Figure 1A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization . Occasionally , additional expression is observed at the posterior tip of the embryo ( asterisk ) . Double labelling by staining for LacZ expression from the CcnoTA allele ( shown in B ) and in situ hybridization for Dand5 , marking crown cells at the circumference of the node . Ccno expression is restricted to ciliated , ventral pit cells of the node as detailed in the transverse section at the indicated plane . C Whole embryo X ‐ Gal staining of E16 CcnoTA / + and wild ‐ type control embryos and indicated section planes of ( D - F ' ) . D - F ' Histological sections of LacZ ‐ stained CcnoTA / + embryo at E16 reveal Ccno expression in the epithelium of ( D ) plexus choroideus and ependyme , ( E ) snout epithelium , and ( F , F ' ) trachea and bronchi . pc , plexus choroideus ; se , snout epithelium ; tr , trachea ; and br , bronchus . G Co ‐ expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X ‐ Gal and immunohistochemistry using a FOXJ1 ‐ specific antibody .

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 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 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

Figure 1A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization. Occasionally, additional expression is observed at the posterior tip of the embryo (asterisk). Double labelling by staining for LacZ expression from the CcnoTA allele (shown in B) and in situ hybridization for Dand5, marking crown cells at the circumference of the node. Ccno expression is restricted to ciliated, ventral pit cells of the node as detailed in the transverse section at the indicated plane.C Whole embryo X‐Gal staining of E16 CcnoTA/+ and wild‐type control embryos and indicated section planes of (D-F').D-F' Histological sections of LacZ‐stained CcnoTA/+ embryo at E16 reveal Ccno expression in the epithelium of (D) plexus choroideus and ependyme, (E) snout epithelium, and (F, F') trachea and bronchi. pc, plexus choroideus; se, snout epithelium; tr, trachea; and br, bronchus.G Co‐expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X‐Gal and immunohistochemistry using a FOXJ1‐specific antibody.


Figure 2A Ccno ‐ deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 ( arrow ) . B , C Cranial MRI analysis ( B ) shows the drastically enlarged ventricular cavity ( marked by V ) and diminished cerebral cortex tissue , ( C ) also seen when brains are cut in coronal orientation . D , D ' Alcian ‐ blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium ( arrows ) . F , F ' Scanning electron microscopy ( SEM ) of P21 adult trachea shows reduced numbers of cilia of Ccno ‐ deficient MCCs . Remaining cilia are found in the central regions of the cell surface , and cell margins frequently lack the ciliary decoration ( arrows ) . G Transmission electron microscopy ( TEM ) of wild ‐ type and CcnoRA / RA MCCs from adult trachea . Ccno ‐ deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface ( arrows ) . Ectopic electron ‐ dense material is found within the cytoplasm of Ccno ‐ deficient MCCs ( arrowheads ) .

O B-PANEL_START 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 B-PANEL_START 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 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 2A Ccno‐deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 (arrow).B, C Cranial MRI analysis (B) shows the drastically enlarged ventricular cavity (marked by V) and diminished cerebral cortex tissue, (C) also seen when brains are cut in coronal orientation.D, D' Alcian‐blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium (arrows).F, F' Scanning electron microscopy (SEM) of P21 adult trachea shows reduced numbers of cilia of Ccno‐deficient MCCs. Remaining cilia are found in the central regions of the cell surface, and cell margins frequently lack the ciliary decoration (arrows).G Transmission electron microscopy (TEM) of wild‐type and CcnoRA/RA MCCs from adult trachea. Ccno‐deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface (arrows). Ectopic electron‐dense material is found within the cytoplasm of Ccno‐deficient MCCs (arrowheads).


Figure 3A - H X ‐ Gal ‐ staining of ( A - C ) control and ( D - H ) CcnoTA / + embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi . Expression is extending to more distal regions , and from E16 staining is also found in bronchioli . I X ‐ Gal ‐ staining indicating LacZ expression from the CcnoTA / + allele in mTEC cultures after 5 days of differentiation ‐ onset by switching to air - liquid interface ( ALI ) conditions ( ALI d5 ) . J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation . mRNA levels from three independent experiments were measured by qRT - PCR and levels of expression set as 1 on day 0 of ALI cultures . Scales for Ccno and Deup1 are indicated on the left , and for Cep152 , Cep63 and Ift57 on the right side . K Relative mRNA expression levels for indicated genes were measured by qRT - PCR in wild ‐ type and Ccno ‐ deficient mTEC cultures at indicated days after differentiation ‐ onset . Relative values were calculated as in ( J ) relative to day 0 of ALI cultures .

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 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 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

Figure 3A-H X‐Gal‐staining of (A-C) control and (D-H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi. Expression is extending to more distal regions, and from E16 staining is also found in bronchioli.I X‐Gal‐staining indicating LacZ expression from the CcnoTA/+ allele in mTEC cultures after 5 days of differentiation‐onset by switching to air-liquid interface (ALI) conditions (ALI d5).J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation. mRNA levels from three independent experiments were measured by qRT-PCR and levels of expression set as 1 on day 0 of ALI cultures. Scales for Ccno and Deup1 are indicated on the left, and for Cep152, Cep63 and Ift57 on the right side.K Relative mRNA expression levels for indicated genes were measured by qRT-PCR in wild‐type and Ccno‐deficient mTEC cultures at indicated days after differentiation‐onset. Relative values were calculated as in (J) relative to day 0 of ALI cultures.


Figure 4A - B ' SEM of tracheae from ( A , A ' ) wild ‐ type and ( B , B ' ) CcnoRA / RA embryos at E17 . In comparison to wild ‐ type MCCs , Ccno ‐ deficient MCCs almost completely lack formation of multiple cilia and only single , or doublet ( arrow ) cilia are observed at E17 . C - E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α ‐ tubulin staining as marker for axonemes in ( D ) Ccno ‐ deficient MCCs of E17 embryonic trachea as quantified in ( E ) . FOV , field of view . F - I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in ( F , F ' , H ) Ccno ‐ proficient and ( G , G ' , I ) Ccno ‐ deficient MCCs . Asterisks in ( F , G ) indicate microvilli that extend from the apical cell surface . ( F ' , G ' ) Deuterosomes in Ccno ‐ deficient MCCs are significantly enlarged ( identical size bars in F ' and G ' ) and show irregular morphology , which is different to the annular shape of wild ‐ type deuterosomes as seen in ( F ' , H ) . Procentrioles ( arrowheads in F ' and I ) that are found at deuterosomes of ( I ) Ccno ‐ deficient MCCs were recurrently found being shorter and appeared less structured in comparison to ( F ' , H ) wild ‐ type cells . J The average length of procentrioles was quantified for wild ‐ type and Ccno ‐ deficient MCCs of E17 tracheae ( 49 procentrioles in n = 3 independent samples for wild ‐ types and 47 procentrioles in n = 3 independent samples for Ccno ‐ deficient MCCs ) . K The diameter of deuterosomes ( 41 deuterosomes in n = 3 independent samples for wild ‐ types ; 38 deuterosomes in n = 5 independent samples for Ccno ‐ deficient MCCs ) was measured in wild ‐ type and Ccno ‐ deficient MCCs of E17 tracheae .

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 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 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

Figure 4A-B' SEM of tracheae from (A, A') wild‐type and (B, B') CcnoRA/RA embryos at E17. In comparison to wild‐type MCCs, Ccno‐deficient MCCs almost completely lack formation of multiple cilia and only single, or doublet (arrow) cilia are observed at E17.C-E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α‐tubulin staining as marker for axonemes in (D) Ccno‐deficient MCCs of E17 embryonic trachea as quantified in (E). FOV, field of view.F-I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in (F, F', H) Ccno‐proficient and (G, G', I) Ccno‐deficient MCCs. Asterisks in (F, G) indicate microvilli that extend from the apical cell surface. (F', G') Deuterosomes in Ccno‐deficient MCCs are significantly enlarged (identical size bars in F' and G') and show irregular morphology, which is different to the annular shape of wild‐type deuterosomes as seen in (F', H). Procentrioles (arrowheads in F' and I) that are found at deuterosomes of (I) Ccno‐deficient MCCs were recurrently found being shorter and appeared less structured in comparison to (F', H) wild‐type cells.J The average length of procentrioles was quantified for wild‐type and Ccno‐deficient MCCs of E17 tracheae (49 procentrioles in n = 3 independent samples for wild‐types and 47 procentrioles in n = 3 independent samples for Ccno‐deficient MCCs).K The diameter of deuterosomes (41 deuterosomes in n = 3 independent samples for wild‐types; 38 deuterosomes in n = 5 independent samples for Ccno‐deficient MCCs) was measured in wild‐type and Ccno‐deficient MCCs of E17 tracheae.


Figure 5Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification ( ALI day 3 ) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS ‐ 6 . Deuterosome numbers are decreased and deuterosome size enlarged in Ccno ‐ deficient MCCs as quantified in ( C ) . Deuterosome ‐ dependent centriole biogenesis is severely reduced in Ccno ‐ deficient MCCs as shown by reduced staining for the early centriole marker SAS ‐ 6 . IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno ‐ deficient MCCs at later stages of centriole biogenesis ( ALI day 7 ) . Deuterosome number and size were quantified in ( C ) . IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7 , which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno ‐ deficient MCCs as seen in z ‐ projection . IFstaining for DEUP1 in ( A , B ) was analysed by Imaris 7 . 7 . 2 software to calculate average deuterosome number of wild ‐ type ( n = 17 ) and Ccno ‐ deficient ( n = 19 ) MCCs at ALI day 3 , and wild ‐ type ( n = 12 ) and Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 . Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3 , and in wild ‐ type ( n = 12 ) and Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 .

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 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 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

Figure 5Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification (ALI day 3) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS‐6. Deuterosome numbers are decreased and deuterosome size enlarged in Ccno‐deficient MCCs as quantified in (C). Deuterosome‐dependent centriole biogenesis is severely reduced in Ccno‐deficient MCCs as shown by reduced staining for the early centriole marker SAS‐6.IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno‐deficient MCCs at later stages of centriole biogenesis (ALI day 7). Deuterosome number and size were quantified in (C). IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7, which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno‐deficient MCCs as seen in z‐projection.IFstaining for DEUP1 in (A, B) was analysed by Imaris 7.7.2 software to calculate average deuterosome number of wild‐type (n = 17) and Ccno‐deficient (n = 19) MCCs at ALI day 3, and wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7. Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3, and in wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7.


Figure 6A , B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164 , which marks maturing centrioles . At ALI day 7 , centrioles of wild ‐ type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane . The fewer centrioles of Ccno ‐ deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z ‐ projection . Lack of CEP164 indicates immature centrioles in Ccno ‐ deficient MCCs . ( B ) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7 . 7 . 2 software . C , D Double IFstaining using Centrin ‐ and CP110 ‐ specific antibodies shows an almost complete absence of CP110 from Centrin ‐ positive centrioles in the majority of wild ‐ type MCCs at day 7 of ALI cultures . Most Ccno ‐ deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z ‐ projection . ( D ) CP110 ‐ positive MCCs were counted in wild ‐ type and Ccno ‐ deficient MTEC cultures and the percentages of CP110 ‐ positive MCCs from total MCCs calculated .

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 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

Figure 6A, B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164, which marks maturing centrioles. At ALI day 7, centrioles of wild‐type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane. The fewer centrioles of Ccno‐deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z‐projection. Lack of CEP164 indicates immature centrioles in Ccno‐deficient MCCs. (B) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7.7.2 software.C, D Double IFstaining using Centrin‐ and CP110‐specific antibodies shows an almost complete absence of CP110 from Centrin‐positive centrioles in the majority of wild‐type MCCs at day 7 of ALI cultures. Most Ccno‐deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z‐projection. (D) CP110‐positive MCCs were counted in wild‐type and Ccno‐deficient MTEC cultures and the percentages of CP110‐positive MCCs from total MCCs calculated.


Figure 1 ( A ) Representative whole mount images of WT and Cep63T / T brains at P60 . Scale bar = 0 . 2 cm . ( B ) Telencephalon area of WT and Cep63T / T brains . WT littermates N = 4 , Cep63T / T N = 7 ; two - tailed Mann - Whitney t - test . ( C ) Representative whole mount images of WT and Sas4cKO brains at P14 . Scale bar : 0 . 2 cm . ( D ) Telencephalon area of WT and Sas4cKO brains . WT littermates N = 8 , Sas4cKO N = 7 ; two - tailed Mann - Whitney t - test . Black circles represent Nestin - Cre + animals . ( E ) Representative images of E14 . 5 WT , Cep63T / T and Sas4cKO cortices stained with antibodies against α - Tubulin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . Magenta arrows indicate dead cells ; white and yellow arrows indicate cells with bipolar and monopolar spindles , respectively . Insets showing zoomed in view of 2 representative cells . Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface . Scale bar = 25 μm . ( F ) Graph showing the percentage of bipolar , monopolar and multipolar cells at the ventricular surface of E14 . 5 cortices . WT n = 201 cells , N = 3 embryos ; Cep63T / T n = 312 cells , N = 3 embryos ; Sas4cKO n = 296 cells , N = 3 embryos ; # , chi - square test with * , post - hoc analysis , comparisons are made to WT . ( G ) E14 . 5 cortices stained with antibodies against Ki67 ( red ) and phosphorylated - Histone H3 ( PH3 , yellow ) . Insets showing zoomed in view of cells in the ventricular zone ( VZ ) and sub - ventricular zone ( SVZ ) . Scale bar = 100 μm . ( H ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Cep63T / T N = 3 , Sas4cKO N = 3 ; two - tailed Mann - Whitney t - test . ( J ) Representative images from live - cell movies of Nuclear - mCherry + WT , Cep63T / T and Sas4cKO NPCs . The timing of mitosis began at nuclear envelope breakdown ( NEBD ) and finished at nuclear envelope reformation . White arrows track the mitotic events of the mother cell from NEBD to the generation of daughter cells following cytokinesis . ( K ) Plot showing the duration of mitosis in WT , Cep63T / T and Sas4cKO NPCs . Triangles represent individual cells ; triangles of the same color represent cells derived from the same embryo . Circles represent the average mitotic duration of cells from each embryo . WT n = 202 cells , N = 4 embryos ; Cep63T / T n = 141 cells , N = 3 embryos ; Sas4cKO n = 138 cells , N = 3 embryos ; two - tailed Welch ' s t - test .

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 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 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 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 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

Figure 1(A) Representative whole mount images of WT and Cep63T/T brains at P60. Scale bar = 0.2 cm. (B) Telencephalon area of WT and Cep63T/T brains. WT littermates N = 4, Cep63T/T N = 7; two-tailed Mann-Whitney t-test.(C) Representative whole mount images of WT and Sas4cKO brains at P14. Scale bar: 0.2 cm. (D) Telencephalon area of WT and Sas4cKO brains. WT littermates N = 8, Sas4cKO N = 7; two-tailed Mann-Whitney t-test. Black circles represent Nestin-Cre+ animals.(E) Representative images of E14.5 WT, Cep63T/T and Sas4cKO cortices stained with antibodies against α-Tubulin (green), γ-Tubulin (red) and DAPI (blue). Magenta arrows indicate dead cells; white and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 25 μm. (F) Graph showing the percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T n = 312 cells, N = 3 embryos; Sas4cKO n = 296 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT.(G) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the ventricular zone (VZ) and sub-ventricular zone (SVZ). Scale bar = 100 μm.(H) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T N = 3, Sas4cKO N = 3; two-tailed Mann-Whitney t-test.(J) Representative images from live-cell movies of Nuclear-mCherry+ WT, Cep63T/T and Sas4cKO NPCs. The timing of mitosis began at nuclear envelope breakdown (NEBD) and finished at nuclear envelope reformation. White arrows track the mitotic events of the mother cell from NEBD to the generation of daughter cells following cytokinesis. (K) Plot showing the duration of mitosis in WT, Cep63T/T and Sas4cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Cep63T/T n = 141 cells, N = 3 embryos; Sas4cKO n = 138 cells, N = 3 embryos; two-tailed Welch's t-test.


Figure 2 ( A - C ) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 mins . WT n = 330 cells , N = 4 embryos ; Cep63T / T n = 260 cells , N = 3 embryos ; Sas4cKO n = 223 cells , N = 3 embryos . ( D - F ) Percentage of proliferating , arrested and apoptotic progeny within each group of the indicated mitotic duration , calculated from data shown in A - C ; groups with n < 20 cells were excluded from this quantification ; # , chi - square test with * , post - hoc analysis , within each genotype , comparisons are made to the 0 - 30min group . ( G ) Genome - wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14 . 5 embryos . Individual cells are represented in rows with copy number states indicated by colors ; arrows on the zoomed in view ( right ) indicate aneuploid cells . WT n = 38 cells , N = 2 embryos ; Cep63T / T n = 40 cells , N = 2 embryos ; Sas4cKO n = 42 cells , N = 2 embryos . ( H ) Percentage of aneuploid cells from the single - cell sequencing data shown in G .

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 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

Figure 2(A-C) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 mins. WT n = 330 cells, N = 4 embryos; Cep63T/T n = 260 cells, N = 3 embryos; Sas4cKO n = 223 cells, N = 3 embryos. (D-F) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in A-C; groups with n < 20 cells were excluded from this quantification; #, chi-square test with *, post-hoc analysis, within each genotype, comparisons are made to the 0-30min group.(G) Genome-wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14.5 embryos. Individual cells are represented in rows with copy number states indicated by colors; arrows on the zoomed in view (right) indicate aneuploid cells. WT n = 38 cells, N = 2 embryos; Cep63T/T n = 40 cells, N = 2 embryos; Sas4cKO n = 42 cells, N = 2 embryos. (H) Percentage of aneuploid cells from the single-cell sequencing data shown in G.


Figure 3 ( A ) Representative whole mount images of WT , Sas4cKO , Sas4cKO ; Trp53bp1 - / - , Sas4cKO ; Usp28cKO animals at P14 . Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia . Scale bar = 0 . 2 cm . ( B ) Telencephalon area of P14 brains of the indicated genotypes . WT littermates N = 8 , Sas4cKO N = 7 , Sas4cKO ; Trp53bp1 - / - N = 6 , Sas4cKO ; Usp28cKO N = 10 , Sas4cKO ; Trp53 - / - N = 5 ; one - way ANOVA with post - hoc analysis . WT and Sas4cKO data are from Figure 1D and shown alongside for comparison . Black circles represent Nestin - Cre + animals . ( C ) Cortical thickness of P14 brains of the indicated genotypes . WT littermates N = 4 , Sas4cKO N = 4 , Sas4cKO ; Trp53bp1 - / - N = 4 , Sas4cKO ; Usp28cKO N = 4 , Sas4cKO ; Trp53 - / - N = 4 ; one - way ANOVA with post - hoc analysis . WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison . Black circles represent Nestin - Cre + animals . ( D ) Representative whole mount images of WT , Cep63T / T , Cep63T / T ; Trp53bp1 - / - , Cep63T / T ; Usp28 - / - animals at P60 . Scale bar = 0 . 2 cm . ( E ) Telencephalon area of P60 brains of the indicated genotypes . WT littermates N = 4 , Cep63T / T N = 7 , Cep63T / T ; Trp53bp1 - / - N = 6 , Cep63T / T ; Usp28 - / - N = 5 ; one - way ANOVA with post - hoc analysis . WT and Cep63T / T data are from Figure 1B and shown alongside for comparison . ( F ) Cortical thickness of P60 brains of the indicated genotypes . WT littermates N = 4 , Cep63T / T N = 5 , Cep63T / T ; Trp53bp1 - / - N = 3 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis . WT and Cep63T / T data are from Figure EV1B and shown alongside for comparison . ( G ) Body weight of P7 animals of the indicated genotypes . WT littermates N = 18 , Cep63T / T N = 16 , Cep63T / T ; Trp53bp1 - / - N = 2 , Cep63T / T ; Usp28 - / - N = 10 ; one - way ANOVA with post - hoc analysis . ( H ) Quantification of total number of cells within a 500 μm - width column of cortices at P60 . WT littermates N = 4 , Cep63T / T N = 4 , Cep63T / T ; Trp53bp1 - / - N = 4 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis . ( I - J ) Quantification of the number of cells in the ( I ) superficial layer ( CUX1 + ) and ( J ) deep layer ( CTIP2 + ) within a 500 μm - width column of P60 cortices . WT littermates N = 3 , Cep63T / T N = 4 , Cep63T / T ; Trp53bp1 - / - N = 4 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis . ( K ) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 ( green ) , superficial layer marker CUX1 ( red ) and DAPI ( blue ) . Scale bar = 200 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 3(A) Representative whole mount images of WT, Sas4cKO, Sas4cKO;Trp53bp1-/-, Sas4cKO;Usp28cKO animals at P14. Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia. Scale bar = 0.2 cm. (B) Telencephalon area of P14 brains of the indicated genotypes. WT littermates N = 8, Sas4cKO N = 7, Sas4cKO;Trp53bp1-/- N = 6, Sas4cKO;Usp28cKO N = 10, Sas4cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure 1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals. (C) Cortical thickness of P14 brains of the indicated genotypes. WT littermates N = 4, Sas4cKO N = 4, Sas4cKO;Trp53bp1-/- N = 4, Sas4cKO;Usp28cKO N = 4, Sas4cKO;Trp53-/- N = 4; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals.(D) Representative whole mount images of WT, Cep63T/T, Cep63T/T;Trp53bp1-/-, Cep63T/T ;Usp28-/- animals at P60. Scale bar = 0.2 cm. (E) Telencephalon area of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 7, Cep63T/T;Trp53bp1-/- N = 6, Cep63T/T;Usp28-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure 1B and shown alongside for comparison. (F) Cortical thickness of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 5, Cep63T/T;Trp53bp1-/- N = 3, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure EV1B and shown alongside for comparison.(G) Body weight of P7 animals of the indicated genotypes. WT littermates N=18, Cep63T/T N=16, Cep63T/T;Trp53bp1-/- N=2, Cep63T/T;Usp28-/- N=10; one-way ANOVA with post-hoc analysis.(H) Quantification of total number of cells within a 500 μm-width column of cortices at P60. WT littermates N = 4, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .(I-J) Quantification of the number of cells in the (I) superficial layer (CUX1+) and (J) deep layer (CTIP2+) within a 500 μm-width column of P60 cortices. WT littermates N = 3, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .(K) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 (green), superficial layer marker CUX1 (red) and DAPI (blue). Scale bar = 200 μm.


Figure 4 ( A ) Cortices from E14 . 5 brains stained with antibodies against the radial glial cell marker PAX6 ( yellow ) , intermediate progenitor marker TBR2 ( red ) and DAPI ( blue ) . Scale bar = 50 μm . ( B and C ) Quantification of the number of ( B ) radial glial cells ( PAX6 + ) and ( C ) intermediate progenitors ( TBR2 + ) within a 250 μm - width column of E14 . 5 cortices . WT N = 3 , Cep63T / T N = 3 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO N = 3 , Sas4cKO ; Usp28cKO N = 3 ; one - way ANOVA with post - hoc analysis . ( D ) Cortices from E14 . 5 embryos stained with antibodies against Cleaved - Caspase 3 ( CC3 , white ) , TP53 ( red ) and DAPI . Scale bar = 50 μm . ( E and F ) Quantification of the number of CC3 + apoptotic cells ( E ) and cells with TP53 activation ( F ) within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Cep63T / T N = 3 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO N = 4 , Sas4cKO ; Usp28cKO N = 4 ; one - way ANOVA with post - hoc analysis .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cortices from E14.5 brains stained with antibodies against the radial glial cell marker PAX6 (yellow), intermediate progenitor marker TBR2 (red) and DAPI (blue). Scale bar = 50 μm. (B and C) Quantification of the number of (B) radial glial cells (PAX6+) and (C) intermediate progenitors (TBR2+) within a 250 μm-width column of E14.5 cortices. WT N=3, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 3, Sas4cKO;Usp28cKO N = 3; one-way ANOVA with post-hoc analysis.(D) Cortices from E14.5 embryos stained with antibodies against Cleaved-Caspase 3 (CC3, white), TP53 (red) and DAPI. Scale bar = 50 μm. (E and F) Quantification of the number of CC3+ apoptotic cells (E) and cells with TP53 activation (F) within a 250 μm-width column of E14.5 cortices. WT N = 5, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 4, Sas4cKO;Usp28cKO N = 4; one-way ANOVA with post-hoc analysis.


Figure 5 ( A ) Representative images of E14 . 5 cortices stained with antibodies against Centrin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . White and yellow arrows indicate cells with bipolar and monopolar spindles , respectively . Insets showing zoomed in view of 2 representative cells . Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface . Scale bar = 10 μm . ( B ) Percentage of bipolar , monopolar and multipolar cells at the ventricular surface of E14 . 5 cortices . WT n = 201 cells , N = 3 embryos ; Cep63T / T ; Usp28 - / - n = 205 cells , N = 3 embryos ; Sas4cKO ; Usp28cKO n = 308 cells , N = 3 embryos ; # , chi - square test with * , post - hoc analysis , comparisons are made to WT . WT data are from Figure 1F and shown alongside for comparison . ( C ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO ; Usp28cKO N = 3 ; two - tailed Mann - Whitney t - test . 3 WT data points are from Figure 1H and shown alongside for comparison . ( D ) Representative images from live - cell movies of Nuclear - mCherry + WT , Sas4cKO and Sas4cKO ; Usp28cKO NPCs . White arrows showing the nuclei of daughter cells following cytokinesis . ( E ) Plot showing quantification of mitotic duration of WT , Sas4cKO and Sas4cKO ; Usp28cKO NPCs . Triangles represent individual cells ; triangles of the same color represent cells derived from the same embryo . Circles represent the average mitotic duration of cells from each embryo . WT n = 202 cells , N = 4 embryos ; Sas4cKO n = 138 cells , N = 3 embryos ; Sas4cKO ; Usp28cKO n = 191 cells , N = 3 embryos ; two - tailed Welch ' s t - test . WT and Sas4cKO data are from Figure 1K and shown alongside for comparison . ( F ) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO ; Usp28cKO E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 min . Sas4cKO ; Usp28cKO n = 271 cells , N = 3 embryos . ( G ) Percentage of proliferating , arrested and apoptotic progeny within each group of the indicated mitotic duration , calculated from data shown in F ; # , chi - square test with * , post - hoc analysis , comparisons are made to the 0 - 30min group . Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 min . ( H and I ) Data from Figures 2C and 2F shown alongside for comparison with F and G .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of E14.5 cortices stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). White and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 10 μm.(B) Percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T;Usp28-/- n = 205 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 308 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT. WT data are from Figure 1F and shown alongside for comparison.(C) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T;Usp28-/- N = 3, Sas4cKO;Usp28cKO N = 3; two-tailed Mann-Whitney t-test. 3 WT data points are from Figure 1H and shown alongside for comparison.(D) Representative images from live-cell movies of Nuclear-mCherry+ WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. White arrows showing the nuclei of daughter cells following cytokinesis. (E) Plot showing quantification of mitotic duration of WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Sas4cKO n = 138 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 191 cells, N = 3 embryos; two-tailed Welch's t-test. WT and Sas4cKO data are from Figure 1K and shown alongside for comparison. (F) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO;Usp28cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. Sas4cKO;Usp28cKO n = 271 cells, N = 3 embryos. (G) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in F; #, chi-square test with *, post-hoc analysis, comparisons are made to the 0-30min group.Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. (H and I) Data from Figures 2C and 2F shown alongside for comparison with F and G.


Figure 6 ( a ) WT and Smc5cKO cortices at E14 . 5 stained with antibodies against Centrin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . Insets showing zoomed in view of 2 representative cells . Scale bar : 25 μm . ( B and C ) Quantification of the number of ( B ) Centrin foci and ( C ) γ - Tubulin foci in mitotic cells in the VZ of E14 . 5 cortices . WT n = 175 cells , N = 4 embryos ; Smc5cKO n = 134 cells , N = 3 embryos . # , chi - square test . WT data are from Figures EV1F and EV1G and shown alongside for comparison . ( D ) E14 . 5 cortices stained with antibodies against Ki67 ( red ) and phosphorylated - Histone H3 ( PH3 , yellow ) . Insets showing zoomed in view of cells in the VZ and SVZ . Scale bar = 100 μm . ( E ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Smc5cKO N = 5 ; two - tailed Mann - Whitney t - test . WT data are from Figure 1H and shown alongside for comparison . ( F Quantification of the number of ( F ) apoptotic cells ( CC3 + ) within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Smc5cKO N = 4 ; two - tailed Mann - Whitney t - test . WT data are from Figures 4E and 4G and shown alongside for comparison . G ) Quantification of the number of ( G ) cells with TP53 activation within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Smc5cKO N = 4 ; two - tailed Mann - Whitney t - test . WT data are from Figures 4E and 4G and shown alongside for comparison . ( H ) Representative whole mount images of WT , Smc5cKO , Smc5cKO ; Usp28cKO , Smc5cKO ; Trp53 - / - brains at P21 . Scale bar = 0 . 2 cm . ( I ) Telencephalon area of P21 brains of the indicated genotypes . WT littermates N = 6 , Smc5cKO N = 5 , Smc5cKO ; Usp28cKO N = 4 , Smc5cKO ; Trp53 - / - N = 5 ; one - way ANOVA with post - hoc analysis .

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 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 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 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

Figure 6(a) WT and Smc5cKO cortices at E14.5 stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). Insets showing zoomed in view of 2 representative cells. Scale bar: 25 μm. (B and C) Quantification of the number of (B) Centrin foci and (C) γ-Tubulin foci in mitotic cells in the VZ of E14.5 cortices. WT n = 175 cells, N = 4 embryos; Smc5cKO n = 134 cells, N = 3 embryos. #, chi-square test. WT data are from Figures EV1F and EV1G and shown alongside for comparison.(D) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the VZ and SVZ. Scale bar = 100 μm.(E) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Smc5cKO N = 5; two-tailed Mann-Whitney t-test. WT data are from Figure 1H and shown alongside for comparison.(F Quantification of the number of (F) apoptotic cells (CC3+) within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.G) Quantification of the number of (G) cells with TP53 activation within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.(H) Representative whole mount images of WT, Smc5cKO, Smc5cKO;Usp28cKO, Smc5cKO;Trp53-/- brains at P21. Scale bar = 0.2 cm. (I) Telencephalon area of P21 brains of the indicated genotypes. WT littermates N = 6, Smc5cKO N = 5, Smc5cKO;Usp28cKO N = 4, Smc5cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis.


Figure 1A . Total cell lysates from wild type ( WT ) and HAT1 knockout ( KO ) Clone 1 HepG2 cells were analyzed for the levels of Ksucc and Kac by Western blot analysis ( left ) . The expression levels of HAT1 , Histone H3 ( H3 ) , and beta - actin ( β - actin ) were measured by Western blot analysis in the cells ( right ) .

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Figure 1A. Total cell lysates from wild type (WT) and HAT1 knockout (KO) Clone 1 HepG2 cells were analyzed for the levels of Ksucc and Kac by Western blot analysis (left). The expression levels of HAT1, Histone H3 (H3), and beta-actin (β-actin) were measured by Western blot analysis in the cells (right).


Figure 2D . HAT1 - catalyzed histone H3 succinylation was analyzed by mixing purified HAT1 , histone H3 , and succinyl - CoA ( 2 μM ) with or without the addition of the indicated concentrations of CoA . Western blot analysis was performed with indicated antibodies . E . HAT1 - mediated histone H3 succinylation was assessed by mixing purified HAT1 , histone H3 , and succinyl - CoA ( 2 μM ) with or without acetyl - CoA ( 2 μM ) . Western blot analysis was performed with the indicated antibodies .

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Figure 2D. HAT1-catalyzed histone H3 succinylation was analyzed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without the addition of the indicated concentrations of CoA. Western blot analysis was performed with indicated antibodies.E. HAT1-mediated histone H3 succinylation was assessed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without acetyl-CoA (2 μM). Western blot analysis was performed with the indicated antibodies.


Figure 3A . The levels of histone H3K122 succinylation , histone H3K122 acetylation , histone H3K27 acetylation , histone H3 , HAT1 , and β - actin were examined by Western blot analysis in HepG2 , HAT1 knockout ( KO ) Clone 1 HepG2 cells , and in HAT1 KO cells reconstituted with either HAT1 or mutant HAT1 ( T188A ) . D . The effect of HAT1 on histone H3K122 succinylation and histone H3K122 acetylation at the indicated gene promoters was determined by ChIP assays with anti - H3K122 succinylation antibody and anti - H3K122 acetylation antibody , and followed by quantitative PCR with primers of the promoter regions of CREBBP , BPTF and RPTOR in WT HepG2 cells , HAT1 KO HepG2 cells and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 ( T188A ) . N = 3 biological replicates . Data were presented as mean ± SD . Student ' s t - test , * * * P < 0 . 001 ; ns , no significance .

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Figure 3A. The levels of histone H3K122 succinylation, histone H3K122 acetylation, histone H3K27 acetylation, histone H3, HAT1, and β-actin were examined by Western blot analysis in HepG2, HAT1 knockout (KO) Clone 1 HepG2 cells, and in HAT1 KO cells reconstituted with either HAT1 or mutant HAT1 (T188A).D. The effect of HAT1 on histone H3K122 succinylation and histone H3K122 acetylation at the indicated gene promoters was determined by ChIP assays with anti-H3K122 succinylation antibody and anti-H3K122 acetylation antibody, and followed by quantitative PCR with primers of the promoter regions of CREBBP, BPTF and RPTOR in WT HepG2 cells, HAT1 KO HepG2 cells and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data were presented as mean ± SD. Student's t-test, ***P < 0.001; ns, no significance.


Figure 4B . Bar graphs depicting the relative ratio of succinylation in HAT1 KO versus WT of the 7 key enzymes in the glycolysis pathway depicted in ( A ) . N = 3 biological replicates . Data were presented as mean ± SD .

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Figure 4B. Bar graphs depicting the relative ratio of succinylation in HAT1 KO versus WT of the 7 key enzymes in the glycolysis pathway depicted in (A). N = 3 biological replicates. Data were presented as mean ± SD.


Figure 5D . HAT1 - catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1 , PGAM1 , and succinyl - CoA / acetyl - CoA in the in vitro succinylation assays . Western blot analysis was performed with indicated antibodies . F . The decrease of glucose and increase of lactate in the culture medium and the relative amounts of 2 - PG and 3 - PG were analyzed by ELISA assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1 mutant ( K99R ) . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - test , * * P < 0 . 01 .

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Figure 5D. HAT1-catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1, PGAM1, and succinyl-CoA/acetyl-CoA in the in vitro succinylation assays. Western blot analysis was performed with indicated antibodies.F. The decrease of glucose and increase of lactate in the culture medium and the relative amounts of 2-PG and 3-PG were analyzed by ELISA assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1 mutant (K99R). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.


Figure 6A . HAT1 levels were assessed by immunohistochemical staining in clinical tissues of liver cancer patients , pancreatic cancer patients , and cholangiocarcinoma patients . Scale bar = 100 μmB . The effect of HAT1 - mediated succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - test , * * P < 0 . 01 . C . The effect of PGAM1 succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - test , * * P < 0 . 01 . HepG2 cells , HAT1 KO HepG2 cells , and HAT1 KO HepG2 cells reconstituted with either wild type HAT1 or mutant HAT1 ( T188A ) were subcutaneously injected into athymic nude mice Tumor volumes and average tumor weight were calculated . N = 6 biological replicates . Scale bar = 10 mm . In the boxplots , boxes extend from the 25th to 75th percentiles ( inter ‐ quartile range ( IQR ) ) , central band represents the median , whiskers indicate the lowest and highest data within 1 . 5 × IQR from the lower and upper quartiles , respectively . Student ' s t - test , * * * P < 0 .

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Figure 6A. HAT1 levels were assessed by immunohistochemical staining in clinical tissues of liver cancer patients, pancreatic cancer patients, and cholangiocarcinoma patients. Scale bar = 100 μmB. The effect of HAT1-mediated succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. C. The effect of PGAM1 succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wild type HAT1 or mutant HAT1 (T188A) were subcutaneously injected into athymic nude mice Tumor volumes and average tumor weight were calculated. N = 6 biological replicates. Scale bar = 10 mm. In the boxplots, boxes extend from the 25th to 75th percentiles (inter‐quartile range (IQR)), central band represents the median, whiskers indicate the lowest and highest data within 1.5 × IQR from the lower and upper quartiles, respectively. Student's t-test, ***P < 0.001


Figure 1 ( B , C ) Exercise capacity of MCK - KO and WT mice from the low intensity regiment was conducted . ( n = 4 WT , n = 5 KO mice , mean ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( E , F ) Exercise capacity of MCK - KO and WT mice from the high intensity regiment ( n = 5 mice , means ± SEM , two - tailed student ' s t - test ) .

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Figure 1(B, C) Exercise capacity of MCK-KO and WT mice from the low intensity regiment was conducted. (n = 4 WT, n = 5 KO mice, mean ±SEM, * p < 0.05, two-tailed student's t-test).(E, F) Exercise capacity of MCK-KO and WT mice from the high intensity regiment (n = 5 mice, means ± SEM, two-tailed student's t-test).


Figure 2 ( A ) The H & E staining of MCK - KO and WT soleus muscle after a bout of low intensity treadmill running ( top 40X , bottom 100X magnifications ) . Black arrow indicates centralized nuclei . ( B ) The percentage of myofibers with centralized nuclei was determined by manual counting 300 myofibers in 20X magnification . ( n = 3 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( C - D ) The analogous set of data is shown with WT and MCK - KO GA muscles . ( n = 3 , means ± SEM , two - tailed student ' s t - test . GA : Gastrocnemius ) Hydrogen peroxide ( H2O2 ) levels were measured in WT and MCK - KO soleus ( E ) at rest and after a bout of exercise ( n = 6 per group , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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Figure 2(A) The H&E staining of MCK-KO and WT soleus muscle after a bout of low intensity treadmill running (top 40X, bottom 100X magnifications). Black arrow indicates centralized nuclei. (B) The percentage of myofibers with centralized nuclei was determined by manual counting 300 myofibers in 20X magnification. (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test). (C-D) The analogous set of data is shown with WT and MCK-KO GA muscles. (n = 3, means ± SEM, two-tailed student's t-test. GA: Gastrocnemius)Hydrogen peroxide (H2O2) levels were measured in WT and MCK-KO soleus (E) at rest and after a bout of exercise (n = 6 per group, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


Figure 3 ( A - D ) Succinate dehydrogenase staining was performed in WT and MCK - KO soleus at sedentary ( A , B ) and after a bout of low - intensity exercise for 50 min ( C , D ) ( 10X , 20X magnifications ) , and the staining intensity was quantified using ImageJ ( n = 6 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( E - H ) The analogous set of data is shown with WT and MCK - KO GA muscles . ( n = 6 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( I - L ) The analogous set of data is shown with WT and MCK - KO EDL muscles . ( n = 6 , means ± SEM , two - tailed student ' s t - test . EDL : Extensor digitorum longus ) ( M , N ) Mitochondrial respiration was measured in WT and MCK - KO soleus tissue after a bout of low - intensity exercise for 50 min under basal conditions and in response to 4 mM oligomycin ( complex V inhibitor ) , 4 mM FCCP ( uncoupler ) , or 4 mM each of rotenone and antimycin A ( complex I inhibitor ) ( n = 8 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) . ( O , P ) Mitochondrial respiration was measured in Dnmt3a knocked down L6 myotubes which were transduced with lentiviral under basal conditions and in response to 4 mM oligomycin ( complex V inhibitor ) , 4 mM FCCP ( uncoupler ) , or 4 mM rotenone and antimycin A ( complex I inhibitor ) ( n = 5 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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Figure 3(A-D) Succinate dehydrogenase staining was performed in WT and MCK-KO soleus at sedentary (A, B) and after a bout of low-intensity exercise for 50 min (C, D) (10X, 20X magnifications), and the staining intensity was quantified using ImageJ (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (E-H) The analogous set of data is shown with WT and MCK-KO GA muscles. (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (I-L) The analogous set of data is shown with WT and MCK-KO EDL muscles. (n = 6, means ± SEM, two-tailed student's t-test. EDL: Extensor digitorum longus) (M, N) Mitochondrial respiration was measured in WT and MCK-KO soleus tissue after a bout of low-intensity exercise for 50 min under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM each of rotenone and antimycin A (complex I inhibitor) (n = 8, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). (O, P) Mitochondrial respiration was measured in Dnmt3a knocked down L6 myotubes which were transduced with lentiviral under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM rotenone and antimycin A (complex I inhibitor) (n = 5, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


Figure 4 ( E , F ) ALDH1L1 protein expression and the quantification in WT vs . MCK - KO soleus , GA and EDL muscles at sedentary and normalizing to GAPDH using ImageJ .

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Figure 4(E, F) ALDH1L1 protein expression and the quantification in WT vs. MCK-KO soleus, GA and EDL muscles at sedentary and normalizing to GAPDH using ImageJ.


Figure 5Single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes were achieved by lentiviral transduction . NADPH levels ( H ) and NOX activity ( I ) were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes . ( n = 3 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) . ( J , K ) H2O2 levels were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes . ( J : n = 3 and K : n = 6 for Control , Aldh1l1 KD , Aldh1l1 KD + Dnmt3a KD , n = 5 for Dnmt3a KD , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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Figure 5Single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes were achieved by lentiviral transduction. NADPH levels (H) and NOX activity (I) were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).(J, K) H2O2 levels were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (J: n = 3 and K: n = 6 for Control, Aldh1l1 KD, Aldh1l1 KD + Dnmt3a KD, n = 5 for Dnmt3a KD, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


Figure 6 ( D , E ) mitochondrial respiration in Dnmt3a knockdown and control L6 myotubes treated with NAC ( 3mM ) or vehicle treatment for 24hrs ( n = 5 Control groups n = 3 Dnmt3a KD , n = 4 Dnmt3a KD with NAC treatment groups . means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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Figure 6(D, E) mitochondrial respiration in Dnmt3a knockdown and control L6 myotubes treated with NAC (3mM) or vehicle treatment for 24hrs (n = 5 Control groups n = 3 Dnmt3a KD, n = 4 Dnmt3a KD with NAC treatment groups. means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


Figure 7 ( D , E ) Exercise capacity of WT and MCK - KO with or without transfection of gCont vs . gAldh1l1 under the low intensity regimen ( n = 4 WT + gCont , n = 9 MCK - KO + gCont , and MCK - KO + gAldh1l1 , * p < 0 . 05 , means ± SEM , two - tailed student ' s t - test and one - way Anova ) .

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Figure 7(D, E) Exercise capacity of WT and MCK- KO with or without transfection of gCont vs. gAldh1l1 under the low intensity regimen (n = 4 WT + gCont, n = 9 MCK- KO + gCont, and MCK- KO + gAldh1l1, *p<0.05, means ± SEM, two-tailed student's t-test and one-way Anova).


f1 ( a ) Western blot analysis of CALM , actin , GAPDH and LC3 - II in several cell lines ( HeLa , HEK , CAD and murine embryonic fibroblast ( MEF ) ) where CALM was knocked down using shRNA or siRNA , as indicated , or knockout with rescue experiment . In all experiments in this paper , we used a scramble siRNA or a luciferase shRNA as controls . The cells were starved in Hanks balanced salt solution ( HBSS ) and treated with Baf A1 as indicated . ( BC , basal conditions ; SE , short exposure ; LE , longer exposure ) . Quantification of LC3 - II / actin or GAPDH ratio is shown in Supplementary Fig . 1A . * Not specific . ( b ) LC3 dot counting in CALM knockdown cells . CALM was knocked down in HeLa cells expressing GFP - LC3 and with or without an siRNA - resistant form of wild - type CALM ( rescue ) , as indicated . The cells were starved in HBSS for 4 h , fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell . The knockdown efficiency and the level of the siRNA - resistant form of CALM are shown on the left on the western blotting . The number of LC3 dots per cell ( shown as mean ± s . d . ) is shown on the graph for each condition ( n ≥ 300 cells per condition ; BC , basal conditions ) . The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right ( * P0 . 01 ; NS , not significant , two - tailed t - test ) . Pictures obtained from automated microscope are shown . Scale bars , 20 μm .

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f1(a) Western blot analysis of CALM, actin, GAPDH and LC3-II in several cell lines (HeLa, HEK, CAD and murine embryonic fibroblast (MEF)) where CALM was knocked down using shRNA or siRNA, as indicated, or knockout with rescue experiment. In all experiments in this paper, we used a scramble siRNA or a luciferase shRNA as controls. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (BC, basal conditions; SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin or GAPDH ratio is shown in Supplementary Fig. 1A. *Not specific.(b) LC3 dot counting in CALM knockdown cells. CALM was knocked down in HeLa cells expressing GFP-LC3 and with or without an siRNA-resistant form of wild-type CALM (rescue), as indicated. The cells were starved in HBSS for 4 h, fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell. The knockdown efficiency and the level of the siRNA-resistant form of CALM are shown on the left on the western blotting. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right (*P0.01; NS, not significant, two-tailed t-test). Pictures obtained from automated microscope are shown. Scale bars, 20 μm.


f2 ( a ) p62 vesicle formation in CALM knockdown HeLa cells . Confocal pictures are shown . # CALM - downregulated cells where p62 vesicles accumulate . Data are representative of three independent experiments and shown as mean ± s . e . m . ( n ≥ 500 cells ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm . ( b ) Western blot analysis of p62 , actin and GAPDH in HeLa cells and HEK cells ( basal conditions , BC , or without serum for 1 h ) where CALM was knocked down using shRNA or siRNA , as indicated . Data are mean ± s . d ( n = 3 experiments for HeLa cells and HEK cells ; * P0 . 05 ; two - tailed t - test ) . ( c ) Percentage of huntingtinQ74 - expressing cells with aggregates in CALM knockdown HeLa cells . Data depict one representative experiments performed in triplicate , out of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . ( d ) Tau - positive tangle formation in CALM knockdown cells . HeLa cells transiently expressing DsRed - tau 4R were treated with Baf A1 for 4 h as indicated . Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody ( green ) . Data represent the number of cells with phosphorylated tau - positive tangles as mean ± s . e . m . ( n = 3 experiments ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm . ( b ) Western blot analysis of p62 , actin and GAPDH in HeLa cells and HEK cells ( basal conditions , BC , or without serum for 1 h ) where CALM was knocked down using shRNA or siRNA , as indicated . Data are mean ± s . d ( n = 3 experiments for HeLa cells and HEK cells ; * P0 . 05 ; two - tailed t - test ) .

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 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 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 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

f2(a) p62 vesicle formation in CALM knockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells where p62 vesicles accumulate. Data are representative of three independent experiments and shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.(b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).(c) Percentage of huntingtinQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).(d) Tau-positive tangle formation in CALM knockdown cells. HeLa cells transiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h as indicated. Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent the number of cells with phosphorylated tau-positive tangles as mean ±s.e.m. (n=3 experiments; *P0.01; two-tailed t-test). Scale bars, 5 μm.(b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).


f3 ( a ) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal ( BC ) and serum starvation conditions . Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions . Data are from one representative experiment , out of three independent experiments . Data shown as mean ± s . e . m . ( n ≥ 500 cells ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm . ( b ) Co - localization between GFP - ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 ( CTX ; 20 min ) in CALM knockdown HeLa cells . Confocal pictures are presented with magnified areas showing the co - localization between ATG16L1 and CTX in greater detail . Scale bars , 5 μm . The Pearson ' s coefficient between ATG16L1 and cholera toxin is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 20 cells ; * P0 . 05 ; two - tailed t - test ) . ( c ) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions . Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two - tailed t - test ) . ( AU , arbitrary unit ) . Scale bars , 5 μm . ( d ) Live - cell imaging of ATG16L1 - GFP in CALM knockdown HeLa cells . ATG16L1 pictures from various time points of a 5 - min movie are shown in inverted grey style . Arrows indicate ATG16L1 vesicles . The number of fusion events per 5 min is shown . Data are representative of five movies and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm . ( e ) In vitro fusion assay of post - nuclear supernatant from HeLa cells expressing either GFP - ATG16L1 or mStrawberry - ATG16L1 in control and CALM knockdown conditions . Confocal pictures are shown where ATG16L1 - mStrawberry signal is shown in purple to enable better visualization . Fused vesicles appear in white . The ATP - negative condition , which prevents SNARE - dependent fusion , is also shown as a control for the reaction . Magnified areas are shown to allow visualization of the vesicles . The percentage of fused vesicles is represented . n = numbers of vesicles scored per field ( a minimum of five fields were analysed per condition ) . Data are representative of two independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ) . Scale bars , 5 μm . ( * P0 . 05 ; two - tailed t - test ) .

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 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 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 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 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

f3(a) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal (BC) and serum starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions. Data are from one representative experiment, out of three independent experiments. Data shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.(b) Co-localization between GFP-ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 (CTX; 20 min) in CALM knockdown HeLa cells. Confocal pictures are presented with magnified areas showing the co-localization between ATG16L1 and CTX in greater detail. Scale bars, 5 μm. The Pearson's coefficient between ATG16L1 and cholera toxin is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥20 cells; *P0.05; two-tailed t-test).(c) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). (AU, arbitrary unit). Scale bars, 5 μm.(d) Live-cell imaging of ATG16L1-GFP in CALM knockdown HeLa cells. ATG16L1 pictures from various time points of a 5-min movie are shown in inverted grey style. Arrows indicate ATG16L1 vesicles. The number of fusion events per 5 min is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.(e) In vitro fusion assay of post-nuclear supernatant from HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1 in control and CALM knockdown conditions. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control for the reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles is represented. n=numbers of vesicles scored per field (a minimum of five fields were analysed per condition). Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test).


f4 ( a ) LC3 levels in CALM knockdown cells . CALM was knocked down in cells expressing or not expressing an siRNA - resistant form of CALM wild type ( wt ) or CALM 219 mutant , as indicated . The cells were lysed and subjected to western blotting . The knockdown efficiency , LC3 - II levels and the level of the siRNA - resistant form of CALM are shown on the western blotting . ( b ) LC3 dots counting in CALM knockdown cells . CALM was knocked down in cells expressing GFP - LC3 with or without an siRNA - resistant form of CALM wild type ( wt ) or CALM mutant ( 219 mutant ) as indicated . The cells were kept in full medium or starved in Hanks balanced salt solution ( HBSS ) for 4 h , fixed and subjected to microscopy to score the number of LC3 dots per cell . The ratio of the number of LC3 dots per cell ( shown as mean ± s . d . ) between starvation and basal conditions is shown on the graph ( * P0 . 01 ; NS , not significant , two - tailed t - test ; n ≥ 300 cells per condition ; BC , basal conditions ) . ( c ) Co - localization between ATG9A and VAMP3 - HA or GFP - LC3 and VAMP8 - HA in control and CALM knockdown HeLa cells stably expressing VAMP3 - HA or VAMP8 - HA . Confocal pictures are shown with magnified areas showing co - localization between ATG9A and VAMP3 or GFP - LC3 and VAMP8 in control cells and no co - localization in CALM knockdown cells . Quantification of ATG9A - VAMP3 or GFP - LC3 - VAMP8 co - localization is shown on the graph as a Pearson ' s coefficient ( data are mean ± s . d . ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm .

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 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

f4(a) LC3 levels in CALM knockdown cells. CALM was knocked down in cells expressing or not expressing an siRNA-resistant form of CALM wild type (wt) or CALM 219 mutant, as indicated. The cells were lysed and subjected to western blotting. The knockdown efficiency, LC3-II levels and the level of the siRNA-resistant form of CALM are shown on the western blotting.(b) LC3 dots counting in CALM knockdown cells. CALM was knocked down in cells expressing GFP-LC3 with or without an siRNA-resistant form of CALM wild type (wt) or CALM mutant (219 mutant) as indicated. The cells were kept in full medium or starved in Hanks balanced salt solution (HBSS) for 4 h, fixed and subjected to microscopy to score the number of LC3 dots per cell. The ratio of the number of LC3 dots per cell (shown as mean ±s.d.) between starvation and basal conditions is shown on the graph (*P0.01; NS, not significant, two-tailed t-test; n≥300 cells per condition; BC, basal conditions).(c) Co-localization between ATG9A and VAMP3-HA or GFP-LC3 and VAMP8-HA in control and CALM knockdown HeLa cells stably expressing VAMP3-HA or VAMP8-HA. Confocal pictures are shown with magnified areas showing co-localization between ATG9A and VAMP3 or GFP-LC3 and VAMP8 in control cells and no co-localization in CALM knockdown cells. Quantification of ATG9A-VAMP3 or GFP-LC3-VAMP8 co-localization is shown on the graph as a Pearson's coefficient (data are mean ±s.d.; *P0.05; two-tailed t-test). Scale bars, 5 μm.


f5 ( a ) Co - localization between ATG12 and VAMP2 - HA in control and CALM knockdown HeLa cells stably expressing VAMP2 - HA . Confocal pictures are shown with magnified areas on the right showing co - localization between ATG12 and VAMP2 in control cells and no co - localization in CALM knockdown cells . Quantification of ATG12 - VAMP2 co - localization is shown on the graph as the Pearson ' s coefficient ( data are mean ± s . d . ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm . ( b ) Western blot analysis of VAMP2 , actin and LC3 - II in HeLa cells where VAMP2 was knocked down , as indicated . The cells were starved in Hanks balanced salt solution ( HBSS ) and treated with Baf A1 as indicated . ( SE , short exposure ; LE , longer exposure . ) Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; NS , not significant , two - tailed t - test ) . ( c ) LC3 dot counting in CALM and VAMP2 knockdown cells . CALM or VAMP2 were knocked down in cells expressing GFP - LC3 . The cells were kept in full medium or starved in HBSS for 4 h , fixed and subjected to automated fluorescence microscopy to score the number of LC3 dots per cell . The number of LC3 dots per cell ( shown as mean ± s . d . ) is shown on the graph for each condition ( n ≥ 300 cells per condition ; BC , basal conditions ) . ( * P0 . 01 ; NS , not significant , two - tailed t - test ) . ( d ) Number of p62 dots per cell in VAMP2 knockdown . HeLa cells where VAMP2 was knocked down were fixed and subjected to microscopy after labelling endogenous p62 using specific antibody . The data represent the number of p62 dots per cell shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ; n ≥ 300 cells per condition ) . ( e ) Percentage of huntingtinQ74 - expressing cells with aggregates in VAMP2 knockdown HeLa cells . Data are from one representative experiments performed in triplicate , out of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . ( f ) Western blot analysis of VAMP2 , actin and LC3 - II in HeLa cells stably expressing VAMP2 - HA wild type or VAMP2 - HA mutated in the CALM - binding site at different levels ( wild - type clone 6 , wt Cl6 : low level ; wild - type clone 11 , wt Cl11 : high level ; mutant clone 11 , AA Cl11 : low level ; mutant clone 12 , AA Cl12 : high level ) . The cells were treated with Baf A1 as indicated . ( SE , short exposure ; LE , longer exposure ) . Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

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 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 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 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 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 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

f5(a) Co-localization between ATG12 and VAMP2-HA in control and CALM knockdown HeLa cells stably expressing VAMP2-HA. Confocal pictures are shown with magnified areas on the right showing co-localization between ATG12 and VAMP2 in control cells and no co-localization in CALM knockdown cells. Quantification of ATG12-VAMP2 co-localization is shown on the graph as the Pearson's coefficient (data are mean ±s.d.;*P0.05; two-tailed t-test). Scale bars, 5 μm.(b) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells where VAMP2 was knocked down, as indicated. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure.) Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; NS, not significant, two-tailed t-test).(c) LC3 dot counting in CALM and VAMP2 knockdown cells. CALM or VAMP2 were knocked down in cells expressing GFP-LC3. The cells were kept in full medium or starved in HBSS for 4 h, fixed and subjected to automated fluorescence microscopy to score the number of LC3 dots per cell. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). (*P0.01; NS, not significant, two-tailed t-test).(d) Number of p62 dots per cell in VAMP2 knockdown. HeLa cells where VAMP2 was knocked down were fixed and subjected to microscopy after labelling endogenous p62 using specific antibody. The data represent the number of p62 dots per cell shown as mean ±s.d. (*P0.05; two-tailed t-test; n≥300 cells per condition).(e) Percentage of huntingtinQ74-expressing cells with aggregates in VAMP2 knockdown HeLa cells. Data are from one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).(f) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells stably expressing VAMP2-HA wild type or VAMP2-HA mutated in the CALM-binding site at different levels (wild-type clone 6, wt Cl6: low level; wild-type clone 11, wt Cl11: high level; mutant clone 11, AA Cl11: low level; mutant clone 12, AA Cl12: high level). The cells were treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


f6 ( a ) Size of endogenous ATG12 vesicles in HeLa cells stably expressing either wild - type or mutant VAMP2 . Confocal pictures are presented with magnified areas showing ATG12 vesicles . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two tail t - test ) . Scale bars , 5 μm . ( b ) Co - localization between ATG12 and LC3 in HeLa cells stably expressing either wild - type clone 11 or mutant clone 12 VAMP2 . Confocal pictures are presented with magnified areas showing ATG12 - LC3 co - localization . The Pearson ' s coefficient between ATG12 and LC3 is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm . ( c ) Size and number of endogenous ATG12 vesicles in VAMP2 - knockdown HeLa cells in starvation condition . Confocal pictures are shown with arrows indicating ATG12 vesicles . Data are representative of three independent experiments and shown as mean ± s . d . for the size of ATG12 vesicle . ( n ≥ 100 vesicles ; * P0 . 05 ; based two - tailed t - test ) . Scale bars , 5 μm . ( d ) Live - cell imaging of ATG16L1 - GFP in VAMP2 - knockdown HeLa cells . Confocal pictures from various time points of a 5 - min movie are shown in inverted greyscale . Arrows indicate ATG16L1null . The number of fusion events per vesicles is shown . Data are representative of five movies and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm . ( e ) In vitro fusion assay of post - nuclear supernatants from control and VAMP2 knockdown HeLa cells expressing either GFP - ATG16L1 or mStrawberry - ATG16L1 . Confocal pictures are shown where ATG16L1 - mStrawberry signal is shown in purple to enable better visualization . Fused vesicles appear in white . The ATP - negative condition , which prevents SNARE - dependent fusion , is also shown as a control of reaction . Magnified areas are shown to allow visualization of the vesicles . The percentage of fused vesicles ( white ) is represented . Data are representative of two independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ) . Scale bars , 5 μm . ( * P0 . 05 ; two - tailed t - test ) . n = numbers of vesicles scored per field .

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 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 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 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 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

f6(a) Size of endogenous ATG12 vesicles in HeLa cells stably expressing either wild-type or mutant VAMP2. Confocal pictures are presented with magnified areas showing ATG12 vesicles. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two tail t-test). Scale bars, 5 μm.(b) Co-localization between ATG12 and LC3 in HeLa cells stably expressing either wild-type clone 11 or mutant clone 12 VAMP2. Confocal pictures are presented with magnified areas showing ATG12-LC3 co-localization. The Pearson's coefficient between ATG12 and LC3 is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). Scale bars, 5 μm.(c) Size and number of endogenous ATG12 vesicles in VAMP2-knockdown HeLa cells in starvation condition. Confocal pictures are shown with arrows indicating ATG12 vesicles. Data are representative of three independent experiments and shown as mean ±s.d. for the size of ATG12 vesicle. (n≥100 vesicles; *P0.05; based two-tailed t-test). Scale bars, 5 μm.(d) Live-cell imaging of ATG16L1-GFP in VAMP2-knockdown HeLa cells. Confocal pictures from various time points of a 5-min movie are shown in inverted greyscale. Arrows indicate ATG16L1null. The number of fusion events per vesicles is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.(e) In vitro fusion assay of post-nuclear supernatants from control and VAMP2 knockdown HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control of reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles (white) is represented. Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test). n=numbers of vesicles scored per field.


f7 ( a ) Western blotting showing downregulation of lap ( i ) or increase in Atg8a - II ( ii ) expression level in adult fly heads on lap downregulation using the UAS - RNAi lines lapGD12732 or lapKK105767 , or the heterozygous allele lap1 . Quantification of lap / actin and Atg8a - II / actin is shown . Genotypes : Control w ; elav - GAL4 / + ; for the RNAi lines : w ; elav - GAL4 / lapKK105767 and w ; elav - GAL4 / + ; lapGD12732 / + ; for the lap1 allele : w ; elav - GAL4 / + ; lap1 / + . ( b ) Western blotting showing the accumulation of tau in Drosophila adult flyheads on lap downregulation using the UAS - RNAi lines lapGD12732 or lapKK105767 , or the heterozygous allele lap1 . Quantification of tau / lapGD12732 is shown . Genotypes : Control w ; elav - GAL4 / + ; for tau - WT : w ; elav - GAL4 / + ; UAS - tau - WT / + ; for RNAi lines : w ; elav - GAL4 / lapKK105767 ; UAS - tau - WT / + and w ; elav - GAL4 / + ; lapGD12732 / tau - WT ; for the lap1 allele : w ; elav - GAL4 / + ; lap1 / + or w ; elav - GAL4 / + ; lap1 / UAS - tau - WT .

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

f7(a) Western blotting showing downregulation of lap (i) or increase in Atg8a-II (ii) expression level in adult fly heads on lap downregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or the heterozygous allele lap1. Quantification of lap/actin and Atg8a-II/actin is shown. Genotypes: Control w; elav-GAL4/+; for the RNAi lines: w; elav-GAL4/ lapKK105767 and w; elav-GAL4/+; lapGD12732/+; for the lap1 allele: w; elav-GAL4/+; lap1/+.(b) Western blotting showing the accumulation of tau in Drosophila adult flyheads on lap downregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or the heterozygous allele lap1. Quantification of tau/lapGD12732 is shown. Genotypes: Control w; elav-GAL4/+; for tau-WT: w; elav-GAL4/+; UAS-tau-WT/+; for RNAi lines: w; elav-GAL4/ lapKK105767; UAS-tau-WT/+ and w; elav-GAL4/+; lapGD12732/tau-WT; for the lap1 allele: w; elav-GAL4/+; lap1/+ or w; elav-GAL4/+; lap1/UAS-tau-WT.