminiferous tubule cross - section was quantified in control Aurka cHet , and Aurka cKO mice . Two technical replicates were performed per genotype , and 100 seminiferous tubule cross sections were analyzed per replicate . I . Representative images of spermatocytes containing monopolar , bipolar , and multipolar spindles immunolabeled against alpha - tubulin ( red ) , centromere ( green ) , and stained with DAPI . J . Spindle pole number per primary spermatocyte was quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO . n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment .

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Figure 2A. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. B. Percent testis to body weight ratios from control (n = 8), Plk1 cHet (n = 4), and Plk1 cKO (n = 7) adult mice.C. TUNEL staining of paraffin embedded testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. D. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Plk1 cHet, and Plk1 cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.E. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Aurka cHet, and Aurka cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. F. Percent testis to body weight ratios from control (n = 5), Aurka cHet (n = 3), and Aurka cKO (n = 5) adult mice.G. TUNEL staining of paraffin embedded testis sections of adult control, Aurka cHet, and Aurka cKO mice. H. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Aurka cHet, and Aurka cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.I. Representative images of spermatocytes containing monopolar, bipolar, and multipolar spindles immunolabeled against alpha-tubulin (red), centromere (green), and stained with DAPI. J. Spindle pole number per primary spermatocyte was quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO. n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment.


Figure 3A . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom left inset . Scale bars = 5 μm . B . Quantification of the number of CETN3 foci per spermatocyte . n = 30 for each meiotic prophase substage . C . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom and top left insets . Scale bars = 5 μm . D . Quantification of the number of CETN3 foci per spermatocyte . n = 30 for each meiotic prophase substage . E . Centrosome ( PCNT ) separation was scored per spermatocyte . Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications P values obtained from two - tailed Student ' s t - test comparing the percent of unseparated centrosomes in each mouse mutant to control . n . s . ( not significant ) , * * * P < 0 . 001 .

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Figure 3A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.B. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage.C. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.D. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage.E. Centrosome (PCNT) separation was scored per spermatocyte. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications P values obtained from two-tailed Student's t-test comparing the percent of unseparated centrosomes in each mouse mutant to control. n.s. (not significant), ***P<0.001 .


Figure 4A . Spermatocytes were immunolabeled against CETN3 ( green ) , CEP164 ( red ) , SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom and top left insets . Scale bars = 5 μm . B . The percent of centrioles containing CEP164 distal appendages was quantified from three biological replicates ( n = 30 total cells ) in control , Aurka cKO , Plk1 cHet , and Plk1 cKO spermatocytes at the pachytene - stage of meiotic prophase and metaphase I . C . Quantification of gamma - tubulin positive centrosomes per metaphase I spermatocyte . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . The total number of cells quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO mice were 137 , 169 , 198 , and 198 , respectively . D . Spermatocytes were immunolabeled against gamma - tubulin ( green ) , alpha - tubulin ( red ) , and stained with DAPI ( blue ) . Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage . Scale bars = 5 μm . Western blots of gamma - tubulin from protein extracts obtained from 19dpp and 22dpp control , Aurka cKO , and Plk1 cKO spermatocytes ( E ) . Total protein stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display protein loading . Western blots of gamma - tubulin from Protein extracts from control and Plk1 cHet spermatocytes at 16 , 19 and 22 dpp ( F ) . Total protein stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display protein loading .

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Figure 4A. Spermatocytes were immunolabeled against CETN3 (green), CEP164 (red), SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.B. The percent of centrioles containing CEP164 distal appendages was quantified from three biological replicates (n = 30 total cells) in control, Aurka cKO, Plk1 cHet, and Plk1 cKO spermatocytes at the pachytene-stage of meiotic prophase and metaphase I.C. Quantification of gamma-tubulin positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 137, 169, 198, and 198, respectively.D. Spermatocytes were immunolabeled against gamma-tubulin (green), alpha-tubulin (red), and stained with DAPI (blue). Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage. Scale bars = 5 μm.Western blots of gamma-tubulin from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (E). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.Western blots of gamma-tubulin from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (F). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


Figure 5A . Spermatocytes were immunolabeled against TACC3 ( green ) , SYCP3 ( red ) , CETN3 ( magenta ) , and stained with DAPI ( blue ) . Zoom images of the centrosomes are presented to the right . Scale bars = 5 μm . B . Quantification of TACC3 positive centrosomes per metaphase I spermatocyte . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . The total number of cells quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO mice were 97 , 103 , 96 , and 105 , respectively . Western blots of TACC3 from protein extracts obtained from 19dpp and 22dpp control , Aurka cKO , and Plk1 cKO spermatocytes ( D ) . Total protein stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display protein loading . Western blots of TACC3 from Protein extracts from control and Plk1 cHet spermatocytes at 16 , 19 and 22 dpp ( C ) . Total protein stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display protein loading . E , F . Spermatocytes were immunolabeled against beta - catenin ( green ) , SYCP3 ( red ) , PCNT ( magenta ) , and stained with DAPI ( blue ) . Middle columns emphasize beta - catenin signal in diplonema and diakinesis spermatocytes . Scale bars = 5 μm . G . Quantification of beta - catenin positive centrosomes per spermatocyte at diakinesis . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . The total number of cells quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO mice were 89 , 106 , 122 , and 93 , respectively .

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Figure 5A. Spermatocytes were immunolabeled against TACC3 (green), SYCP3 (red), CETN3 (magenta), and stained with DAPI (blue). Zoom images of the centrosomes are presented to the right. Scale bars = 5 μm.B. Quantification of TACC3 positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 97, 103, 96, and 105, respectively.Western blots of TACC3 from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (D). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.Western blots of TACC3 from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (C). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.E, F. Spermatocytes were immunolabeled against beta-catenin (green), SYCP3 (red), PCNT (magenta), and stained with DAPI (blue). Middle columns emphasize beta-catenin signal in diplonema and diakinesis spermatocytes. Scale bars = 5 μm. G. Quantification of beta-catenin positive centrosomes per spermatocyte at diakinesis. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 89, 106, 122, and 93, respectively.


Figure 6A . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom left inset . Scale bars = 5 μm . B . Quantification of the number of CETN3 foci per spermatocyte ( n = 30 ) at each meiotic prophase substage . C . Centrosome ( PCNT ) separation was assessed in Aurka ; Plk1 dKO spermatocytes . Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation

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Figure 6A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.C. Centrosome (PCNT) separation was assessed in Aurka; Plk1 dKO spermatocytes. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications


Figure 1 ( E ) Fold increase of Oct4 - GFP + colony number over control under forced expression of individual factors . n ≥ 5 , where each dot indicates an independent experiment . Box - plots show median , 1st and 3rd quartile values . One - sample Wilcoxon test p - values are as indicated , with p values < 0 . 05 shown in

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Figure 1(E) Fold increase of Oct4-GFP+ colony number over control under forced expression of individual factors. n ≥ 5, where each dot indicates an independent experiment. Box-plots show median, 1st and 3rd quartile values. One-sample Wilcoxon test p-values are as indicated, with p values < 0.05 shown in red


Figure 2 ( B ) Heatmap of average gene expression normalised to β - actin over an EpiSC resetting time course in 2i + LIF . Each row is coloured according to the unique minimum and maximum for that gene . The values shown are average expression of four independent experiments ( C ) Gene expression for Stat3 , Klf2 , Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs . β - actin serves as an internal control . Mean + / - SEM , n = 4 independent experiments . * = p < 0 . 05 Student " s

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Figure 2(B) Heatmap of average gene expression normalised to β-actin over an EpiSC resetting time course in 2i+LIF. Each row is coloured according to the unique minimum and maximum for that gene. The values shown are average expression of four independent experiments(C) Gene expression for Stat3, Klf2, Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs. β-actin serves as an internal control. Mean +/- SEM, n=4 independent experiments. * = p<0.05 Student"s t


Figure 3 ( E ) Representative confocal images ( top ) and quantification ( bottom ) of Oct4 - GFP reporter mean intensity . The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6 . Mean + / - SD of 9 technical replicates . One representative experiment of two is shown . Scale bars = 300µm ( F ) Representative alkaline phosphatase ( AP ) staining images ( left ) and quantification ( right ) of AP + colonies after clonal replating , as described in

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Figure 3(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm(F) Representative alkaline phosphatase (AP) staining images (left) and quantification (right) of AP+ colonies after clonal replating, as described in panel