Representative kinetics ( G ) and analysis ( H ) of aequorin - based [ Ca2 + ] m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid ( CPA ) in the presence of 100 μM EGTA ( n = 3 independent experiments )

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Representative kinetics (G) and analysis (H) of aequorin-based [Ca2+]m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)


Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR / Cas9 - mediated genome editing . The results for 12 of the 36 clones that were examined are

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Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR/Cas9-mediated genome editing. The results for 12 of the 36 clones that were examined are shown


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial - targeted GCaMP6m , in MICU1 - KO cells generated using the CRISPR / Cas9 technique and transfected with the indicated constructs ( n = 3 independent experiments ; 30 - 56 cells )

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in MICU1-KO cells generated using the CRISPR/Cas9 technique and transfected with the indicated constructs (n=3 independent experiments; 30-56 cells)


Representative kinetics ( K ) and analysis ( L ) of aequorin - based [ Ca2 + ] m measurements in intact MICU1 - KO cells generated using the CRISPR / Cas9 technique , transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2 + ( n = 3 independent experiments . )

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

Representative kinetics (K) and analysis (L) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2+ (n=3 independent experiments.)


Representative kinetics ( M ) and analysis ( N ) of aequorin - based [ Ca2 + ] m measurements in intact MICU1 - KO cells generated using the CRISPR / Cas9 technique , transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA ( n = 3 independent experiments ) .

Representative kinetics (M) and analysis (N) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA (n=3 independent experiments).


HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated ( S473 ) Akt ( p - Akt ) or HSP60 ( mitochondrial marker ) . Merged images are indicated ( merge ) . Scale bar 10

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HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm


Analysis of the number of cells , expressed as a percentage , showing obvious mitochondrial staining of activated ( S473 phosphorylated ) Akt ( p - Akt ) ( n = 3 independent experiments ; 350 - 355 cells )

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Analysis of the number of cells, expressed as a percentage, showing obvious mitochondrial staining of activated (S473 phosphorylated) Akt (p-Akt) (n=3 independent experiments; 350-355 cells)


Hek293T cells treated with vehicle or 1 μM rapamycin ( Rapa . ) for 4 h were fractionated into Cytosol ( Cyt . ) or mitochondrial ( Mito . ) extracts and analyzed by western blotting . E - cadherin : plasma membrane marker ; β - tubulin : cytosolic marker ; VDAC : mitochondrial marker ; Homo : cell

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Hek293T cells treated with vehicle or 1 μM rapamycin (Rapa.) for 4 h were fractionated into Cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate


Mouse livers treated with vehicle or 1 μM rapamycin ( Rapa . ) for 16 h by an intraperitoneal injection were fractionated into cytosol ( Cyt . ) or mitochondrial ( Mito . ) extracts and analyzed by western blotting . Phosphorylated ( S2468 ) mTOR ( p - mTOR ) was used to assess the rapamycin activity . E - cadherin : plasma membrane marker ; β - tubulin : cytosolic marker ; VDAC : mitochondrial marker ; Homo : cell

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Mouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate


Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt , phosphorylated ( S473 ) Akt ( p - Akt ) , and mitochondrial proteins with known localizations . Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture . OMM : outer mitochondrial membrane ; IMS : intermembrane

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Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt, phosphorylated (S473) Akt (p-Akt), and mitochondrial proteins with known localizations. Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture. OMM: outer mitochondrial membrane; IMS: intermembrane space


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of vehicle - or rapamycin ( Rapa . , 1 μM for 4 h ) - treated Hek293T cell mitochondria following carbonate extraction at pH 11 . 5 . Akt , phosphorylated ( S473 ) Akt ( p - Akt ) , MICU1 , the established integral membrane proteins TIM23 and MCU , and the soluble protein cytochrome c ( Cyt . c ) was

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Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of vehicle- or rapamycin (Rapa., 1 μM for 4 h)-treated Hek293T cell mitochondria following carbonate extraction at pH 11.5. Akt, phosphorylated (S473) Akt (p-Akt), MICU1, the established integral membrane proteins TIM23 and MCU, and the soluble protein cytochrome c (Cyt. c) was analyzed


Hek293T cells were transfected with the GFP - tagged wild - type ( WT ) MICU1 and then treated with 1 μM rapamycin ( Rapa . ) alone for 4 h or in combination with the Akt inhibitor triciribine ( 10 μM ) . GFP - MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) and phosphorylated ( S473 ) Akt ( p - Akt ) antibodies by western

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Hek293T cells were transfected with the GFP-tagged wild-type (WT) MICU1 and then treated with 1 μM rapamycin (Rapa.) alone for 4 h or in combination with the Akt inhibitor triciribine (10 μM). GFP-MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) and phosphorylated (S473) Akt (p-Akt) antibodies by western blotting


Hek293T cells were transfected with either the GFP - tagged wild - type ( WT ) MICU1 or MICU1 S124A - GFP mutant . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western blotting .

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Hek293T cells were transfected with either the GFP-tagged wild-type (WT) MICU1 or MICU1 S124A-GFP mutant. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting.


Mitochondrial preparations of both Hek293T ( left ) and HeLa ( right ) cells transiently transfected with an HA - tagged constitutively active Akt T308D / S473D ( Akt D / D ) construct were analyzed by western blotting . GAPDH : cytosolic marker ; TIM23 : mitochondrial

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Mitochondrial preparations of both Hek293T (left) and HeLa (right) cells transiently transfected with an HA-tagged constitutively active Akt T308D/S473D (Akt D/D) construct were analyzed by western blotting. GAPDH: cytosolic marker; TIM23: mitochondrial marker


Resting mitochondrial calcium levels in the control ( ctrl ) and Akt D / D - expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial - targeted GCaMP6m ( n = 4 independent experiments ; 44 - 49 cells )

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Resting mitochondrial calcium levels in the control (ctrl) and Akt D/D-expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 44-49 cells)


Schematic model of the HA - tagged constitutively active Akt form targeted to the mitochondrial intermembrane space ( mt - Akt D / D ) . Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence . TOM20 was used as a mitochondrial marker . Merged images are indicated ( merge ) . Scale bar 10

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Schematic model of the HA-tagged constitutively active Akt form targeted to the mitochondrial intermembrane space (mt-Akt D/D). Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence. TOM20 was used as a mitochondrial marker. Merged images are indicated (merge). Scale bar 10 μm


Hek293T cells were transfected with the indicated constructs . The GFP - tagged MICU1 was immunoprecipitated from the whole - cell lysate , and the precipitated proteins were immunoblotted with the HA and phosphorylated ( S473 ) Akt ( p - Akt )

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Hek293T cells were transfected with the indicated constructs. The GFP-tagged MICU1 was immunoprecipitated from the whole-cell lysate, and the precipitated proteins were immunoblotted with the HA and phosphorylated (S473) Akt (p-Akt) antibodies


Hek293T cells were transfected with the indicated constructs . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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

Hek293T cells were transfected with the indicated constructs. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Resting mitochondrial calcium levels in control ( ctrl ) and mitochondrial - targeted Akt D / D ( mt - Akt D / D ) - expressing HeLa cells , evaluated through ratiometric imaging of the mitochondrial - targeted GCaMP6m ( n = 4 independent experiments ; 43 - 47 cells )

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Resting mitochondrial calcium levels in control (ctrl) and mitochondrial-targeted Akt D/D (mt-Akt D/D)-expressing HeLa cells, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 43-47 cells)


Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m ( n = 3 independent experiments ; 38 - 50 cells )

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Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m (n=3 independent experiments; 38-50 cells)


Representative kinetics ( H ) and analysis ( I ) of aequorin - based [ Ca2 + ] m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [ Ca2 + ] ( n = 3 independent experiments ) .

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

Representative kinetics (H) and analysis (I) of aequorin-based [Ca2+]m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [Ca2+] (n=3 independent experiments).


Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs . pre - MICU1 : precursor form of MICU1 ; m - MICU1 : mature form of

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Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. pre-MICU1: precursor form of MICU1; m-MICU1: mature form of MICU1


Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs . Where indicated , the cells were treated with 5 μM MG132 for 4 h . pre : precursor ; int : intermediate ; m : mature

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Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. Where indicated, the cells were treated with 5 μM MG132 for 4 h. pre: precursor; int: intermediate; m: mature form


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs , following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markers . The cells were treated with 5 μM MG132 for 4 h before

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Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs, following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. The cells were treated with 5 μM MG132 for 4 h before fractionation


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT - HA or MICU1 SD - HA , together with MCU - flag and EMRE constructs , following carbonate extraction at pH 7 . 4 , pH 10 . 5 , pH 11 . 5 or pH 12 . 5 . The cells were treated with 5 μM MG132 for 4 h before

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT-HA or MICU1 SD-HA, together with MCU-flag and EMRE constructs, following carbonate extraction at pH 7.4, pH 10.5, pH 11.5 or pH 12.5. The cells were treated with 5 μM MG132 for 4 h before fractionation


ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs . Cells were incubated with 50 μg / ml cycloheximide ( CHX ) for the indicated times , collected and subjected to western blotting analysis as indicated . In the graph , the amount of HA ( MICU1 ) , both WT and mutants , is represented relative to the amount at time 0 . ( n = 3 independent experiments )

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ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1), both WT and mutants, is represented relative to the amount at time 0. (n=3 independent experiments)


ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs . Cells were harvested , and total protein was extracted and subjected to western blotting analysis with the indicated antibodies . Immunoblotting was performed under reducing ( with the presence of DTT ) or non - reducing ( non - red ) conditions ( without DTT ) . Cells were treated with 5 μM MG132 for 4 h before

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ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were harvested, and total protein was extracted and subjected to western blotting analysis with the indicated antibodies. Immunoblotting was performed under reducing (with the presence of DTT) or non-reducing (non-red) conditions (without DTT). Cells were treated with 5 μM MG132 for 4 h before lysis


Quantification of HA levels in non - reducing condition showed in ( E ) . ( n = 3 independent experiments )

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Quantification of HA levels in non-reducing condition showed in (E). (n=3 independent experiments)


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs ( n = 3 independent experiments ; 46 - 54 cells ) .

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=3 independent experiments; 46-54 cells).


Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and / or IMMP2L . int : intermediate ; m : mature

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Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and/or IMMP2L. int: intermediate; m: mature form


Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and / or IMMP2L . int : intermediate ; m : mature form . Cells were treated with 5 μM MG132 for 4 h before

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Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and/or IMMP2L. int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of control or IMMP1L - silenced HeLa cell mitochondria following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of control or IMMP1L - silenced HeLa cell mitochondria transfected with HA - tagged MICU1 WT following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markers . pre : precursor ; int : intermediate ; m : mature form . Cells were treated with 5 μM MG132 for 4 h before

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria transfected with HA-tagged MICU1 WT following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. pre: precursor; int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis


Control or IMMP1L - silenced HeLa cells were transfected with HA - tagged MICU1 WT . Cells were incubated with 50 μg / ml cycloheximide ( CHX ) for the indicated times , collected and subjected to western blotting analysis as indicated . In the graph , the amount of HA ( MICU1 ) is represented relative to the amount at time 0 . ( n = 3 independent experiments )

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Control or IMMP1L-silenced HeLa cells were transfected with HA-tagged MICU1 WT. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1) is represented relative to the amount at time 0. (n=3 independent experiments)


Representative kinetics ( F ) and analysis ( G ) of aequorin - based [ Ca2 + ] m measurements in control or IMMP1L - silenced HeLa cells challenged with 10 μM cyclopiazonic acid ( CPA ) in the presence of 100 μM EGTA ( n = 3 independent experiments )

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Representative kinetics (F) and analysis (G) of aequorin-based [Ca2+]m measurements in control or IMMP1L-silenced HeLa cells challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in control or IMMP1L - silenced HeLa cells ( n = 3 independent experiments ; 37 - 55 cells ) .

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in control or IMMP1L-silenced HeLa cells (n=3 independent experiments; 37-55 cells).


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Western blot analysis of MICU1 and MICU2 levels in lung cancer cells with low or high Akt activity


Lung cancer cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS ( phospho - Akt substrate ) antibody by western

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Lung cancer cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS (phospho-Akt substrate) antibody by western blotting


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Western blot analysis of MICU1 and MICU2 levels in melanoma cells with low or high Akt activity


Melanoma cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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Melanoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Western blot analysis of MICU1 and MICU2 levels in glioblastoma cells with low or high Akt activity


Glioblastoma cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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Glioblastoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Number of soft agar colonies ( n = 3 independent experiments formed by Rat2 cells stably expressing the indicated

Number of soft agar colonies (n=3 independent experiments formed by Rat2 cells stably expressing the indicated constructs


xenograft tumor volumes ( H ) and tumor weights at day 21 ( n = 8 mice for each group ) ( I ) formed by Rat2 cells stably expressing the indicated

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xenograft tumor volumes (H) and tumor weights at day 21 (n=8 mice for each group) (I) formed by Rat2 cells stably expressing the indicated constructs


Representative images ( J ) and analysis ( K ) of MitoSOX - based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D ( SD ) or MICU1 S124A ( SA ) ( n = 3 independent experiments ) . Scale bar 10 μm . f . a . u . : fluorescence arbitrary

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Representative images (J) and analysis (K) of MitoSOX-based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) (n=3 independent experiments). Scale bar 10 μm. f.a.u.: fluorescence arbitrary unit


ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D ( SD ) or MICU1 S124A ( SA ) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated . l . e . : long exposure .

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ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated. l. e.: long exposure.


Isolated sporozoites from the hemolymph ( HL ) and salivary glands ( SG ) of infected mosquitoes stained with Hoechst and SiR - tubulin . Note the similar staining of WT and α1 - tubulin ( - ) complemented lines . Scale bars : 5 µm .

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Isolated sporozoites from the hemolymph (HL) and salivary glands (SG) of infected mosquitoes stained with Hoechst and SiR-tubulin. Note the similar staining of WT and α1-tubulin(-) complemented lines. Scale bars: 5 µm.


SiR - tubulin staining ( green , I and II ) and TEM ( III ) show that microtubules shrink after wild type sporozoite formation . During budding microtubules can be up to 15 µm long ( I ) , while after formation they measure on average only 6 µm ( graph ) . Inset in II shows sporozoite cross - section at the sporoblast membrane ; note the SiR - tubulin fluorescence ( green , arrowheads ) next to the nuclear Hoechst stain ( blue ) . ( III ) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule ( white line , length ) . Green arrowheads point to microtubules close to the sporoblast . Rh : rhoptry ; N : nucleus ; ER : endoplasmic reticulum ; Mi : mitochondrion ; Sb : sporoblast . * * * * indicate p < 0 . 0001 ; Kruskal - Wallis - test . Scale bars : I & II : 5 µm , magnification box in II : 1 µm , III : 1 µm , magnification box in III : 0 . 1 µm .

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SiR-tubulin staining (green, I and II) and TEM (III) show that microtubules shrink after wild type sporozoite formation. During budding microtubules can be up to 15 µm long (I), while after formation they measure on average only 6 µm (graph). Inset in II shows sporozoite cross-section at the sporoblast membrane; note the SiR-tubulin fluorescence (green, arrowheads) next to the nuclear Hoechst stain (blue). (III) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule (white line, length). Green arrowheads point to microtubules close to the sporoblast. Rh: rhoptry; N: nucleus; ER: endoplasmic reticulum; Mi: mitochondrion; Sb: sporoblast. **** indicate p<0.0001; Kruskal-Wallis-test. Scale bars: I&II: 5 µm, magnification box in II: 1 µm, III: 1 µm, magnification box in III: 0.1 µm.


Comparison of sporozoite development in oocysts of WT , α1 - tubulin ( - ) and complemented lines . Hoechst ( blue ) and SiR - tubulin ( green ) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls , while SiR tubulin staining is absent from α1 - tubulin ( - ) oocysts . mCherry stains the cytoplasm of the parasite . Scale bars : 5 µm .

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Comparison of sporozoite development in oocysts of WT, α1-tubulin(-) and complemented lines. Hoechst (blue) and SiR-tubulin (green) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls, while SiR tubulin staining is absent from α1-tubulin(-) oocysts. mCherry stains the cytoplasm of the parasite. Scale bars: 5 µm.


Transmission electron micrographs of developing sporozoites in WT and α1 - tubulin ( - ) parasite lines . Note the slender sporozoites in the WT oocysts , which are absent in the mutant . Sb : sporoblast ; scale bars : 5 µm .

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Transmission electron micrographs of developing sporozoites in WT and α1-tubulin(-) parasite lines. Note the slender sporozoites in the WT oocysts, which are absent in the mutant. Sb: sporoblast; scale bars: 5 µm.


TEM cross sections of WT and α1 - tubulin ( - ) parasites showing the aberrant structure of the latter . Subpellicular and centriolar plaque ( spindle pole body ) associated ( hemispindle ) microtubules are readily recognizable in the WT sporozoites ( green arrowheads in insert ) but absent in the mutant , while the inner membrane complex is formed below the plasma membrane of both parasites ( white arrowheads ) . Note the different scale chosen to highlight the malformed shapes in the mutant . N : nucleus ; scale bars : 0 . 2 µm , magnification boxes : 0 . 1 µm , .

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TEM cross sections of WT and α1-tubulin(-) parasites showing the aberrant structure of the latter. Subpellicular and centriolar plaque (spindle pole body) associated (hemispindle) microtubules are readily recognizable in the WT sporozoites (green arrowheads in insert) but absent in the mutant, while the inner membrane complex is formed below the plasma membrane of both parasites (white arrowheads). Note the different scale chosen to highlight the malformed shapes in the mutant. N: nucleus; scale bars: 0.2 µm, magnification boxes: 0.1 µm,.


Electron micrographs showing different stages of development in WT and α1 - tubulin ( - ) parasites . Key structural elements are highlighted in the following colors : blue : plasma membrane ; yellow : inner membrane complex ; green : microtubules ; light blue : micronemes ; lilac : rhoptries ; brown : rootlet fiber ; cyan : nucleus . Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1 - tubulin ( - ) parasites . The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque ( dark structure in nuclear envelope ) . Panel III corresponds to Fig 1D . Scale bars : 200 nm .

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Electron micrographs showing different stages of development in WT and α1-tubulin(-) parasites. Key structural elements are highlighted in the following colors: blue: plasma membrane; yellow: inner membrane complex; green: microtubules; light blue: micronemes; lilac: rhoptries; brown: rootlet fiber; cyan: nucleus. Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1-tubulin(-) parasites. The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque (dark structure in nuclear envelope). Panel III corresponds to Fig 1D. Scale bars: 200 nm.


α1 - tubulin ( - ) parasites show multiple attachments ( arrowheads ) with the sporoblast membrane at day 17 post infection . Note the highly branched nature of the budding parasite ( same parasite as in panel C ) shown in two views in I and II . Right : slices through the tomogram in grey scale with the outlines used for the 3D model indicated . Two connections with the sporoblast are indicated by 1 and 2 . Scale bars : 1 µm .

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α1-tubulin(-) parasites show multiple attachments (arrowheads) with the sporoblast membrane at day 17 post infection. Note the highly branched nature of the budding parasite (same parasite as in panel C) shown in two views in I and II. Right: slices through the tomogram in grey scale with the outlines used for the 3D model indicated. Two connections with the sporoblast are indicated by 1 and 2. Scale bars: 1 µm.


Life imaging of wild - type ( WT ) and α1 - tubulin ( - ) oocysts expressing ef1α : GFP ( early and late oocysts ) and csp : mCherry ( late oocysts ) to locate the oocyst cytoplasm . Microtubules were labeled with SiR - tubulin ( green ) and DNA with Hoechst ( blue ) . Oocysts are shown in a chronological order from early ( day 4 ) to late ( day 12 ) development . ( A , B ) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage ( A ) and subsequent DNA replication ( B ) , where some but not all DNA is co - stained by microtubules in the mutant . ( C ) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites . Note the strong SiR - tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant . Sb : sporoblast . ( D ) Oocyst ready to burst with fully formed sporozoites in WT oocysts . Note that α1 - tubulin ( - ) oocysts retained sporozoite nuclei predominantly within the sporoblast ( Sb ) during budding . Scale bars : 5 μm , magnification box in B : 1 µm , .

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Life imaging of wild-type (WT) and α1-tubulin(-) oocysts expressing ef1α:GFP (early and late oocysts) and csp:mCherry (late oocysts) to locate the oocyst cytoplasm. Microtubules were labeled with SiR-tubulin (green) and DNA with Hoechst (blue). Oocysts are shown in a chronological order from early (day 4) to late (day 12) development. (A, B) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage (A) and subsequent DNA replication (B), where some but not all DNA is co-stained by microtubules in the mutant. (C) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites. Note the strong SiR-tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant. Sb: sporoblast. (D) Oocyst ready to burst with fully formed sporozoites in WT oocysts. Note that α1-tubulin(-) oocysts retained sporozoite nuclei predominantly within the sporoblast (Sb) during budding. Scale bars: 5 μm, magnification box in B: 1 µm,.


3D reconstructions from spinning disc confocal microscopy z - stacks across an early oocyst labelled with the DNA dye Hoechst ( left ) . 50 and 19 images with a z - distance of 0 . 5 µm and 1 µm were collated for WT and α1 - tubulin ( - ) oocysts , respectively . Right : 3D reconstructions from late WT and α1 - tubulin ( - ) oocysts labelled with Hoechst ( blue ) and SiR - tubulin ( green ) . Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites . 10 and 28 images with a z - distance of 1 , 5 µm and 1 µm were collated for WT and α1 - tubulin ( - ) oocysts , respectively . Sb : sporoblast ; scale bars : 5 µm .

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3D reconstructions from spinning disc confocal microscopy z-stacks across an early oocyst labelled with the DNA dye Hoechst (left). 50 and 19 images with a z-distance of 0.5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Right: 3D reconstructions from late WT and α1-tubulin(-) oocysts labelled with Hoechst (blue) and SiR-tubulin (green). Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites. 10 and 28 images with a z-distance of 1,5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Sb: sporoblast; scale bars: 5 µm.


TEM cross sections of WT and α1 - tubulin ( - ) oocysts during nuclear division . Spindle microtubules ( arrowheads ) emerging from centriolar plaques ( M ) were seen in WT oocysts . Although individual nuclei ( N ) were visible in α1 ( - ) oocysts , microtubules were absent even in dividing centriolar plaques ( M ) . Scale bars : 0 . 2 μm .

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TEM cross sections of WT and α1-tubulin(-) oocysts during nuclear division. Spindle microtubules (arrowheads) emerging from centriolar plaques (M) were seen in WT oocysts. Although individual nuclei (N) were visible in α1(-) oocysts, microtubules were absent even in dividing centriolar plaques (M). Scale bars: 0.2 μm.


Fluorescence images showing the SiR - tubulin stained microtubules , nuclei ( Hoechst ) and cytoplasmic shape ( mCherry ) of the parasite lines . Note the small amount of DNA ( black arrowhead ) and the reduced level of SiR - tubulin fluorescence in the α1cm & ∆ introns - expressing sporozoites ( lower graph ) . See also Fig S6 . Quantitative data was derived from 96 - 120 sporozoites per line . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . Scale bar : 5 μm . * * * * indicates p < 0 . 0001 , Kruskal - Wallis - test . Note that α1cm & ∆ introns - expressing sporozoites do not express mCherry ; their autofluorescence ( auto - fluo ) indicates an aberrant shape .

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Fluorescence images showing the SiR-tubulin stained microtubules, nuclei (Hoechst) and cytoplasmic shape (mCherry) of the parasite lines. Note the small amount of DNA (black arrowhead) and the reduced level of SiR-tubulin fluorescence in the α1cm&∆introns-expressing sporozoites (lower graph). See also Fig S6. Quantitative data was derived from 96-120 sporozoites per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. Scale bar: 5 μm. **** indicates p<0.0001, Kruskal-Wallis-test. Note that α1cm&∆introns-expressing sporozoites do not express mCherry; their autofluorescence (auto-fluo) indicates an aberrant shape.


qRT - PCR analysis of the three parasite lines at day 7 and 12 after infection . Error bars correspond to the standard deviation from two biological replicates .

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qRT-PCR analysis of the three parasite lines at day 7 and 12 after infection. Error bars correspond to the standard deviation from two biological replicates.


TEM reveals α1cm & ∆ introns - expressing sporozoites with smaller ( A and B ) and larger ( C ) diameters than wild type sporozoites . See also Fig S7 . Scale bar : 0 . 2 µm .

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TEM reveals α1cm&∆introns-expressing sporozoites with smaller (A and B) and larger (C) diameters than wild type sporozoites. See also Fig S7. Scale bar: 0.2 µm.


α1cm & ∆ introns - expressing sporozoites show a reduced number of subpellicular microtubules ( green arrowheads ) by TEM .

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α1cm&∆introns-expressing sporozoites show a reduced number of subpellicular microtubules (green arrowheads) by TEM.


Expression of different α1 - α2 chimeric α - tubulins instead of α1 - tubulin leads to sporozoite shape changes as revealed by SEM . See also Fig EV5 . Scale bar : 1 µm . α1 ∆ c - term lacks the last 3 amino acids of α1 - tubulin . α2 + parasites contain an α1 - tubulin with all amino acids that are different between the two α - tubulins changed into those of α2 - tubulin ; see also Fig S4 . α2 + + parasites contain α2 - tubulin ( exons , introns , 3 ' UTR ) and α2 + + + parasites express the exons of α2 - tubulin and introns of α1 - tubulin from the α1 - tubulin promoter .

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Expression of different α1-α2 chimeric α-tubulins instead of α1-tubulin leads to sporozoite shape changes as revealed by SEM. See also Fig EV5. Scale bar: 1 µm. α1∆c-term lacks the last 3 amino acids of α1-tubulin. α2+ parasites contain an α1-tubulin with all amino acids that are different between the two α-tubulins changed into those of α2-tubulin; see also Fig S4. α2++ parasites contain α2-tubulin (exons, introns, 3'UTR) and α2+++ parasites express the exons of α2-tubulin and introns of α1-tubulin from the α1-tubulin promoter.


SiR - tubulin staining reveals differences in microtubule lengths and intensity . Between 69 and 156 sporozoites were analysed per line . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * * * , and * * * * indicate p < 0 . 001 and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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SiR-tubulin staining reveals differences in microtubule lengths and intensity. Between 69 and 156 sporozoites were analysed per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. ***, and **** indicate p<0.001 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


qRT - PCR analysis of the parasite lines at day 12 after infection shows different expression level . Note the subtly higher expression of α2 + + compared to α2 + + + . Error bars show standard deviation from the mean of two biological replicates .

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qRT-PCR analysis of the parasite lines at day 12 after infection shows different expression level. Note the subtly higher expression of α2++ compared to α2+++. Error bars show standard deviation from the mean of two biological replicates.


TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α - tubulin chimeras . Green arrowheads point to microtubules . See also Fig S8 and S9 . Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier . Scale bars : 0 . 2 µm .

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TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α-tubulin chimeras. Green arrowheads point to microtubules. See also Fig S8 and S9. Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier. Scale bars: 0.2 µm.


TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines . Each dot represents the numbers of microtubules counted per section . Red lines indicate median from 23 to 40 analysed sections per line .

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TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines. Each dot represents the numbers of microtubules counted per section. Red lines indicate median from 23 to 40 analysed sections per line.


Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2 + and α1 ∆ c - term parasites compared to wild type sporozoites .

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Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2+ and α1∆c-term parasites compared to wild type sporozoites.


Semilog correlation of gene expression as determined by qRT - PCR with microtubule numbers as determine by TEM . Bars show range of values in x and standard deviation in y around the mean ( dots ) of two biological replicates ( x ) and all determined values ( y ) .

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Semilog correlation of gene expression as determined by qRT-PCR with microtubule numbers as determine by TEM. Bars show range of values in x and standard deviation in y around the mean (dots) of two biological replicates (x) and all determined values (y).


The microtubule number increases with the increase in the concentration of tubulin dimers . Microtubule number scales with tubulin concentration at low expression . At high expression , microtubule number is limited by the number of nucleation sites . Shown are mean + / - standard deviation from all determined values .

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The microtubule number increases with the increase in the concentration of tubulin dimers. Microtubule number scales with tubulin concentration at low expression. At high expression, microtubule number is limited by the number of nucleation sites. Shown are mean +/- standard deviation from all determined values.


The average microtubule length remains close to the maximum length 6 μm . The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data ( blue stars are for ( α2 + , α2 + + , α2 + + + ) strains ) . The error bar shows the standard deviation in the data and simulation results .

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The average microtubule length remains close to the maximum length 6 μm. The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data (blue stars are for (α2+, α2++, α2+++) strains). The error bar shows the standard deviation in the data and simulation results.


For the WT expression level ( c0 = 36 . 6 ( m0 + p + n ) ) , the rate of nucleation is faster than the microtubule growth . Then the microtubule number is determined by the number of nucleation sites . The average microtubule length is around 1 μm ( shown by dashed arrow ) when the last nucleus forms . At intermediate tubulin numbers ( c0 = 16 ( m0 + p + n ) ) , corresponding to the α1c - term mutant , the microtubule formation rate is comparable to nucleation rate . The average microtubule length is around 4 μm ( shown by dashed arrow ) when the last nucleus forms .

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For the WT expression level (c0 = 36.6 (m0+p+n)), the rate of nucleation is faster than the microtubule growth. Then the microtubule number is determined by the number of nucleation sites. The average microtubule length is around 1 μm (shown by dashed arrow) when the last nucleus forms. At intermediate tubulin numbers (c0 = 16 (m0+p+n)), corresponding to the α1c-term mutant, the microtubule formation rate is comparable to nucleation rate. The average microtubule length is around 4 μm (shown by dashed arrow) when the last nucleus forms.


Quantitative analysis of salivary gland sporozoite lengths from the different lines . n indicates numbers of investigated sporozoites . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * * * and * * * * indicate p < 0 . 001 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test ; scale bar : 1 µm .

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Quantitative analysis of salivary gland sporozoite lengths from the different lines. n indicates numbers of investigated sporozoites. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. *** and **** indicate p<0.001, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test; scale bar: 1 µm.


Quantification of SiR - tubulin fluorescence from 69 - 127 salivary gland derived sporozoites . Note the decrease in the fluorescence intensity of sporozoites expressing α1 - tubulin without introns as well as the decreased intensity and length of the chimeras . Linear correlation of sporozoite length and microtubule length reveals a R2 = 0 . 94 . * , * * , and * * * * indicate p < 0 . 05 , p < 0 . 01 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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Quantification of SiR-tubulin fluorescence from 69-127 salivary gland derived sporozoites. Note the decrease in the fluorescence intensity of sporozoites expressing α1-tubulin without introns as well as the decreased intensity and length of the chimeras. Linear correlation of sporozoite length and microtubule length reveals a R2=0.94. *, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass . Scale bar : 20 µm . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * and * * * * indicate p < 0 . 05 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass. Scale bar: 20 µm. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. * and **** indicate p<0.05, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


Sporozoites with fewest microtubules ( α2 + + + ) show a motility defect . n indicates numbers of investigated sporozoites ; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites .

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Sporozoites with fewest microtubules (α2+++) show a motility defect. n indicates numbers of investigated sporozoites; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites.


Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise ( CW ) direction . Numbers on black bars indicate percentage of CW moving sporozoites ; numbers in movie stills indicate time in seconds . CW moving sporozoites move at lower speed than CCW moving ones .

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Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise (CW) direction. Numbers on black bars indicate percentage of CW moving sporozoites; numbers in movie stills indicate time in seconds. CW moving sporozoites move at lower speed than CCW moving ones.


Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2 + + infected salivary glands . Green arrowheads point to microtubules . Scale bar : 0 . 2 µm . Error bars show the full range and boxes 50 % of values . Red crosses indicate the mean , red lines the median . Fisher ' s exact test ( two - tailed ) .

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Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2++ infected salivary glands. Green arrowheads point to microtubules. Scale bar: 0.2 µm. Error bars show the full range and boxes 50% of values. Red crosses indicate the mean, red lines the median. Fisher's exact test (two-tailed).


( C ) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4 - hydroxy tamoxifen ( 4OHT ) for 14 days to induce Cre , compared to Ethanol treated control cells ( See also Appendix figure S1A ) .

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(C) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4-hydroxy tamoxifen (4OHT) for 14 days to induce Cre, compared to Ethanol treated control cells (See also Appendix figure S1A).


( D ) Shallow whole genome sequencing ( copy number profiles ) of large T antigen immortalized , MEFs after exposure to Cre recombinase and sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosomes 12 , 14 and 9 and 10 , without any in vitro culturing , post FACS sort .

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(D) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10, without any in vitro culturing, post FACS sort.


( A ) Shallow whole genome sequencing ( copy number profiles ) of large T antigen immortalized , early passage MEFs after exposure to Cre recombinase and sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosomes 12 , 14 and 9 and 10 .

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(A) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, early passage MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10.


( B ) Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice ( n = 5 per group and error bars denote SEM , p < 0 . 05 for Ch9 and Ch14 ) .

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(B) Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SEM, p<0.05 for Ch9 and Ch14).


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( A ) Ch12 Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

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Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (A) Ch12 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( B ) Ch14 . Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

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Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (B) Ch14. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( C ) Ch9 Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (C) Ch9 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( D ) Ch10 . Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (D) Ch10. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch12 ( A ) . All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch12 (A). All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch10 ( B ) All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch10 (B) All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch14 ( C ) All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch14 (C) All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch9 ( D ) lines . All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch9 (D) lines. All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


( A ) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice ( n = 5 per group and error bars denote SD , p < 0 . 0005 for Ch9 , p < 0 . 05 for Ch10 and p < 0 . 005 for Ch14 ) .

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(A) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SD, p<0.0005 for Ch9, p<0.05 for Ch10 and p<0.005 for Ch14).


( B ) Tumors sections were stained with H & E to assess tumor histology ( scale bars denote 5mm for the inset and 200μm for the zoomed image ) Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue .

(B) Tumors sections were stained with H&E to assess tumor histology (scale bars denote 5mm for the inset and 200μm for the zoomed image) Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue.


( C ) Tumor growth curve after later passage ICL cells were injected into flanks of NOD / SCID mice ( n = 5 per group and error bars denote SD p < 0 . 0005 for Ch10 and p < 0 . 005 for Ch14 ) .

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(C) Tumor growth curve after later passage ICL cells were injected into flanks of NOD/SCID mice (n = 5 per group and error bars denote SD p<0.0005 for Ch10 and p<0.005 for Ch14).


( A ) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions ( n = 3 for each data set and error bars denote SD , p < 0 . 005 for Ch10 and p < 0 . 0001 for Ch14 ) . ( See also Appendix Figure S3A )

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(A) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions (n=3 for each data set and error bars denote SD, p<0.005 for Ch10 and p<0.0001 for Ch14). (See also Appendix Figure S3A)


( B ) Colony formation in ICL and control MEFs . 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days . Representative colony formation images in Ch10 and Ch14 are shown ( left ) and quantification of colonies formed ( right ) in the 4 ICL lines ( n = 3 for each data set and error bars denote SD , p < 0 . 005 for Ch10 , Ch12 and Ch14 and ns for Ch9 ) .

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(B) Colony formation in ICL and control MEFs. 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days. Representative colony formation images in Ch10 and Ch14 are shown (left) and quantification of colonies formed (right) in the 4 ICL lines (n=3 for each data set and error bars denote SD, p<0.005 for Ch10, Ch12 and Ch14 and ns for Ch9).


( C ) Quantification of growth in ultra - low adherent , sphere forming conditions for the 4 ICL lines ( n = 3 for Ch10 and Ch14 , n = 6 for Ch9 and Ch12 and error bars denote SD , p < 0 . 0001 for Ch9 , p < 0 . 001 for Ch10 and p < 0 . 05 for Ch12 and Ch14 ) .

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(C) Quantification of growth in ultra-low adherent, sphere forming conditions for the 4 ICL lines (n=3 for Ch10 and Ch14, n=6 for Ch9 and Ch12 and error bars denote SD, p<0.0001 for Ch9, p<0.001 for Ch10 and p<0.05 for Ch12 and Ch14).


( D ) Analysis of metabolites ( glucose consumed , lactate produced and glutamine consumed ) for the ICL lines . Spent media was analyzed after each ICL & control line was grown in culture for three days . Media in identical culture conditions , but with no plated cells , was used as a baseline for all samples .

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(D) Analysis of metabolites (glucose consumed, lactate produced and glutamine consumed) for the ICL lines. Spent media was analyzed after each ICL & control line was grown in culture for three days. Media in identical culture conditions, but with no plated cells, was used as a baseline for all samples.


( A ) Flow cytometry analysis of tail blood from Ch10 / + ( control ) at 30 days post tamoxifen administration , Ch10 / + ; Id1 Cre / + at 30 days post tamoxifen administration , and Ch10 / + ; Id1Cre / + at 80 days post tamoxifen administration .

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(A) Flow cytometry analysis of tail blood from Ch10/+ (control) at 30 days post tamoxifen administration, Ch10/+; Id1 Cre/+ at 30 days post tamoxifen administration, and Ch10/+; Id1Cre/+ at 80 days post tamoxifen administration.


( B ) Analysis from the Mitelman database for the fraction of human solid tumors that have lost ( top ) or gained ( bottom ) individual chromosomes , that have modal chromosome number > 2N or ~ 2N ( top , p < 0 . 0001 for chromosome 1 - 22 , X and p < 0 . 05 for chromosome Y ; bottom , p < 0 . 0001 for all chromosomes ) .

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(B) Analysis from the Mitelman database for the fraction of human solid tumors that have lost (top) or gained (bottom) individual chromosomes, that have modal chromosome number >2N or ~2N (top, p<0.0001 for chromosome 1-22, X and p<0.05 for chromosome Y; bottom, p<0.0001 for all chromosomes).


( C ) Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome , that have modal chromosome number > 2N or ~ 2N ( p < 0 . 0001 for both modal number > 2N and ~ 2N ) .

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(C) Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome, that have modal chromosome number >2N or ~2N (p<0.0001 for both modal number >2N and ~2N).


( A ) Chromosomal instability readouts , of later passage lines , assessed by average chromosomes loss per metaphase , average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes ( n = 20 for each dataset and error bars denote SD ) .

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(A) Chromosomal instability readouts, of later passage lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n=20 for each dataset and error bars denote SD).


( B ) Determination of number of γH2AX foci in ICL and control nuclei and quantification ( scale bars denote 10μm , p < 0 . 0001 for Ch9 , Ch10 and Ch12 , p < 0 . 05 for Ch14 ; n = 25 for each dataset and error bars denote SD ) . ( See also Figures S5A and S5B )

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(B) Determination of number of γH2AX foci in ICL and control nuclei and quantification (scale bars denote 10μm, p<0.0001 for Ch9, Ch10 and Ch12, p<0.05 for Ch14; n=25 for each dataset and error bars denote SD). (See also Figures S5A and S5B)


( C ) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification ( Scale bars denote 20μm , p < 0 . 0001 for Ch10 and p < 0 . 05 for Ch14 ) .

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(C) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification (Scale bars denote 20μm, p<0.0001 for Ch10 and p<0.05 for Ch14).


( A , B ) Airyscan confocal analysis of PC1 ( 568 , red ) co - localization with GFP - LC3 ( green ) in ( A ) MEF ( B ) Saos2 . Scale bars = 10 µm . ( C ) Airyscan confocal analysis of PC2 ( 647 , red ) co - localization with LC3 ( 488 , green ) in RCS cells . Scale bars = 10 µm . ( A - C ) The insets show higher magnification ( A = x4 . 68 ; B = x6 . 76 ; C = x7 . 33 ) and single colour channels of the boxed area . ( D ) Quantification of GFP ( A , B ) or LC3 ( C ) vesicles positive for PC1 or PC2 , expressed as % of total LC3 ( mean + / - SEM ) , n = 18 cells ( MEFs and Saos2 ) ; n = 12 ( RCS ) from 3 independent experiments .

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(A, B) Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 µm. (C) Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 µm. (A-C) The insets show higher magnification (A = x4.68; B = x6.76; C = x7.33) and single colour channels of the boxed area. (D) Quantification of GFP (A,B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean +/- SEM), n = 18 cells (MEFs and Saos2); n=12 (RCS) from 3 independent experiments.


( E - G ) Scanning confocal microscopy analysis of MEFs , Saos2 and RCS cells treated with BafA1 , immunolabeled for PC1 or PC2 and LAMP1 . Nuclei were stained with Hoechst . ( E , F ) Scale bars = 10 µm , ( G ) Scale bars = 5 µm . The insets show higher magnification ( E = x4 . 99 ; F = x6 . 49 ; G = x2 . 01 ) and single colour channels of the boxed area .

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(E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area.


( H ) Transmission EM analysis in Saos2 cells , treated with BafA1 , showing in detail a lysosome which contains immunolabeled PC1 ( with nanogold particles ) , as indicated by arrows .

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(H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows.


( I ) Scanning confocal microscopy analysis of Saos2 WT and CRISPR - Cas9 IDUA Saos2 at steady state , immunolabeled for PC1 and LAMP1 . Nuclei were stained with Hoechst . Scale bar = 10 µm . The insets show higher magnification ( left = x3 . 09 ; right = x3 . 12 ) and single colour channels of the boxed area . Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell ( mean + / - SEM ) . n = 31 WT cells , n = 33 CRISPR cells counted ; 3 independent experiments . Student ' s unpaired , two - tailed T - Test * * * P < 0 . 0001 .

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(I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001.


( J ) WT and CRISPR IDUA Saos2 lysed and analysed by western blot . Data are representative of 3 independent experiments .

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(J) WT and CRISPR IDUA Saos2 lysed and analysed by western blot. Data are representative of 3 independent experiments.


U2OS expressing ( A ) GFP - LC3 mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification ( A = x3 . 93 from the boxed region are shown on the right . Scale bar = 10 µm .

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U2OS expressing (A) GFP-LC3 mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93 from the boxed region are shown on the right. Scale bar = 10 µm.


U2OS expressing ( B ) GFP - 2 - FYVE ( green ) , mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification B = x3 . 42 from the boxed region are shown on the right . Scale bar = 10 µm .

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U2OS expressing (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification B = x3.42 from the boxed region are shown on the right. Scale bar = 10 µm.


( C ) Airyscan analysis of Saos2 cells expressing GFP - LC3 ( green ) and immunolabeled for PC1 ( 405 , blue ) and CANX ( 647 , red ) . The insets show higher magnification ( x5 . 26 ) and single colour channels of the boxed area . Scale bar = 10 µm .

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(C) Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabeled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 µm.


( D ) Correlative Light Electron Microscopy ( CLEM ) and Electron Tomography of Saos2 cells transfected with GFP - LC3 ( green ) and labelled for PC1 ( 568 , red and nanogold particles ) and CANX ( 647 , blue ) . Cells were first imaged by confocal microscopy ( top left panel ) and then the same region was retraced in EM ( upper middle panel ) and overlay is shown ( upper right panel ) . Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules .

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(D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules.


( E ) Single tomography slice ( left panel , taken from boxed are in D at a magnification of x2 . 84 ) , overlay with immunofluorescence ( IF ) ( central panel ) and IF 3D rendering of AV ( green ) and the CANX positive vesicle containing gold particles of labelled collagen ( blue and white respectively ) inside an AV ( right panel ) .

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(E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).


( A ) . Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of total number of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h . n = 20 cells per condition ; three independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * * P < 0 . 0001 .

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(A). Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of total number of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h. n=20 cells per condition; three independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ***P<0.0001.


( B ) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1 , fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . Scale bar = 10 µm . Insets show magnification of the boxed area . Bar graph ( on the left ) shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 12 , 10 , 12 , 10 , 7 , 10 cells per genotype respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . ns ≥ 0 . 05 , * * * P < 0 . 0001 .

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(B) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1, fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). Scale bar = 10 µm. Insets show magnification of the boxed area. Bar graph (on the left) shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=12, 10, 12, 10, 7, 10 cells per genotype respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. ns ≥0.05, *** P<0.0001.


( A ) WT and CRISPR - Cas9 Fam134b knockout MEFs were treated as indicated , lysed and analysed by western blot with the indicated antibodies . Western blots are representative of 4 independent experiments .

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(A) WT and CRISPR-Cas9 Fam134b knockout MEFs were treated as indicated, lysed and analysed by western blot with the indicated antibodies. Western blots are representative of 4 independent experiments.


( B ) WT and CRISPR - Cas9 Fam134b MEFs were immunolabeled for PC1 ( 568 , red ) , nuclei stained with Hoechst ( blue ) and analysed by scanning confocal microscopy . Scale bar = 10 µm .

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(B) WT and CRISPR-Cas9 Fam134b MEFs were immunolabeled for PC1 (568, red), nuclei stained with Hoechst (blue) and analysed by scanning confocal microscopy. Scale bar = 10 µm.


( C ) CRISPR FAM134B MEF mock , wild type FAM134B - HA or FAM134Blir - HA transfected were immunolabeled for PC1 ( 568 , red ) , LAMP1 ( 488 , green ) and HA ( 647 , violet ) and analysed by scanning confocal microscopy . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , quantification of n = 10 cells per treatment ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test was performed . ns ≥ 0 . 05 , * * * P < 0 . 0001 .

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(C) CRISPR FAM134B MEF mock, wild type FAM134B-HA or FAM134Blir-HA transfected were immunolabeled for PC1 (568, red), LAMP1 (488, green) and HA (647, violet) and analysed by scanning confocal microscopy. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), quantification of n=10 cells per treatment; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed. ns ≥0.05, *** P<0.0001.


( A ) Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes , treated with 100 nM BafA1 for 9h . n = 18 cells / treatment ; 3 independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * P < 0 . 005 , * * * P < 0 . 0001 .

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

(A) Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes, treated with 100 nM BafA1 for 9h. n=18 cells/treatment; 3 independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ** P<0.005, *** P<0.0001.


( B ) WT and Canx - / - MEFs were untreated or treated with BafA1 ( 10 μM ) for 6 h , lysed and analysed by western blot with indicated antibodies . Filamin and β - actin were used as loading control . Dashed line indicates that unnecessary lanes were removed . Western blot is representative of 3 independent experiments .

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(B) WT and Canx-/- MEFs were untreated or treated with BafA1 (10 μM) for 6 h, lysed and analysed by western blot with indicated antibodies. Filamin and β-actin were used as loading control. Dashed line indicates that unnecessary lanes were removed. Western blot is representative of 3 independent experiments.


( C ) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . CST was added where indicated . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 8 , 8 , 12 , 8 , 8 , 8 cells respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . * * * P < 0 . 0001 .

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

(C) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). CST was added where indicated. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=8, 8, 12, 8, 8, 8 cells respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. *** P<0.0001.


( A , B ) Volcano plot comparing protein fold changes between WT versus Fam134b - / - ( A ) and WT versus Canx - / - MEFs ( B ) . Significantly regulated proteins are labelled in red ( log2 fold change > 1 , - log10 p > 1 . 3 ) . Red dots with blue ring indicate collagen 1 peptides . Graphs represent statistics from three separate experiments for each genotype .

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(A, B) Volcano plot comparing protein fold changes between WT versus Fam134b-/- (A) and WT versus Canx-/- MEFs (B). Significantly regulated proteins are labelled in red (log2 fold change > 1, -log10 p>1.3). Red dots with blue ring indicate collagen 1 peptides. Graphs represent statistics from three separate experiments for each genotype.


( B ) U2OS cells were transfected with empty vector control ( pcDNA3 ) , FAM134B - HA WT or mutant constructs as indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against CANX , FAM134B 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

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(B) U2OS cells were transfected with empty vector control (pcDNA3), FAM134B-HA WT or mutant constructs as indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against CANX, FAM134B 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


U2OS cells were transfected with HALO - PC2 , FAM134B - HA or FAM134Blir - HA constructs , treated with 100 nM BafA1 for 6h and with CST where indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against HALO , CANX , FAM134B , LC3 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

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U2OS cells were transfected with HALO-PC2, FAM134B-HA or FAM134Blir-HA constructs, treated with 100 nM BafA1 for 6h and with CST where indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against HALO, CANX, FAM134B, LC3 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild - type ( WT ) , heterozygous ( HET ) and null erythroid progenitors ( EPs ) isolated from fetal livers . Actin was used as loading control . * * indicates nonspecific band . C Quantification of mean protein levels from ( B ) . Numbers in brackets correspond to number of embryos analyzed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos ( C , Statistical significances were determined by One - way ANOVA test with Tukey ' s ( C , test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild-type (WT), heterozygous (HET) and null erythroid progenitors (EPs) isolated from fetal livers. Actin was used as loading control. ** indicates nonspecific band. C Quantification of mean protein levels from (B). Numbers in brackets correspond to number of embryos analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (C, Statistical significances were determined by One-way ANOVA test with Tukey's (C, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


D Immunofluorescence images of Cdk5rap2 WT , HET and null erythroid progenitors isolated from fetal livers . Progenitors were stained for CDK5RAP2 ( grey ) , γ - tubulin ( magenta ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 3 μm . Insets show higher magnification of centrosomes . Scale bar , 1 μm . E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ - tubulin from ( D ) . Numbers in brackets correspond to number of embryos analyzed with a total number of 470 ( WT ) , 406 ( HET ) and 379 ( null ) progenitors . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos E , Statistical significances were determined by One - way ANOVA test with Tukey ' s E , test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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D Immunofluorescence images of Cdk5rap2 WT, HET and null erythroid progenitors isolated from fetal livers. Progenitors were stained for CDK5RAP2 (grey), γ-tubulin (magenta) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 3 μm. Insets show higher magnification of centrosomes. Scale bar, 1 μm. E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ-tubulin from (D). Numbers in brackets correspond to number of embryos analyzed with a total number of 470 (WT), 406 (HET) and 379 (null) progenitors. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos E, Statistical significances were determined by One-way ANOVA test with Tukey's E, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.