Figure 6Macrophages treated with MDP after M . tuberculosis infection develop autophagy vacuoles . Alveolar macrophages were infected with M . tuberculosis H37Rv at an MOI of 5 for 1 h . Nonphagocytosed bacteria were washed away , and the macrophages were incubated for an additional hour . The cells were then treated with 10 μg / mL of MDP for 24 h . Autophagy indicators ( arrowheads ) such as ( A ) vesicles containing organelles ( TEM magnification × 20 , 000 ) and ( B ) onion skin ‐ like lamellar multivesicular bodies ( TEM magnification × 16 , 000 ) were identified by ultrastructural analysis . . ( C , D ) Autophagosomes containing mycobacteria ( Mtb ) are indicated with arrows ( C , TEM magnification × 40 , 000 ; D , TEM magnification × 12 , 000 ) . Micrographs were obtained from one experiment . Bar represents 300 nm .

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 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

Figure 6Macrophages treated with MDP after M. tuberculosis infection develop autophagy vacuoles. Alveolar macrophages were infected with M. tuberculosis H37Rv at an MOI of 5 for 1 h. Nonphagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour. The cells were then treated with 10 μg/mL of MDP for 24 h. Autophagy indicators (arrowheads) such as (A) vesicles containing organelles (TEM magnification ×20,000) and (B) onion skin‐like lamellar multivesicular bodies (TEM magnification ×16,000) were identified by ultrastructural analysis..(C, D) Autophagosomes containing mycobacteria (Mtb) are indicated with arrows (C, TEM magnification ×40,000; D, TEM magnification ×12,000). Micrographs were obtained from one experiment. Bar represents 300 nm.


figf1 ( a ) Chemical structure of PIK - III . ( b ) The indicated kinases were incubated with different doses of PIK - III and IC50 values were determined . ( c ) U2OS cells expressing GFP - FYVE were incubated with the indicated compounds for 2 h , fixed , stained with Hoechst 33342 and imaged by automated epifluorescence microscopy . GFP - positive puncta per cell were calculated and used to determine IC50 values for each compound . Data points represent values from three wells of one experiment . ( d ) U2OS cells expressing GFP - FOXO3A were incubated with the indicated compounds for 2 h , fixed , stained with Hoechst 33342 and imaged by automated epifluorescence microscopy . The ratio of nuclear to cellular GFP fluorescence intensity was calculated and used to determine IC50 values for each compound . Data points represent values from three wells of one experiment .

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 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

figf1(a) Chemical structure of PIK-III. (b) The indicated kinases were incubated with different doses of PIK-III and IC50 values were determined.(c) U2OS cells expressing GFP-FYVE were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. GFP-positive puncta per cell were calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment.(d) U2OS cells expressing GFP-FOXO3A were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. The ratio of nuclear to cellular GFP fluorescence intensity was calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment.


figf2 ( a ) General domain structure of human VPS34 kinase comprising the helical domain ( yellow ) and the kinase domain ( green ) . The ATP - binding pocket and active site lie between the amino - terminal lobe and the carboxy - terminal lobe of the kinase domain . ( b ) Close - up view of the VPS34 active site with PIK - III bound in the ATP - binding pocket . PIK - III forms two hydrogen - bond interactions between the compound ' s donor / acceptor moiety and the backbone amide and carbonyl oxygen of residue Ile 685 . A prominent hydrophobic pocket , bounded in part by the side chains of Phe 612 , Pro 618 and Phe 684 accommodates the cyclopropyl functional group . An extended solvent - mediated hydrogen - bonding network bridges interactions between the PIK - III aminopyrimidine moiety and the side chains of Asp 671 and Asp 644 . ( c ) The binding properties surface of the VPS34 active site is superimposed on the solvent - accessible surface . Green surfaces are regions with hydrophobic binding properties ; note the hydrophobic nature of the active site floor and the deep hydrophobic pocket in which the cyclopropyl moiety fits . Blue and red surfaces represent hydrogen bond donor and acceptor regions , respectively . PIK - III is shown bound in the active site . ( d ) Superposition of the PIK - III - bound VPS34 pocket surface with a mesh ( white ) mapping the human PI ( 3 ) Kα active site . The displacement of the PI ( 3 ) Kα hydrophobic pocket away from the hinge ( relative to that of VPS34 ) is readily apparent .

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

figf2(a) General domain structure of human VPS34 kinase comprising the helical domain (yellow) and the kinase domain (green). The ATP-binding pocket and active site lie between the amino-terminal lobe and the carboxy-terminal lobe of the kinase domain. (b) Close-up view of the VPS34 active site with PIK-III bound in the ATP-binding pocket. PIK-III forms two hydrogen-bond interactions between the compound's donor/acceptor moiety and the backbone amide and carbonyl oxygen of residue Ile 685. A prominent hydrophobic pocket, bounded in part by the side chains of Phe 612, Pro 618 and Phe 684 accommodates the cyclopropyl functional group. An extended solvent-mediated hydrogen-bonding network bridges interactions between the PIK-III aminopyrimidine moiety and the side chains of Asp 671 and Asp 644. (c) The binding properties surface of the VPS34 active site is superimposed on the solvent-accessible surface. Green surfaces are regions with hydrophobic binding properties; note the hydrophobic nature of the active site floor and the deep hydrophobic pocket in which the cyclopropyl moiety fits. Blue and red surfaces represent hydrogen bond donor and acceptor regions, respectively. PIK-III is shown bound in the active site. (d) Superposition of the PIK-III-bound VPS34 pocket surface with a mesh (white) mapping the human PI(3)Kα active site. The displacement of the PI(3)Kα hydrophobic pocket away from the hinge (relative to that of VPS34) is readily apparent.


figf3 ( a ) H4 cells expressing mCherry - GFP - LC3 were treated overnight with the indicated compounds , fixed , stained with Hoechst 33342 and imaged by automated acquisition . Representative images of an overlay of GFP ( green ) , mCherry ( red ) and Hoechst ( blue ) are shown with a scale bar equivalent to 10 μm . Bars represent the mean GFP - and mCherry - positive LC3 puncta per cell and data points represent values from 18 imaged fields in two wells from one experiment . b ) HeLa cells expressing GFP - Parkin were treated with PIK - III for 12 h followed by the addition of CCCP for 12 h , fixed , stained for endogenous Tom20 and imaged . Representative images of an overlay of Tom20 ( red ) and Hoechst ( blue ) are shown with a scale bar equivalent to 50 μm . Bars represent the mean cellular Tom20 staining intensity per cell and data points represent values from four wells from one experiment . ( c ) H4 cells expressing GFP - p62 were treated overnight with the indicated compounds , fixed , stained with Hoechst 33342 and imaged . Representative images of an overlay of GFP ( green ) and Hoechst ( blue ) are shown with a scale bar equivalent to 10 μm . Bars represent the mean GFP - positive puncta area per cell and data points represent values from 18 imaged fields in 2 wells from one experiment . ( d ) Steady - state levels of autophagy markers visualized by western blotting in H4 GFP - p62 cells treated overnight with 0 . 5 μM AZD8055 or 5 μM PIK - III . ( e ) Steady - state levels of p62 , LC3 and GAPDH visualized by western blotting in PSN - 1 cells treated for 48 h with the indicated doses of PIK - III . ( f ) Steady - state levels of p62 , LC3 and GAPDH visualized by western blotting in Panc10 . 05 cells treated for 48 h with the indicated doses of PIK - III . ( g ) RKO cells expressing inducible HA - ATG4B - C74A were treated with doxycycline ( DOX induction ) for five days and then incubated in DOX - free medium for 24 h ( DOX washout ) . Bafilomycin A1 was added with or without PIK - III at the beginning of the washout period ( lanes 5 and 6 ) . Steady - state levels of ATG4B , LC3 and GAPDH were visualized by western blotting . Uncropped images of blots are shown in Supplementary Fig . 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

figf3(a) H4 cells expressing mCherry-GFP-LC3 were treated overnight with the indicated compounds, fixed, stained with Hoechst 33342 and imaged by automated acquisition. Representative images of an overlay of GFP (green), mCherry (red) and Hoechst (blue) are shown with a scale bar equivalent to 10 μm. Bars represent the mean GFP- and mCherry-positive LC3 puncta per cell and data points represent values from 18 imaged fields in two wells from one experiment.b) HeLa cells expressing GFP-Parkin were treated with PIK-III for 12 h followed by the addition of CCCP for 12 h, fixed, stained for endogenous Tom20 and imaged. Representative images of an overlay of Tom20 (red) and Hoechst (blue) are shown with a scale bar equivalent to 50 μm. Bars represent the mean cellular Tom20 staining intensity per cell and data points represent values from four wells from one experiment.(c) H4 cells expressing GFP-p62 were treated overnight with the indicated compounds, fixed, stained with Hoechst 33342 and imaged. Representative images of an overlay of GFP (green) and Hoechst (blue) are shown with a scale bar equivalent to 10 μm. Bars represent the mean GFP-positive puncta area per cell and data points represent values from 18 imaged fields in 2 wells from one experiment.(d) Steady-state levels of autophagy markers visualized by western blotting in H4 GFP-p62 cells treated overnight with 0.5 μM AZD8055 or 5 μM PIK-III.(e) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in PSN-1 cells treated for 48 h with the indicated doses of PIK-III. (f) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in Panc10.05 cells treated for 48 h with the indicated doses of PIK-III.(g) RKO cells expressing inducible HA-ATG4B-C74A were treated with doxycycline (DOX induction) for five days and then incubated in DOX-free medium for 24 h (DOX washout). Bafilomycin A1 was added with or without PIK-III at the beginning of the washout period (lanes 5 and 6). Steady-state levels of ATG4B, LC3 and GAPDH were visualized by western blotting. Uncropped images of blots are shown in Supplementary Fig. 7.


figf4 . ( b ) Proteins represented by unique ubiquitylation sites with > 1 . 5 log2 ( fold change ) in both replicates after diglycyl - peptide enrichment were searched for > 1 . 5 log2 ( fold change ) at the proteome level in both replicates . The resulting 15 proteins are listed to the right in descending order of the average protein fold change in the flow - through replicate analyses . Two independent biological replicates are shown in crossplots . ( c ) Steady - state levels of NCOA4 in DLD1 cells treated with bafilomycin A1 or PIK - III for 20 h . ( d ) The levels of NCOA4 and p62 in ATG7 wild - type ( + / + ) , heterozygous ( + / − ) or homozygous knockout ( − / − ) DLD1 clones 1 and 2 were determinede ) DLD1 cells cultured in rich medium ( Fed ) were deprived of serum and amino acids ( Starve ) for the indicated times ( hours , h ) and levels of NCOA4 , p62 , LC3 and GAPDH were determined by western blotting . ( f ) Confocal microscopy was used to evaluate the cellular distribution of endogenous NCOA4 , LC3 , LAMP2 and EEA1 in cells treated overnight with bafilomycin A1 or PIK - III . Regions outlined with white dashed lines are magnified to the right of each panel . Scale bars in full panels on left , and zoomed panels on right correspond to 10 and 2 μm respectively . Uncropped images of blots are shown in Supplementary Fig . 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4. (b) Proteins represented by unique ubiquitylation sites with >1.5 log2 (fold change) in both replicates after diglycyl-peptide enrichment were searched for >1.5 log2 (fold change) at the proteome level in both replicates. The resulting 15 proteins are listed to the right in descending order of the average protein fold change in the flow-through replicate analyses. Two independent biological replicates are shown in crossplots.(c) Steady-state levels of NCOA4 in DLD1 cells treated with bafilomycin A1 or PIK-III for 20 h.(d) The levels of NCOA4 and p62 in ATG7 wild-type (+/+), heterozygous (+/−) or homozygous knockout (−/−) DLD1 clones 1 and 2 were determinede) DLD1 cells cultured in rich medium (Fed) were deprived of serum and amino acids (Starve) for the indicated times (hours, h) and levels of NCOA4, p62, LC3 and GAPDH were determined by western blotting.(f) Confocal microscopy was used to evaluate the cellular distribution of endogenous NCOA4, LC3, LAMP2 and EEA1 in cells treated overnight with bafilomycin A1 or PIK-III. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full panels on left, and zoomed panels on right correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.


figf5 ( a ) Endogenous immunoprecipitation of NCOA4 with ferritin heavy ( FTH1 ) and light ( FTL ) chains from DLD1 cell lysates . b ) Steady - state levels of FTH1 and FTL expressed in DLD1 cells growing in rich medium or starved for the times indicated ( hours , h ) . ( c ) DLD1 cells were cultured in the presence or absence of bafilomycin A1overnight , fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous FTH1 or FTL with LC3 , LAMP2 or EEA1 . Regions outlined with white dashed lines are magnified to the right of each panel . Scale bars in full and zoomed panels correspond to 10 and 2 μm respectively . ( d ) DLD1 parental or NCOA4 − / − cells ( clones 1 and 2 ; KO - 1 , KO - 2 ) were starved for 6 h and the levels of NCOA4 , FTH1 , FTL and GAPDH determined . ( e ) DLD1 parental or NCOA4 − / − cells were cultured in the presence or absence of bafilomycin A1overnight , fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous ferritin ( FTH1 - FTL ) and LAMP2 . Regions outlined with white dashed lines are magnified to the right of each panel . Scale bars of full and zoomed panels correspond to 10 and 2 μm respectively . Uncropped images of blots are shown in Supplementary Fig . 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 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 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 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

figf5(a) Endogenous immunoprecipitation of NCOA4 with ferritin heavy (FTH1) and light (FTL) chains from DLD1 cell lysates.b) Steady-state levels of FTH1 and FTL expressed in DLD1 cells growing in rich medium or starved for the times indicated (hours, h).(c) DLD1 cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous FTH1 or FTL with LC3, LAMP2 or EEA1. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full and zoomed panels correspond to 10 and 2 μm respectively.(d) DLD1 parental or NCOA4−/− cells (clones 1 and 2; KO-1, KO-2) were starved for 6 h and the levels of NCOA4, FTH1, FTL and GAPDH determined.(e) DLD1 parental or NCOA4−/− cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous ferritin (FTH1-FTL) and LAMP2. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars of full and zoomed panels correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.


figf6 ( a ) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot . Proteins elute in high - to low - molecular - weight fractions from 18 to 33 , respectively . Dashed lines are for visual comparison only . ( b ) DLD1 parental or NCOA4 − / − cell extracts were resolved using SEC and each fraction was analysed for the presence of NCOA4 , FTH1 and FTL by western blot . ( c ) NCOA4 − / − cells were transfected with control or SBP - NCOA4 cDNA for 36 h and incubated overnight in the presence or absence of the indicated inhibitors , lysed and the occurrence of the faster - migrating FTH1 band was determined . ( d ) Endogenous NCOA4 immunoprecipitations from DLD1 parental , FTH1 − / − or FTL − / − ( clones 1 and 2 for each genotype ) protein extracts . The presence of FTH1 and FTL was detected by western blot . Uncropped images of blots are shown in Supplementary Fig . 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot. Proteins elute in high- to low-molecular-weight fractions from 18 to 33, respectively. Dashed lines are for visual comparison only.(b) DLD1 parental or NCOA4−/− cell extracts were resolved using SEC and each fraction was analysed for the presence of NCOA4, FTH1 and FTL by western blot.(c) NCOA4−/− cells were transfected with control or SBP-NCOA4 cDNA for 36 h and incubated overnight in the presence or absence of the indicated inhibitors, lysed and the occurrence of the faster-migrating FTH1 band was determined.(d) Endogenous NCOA4 immunoprecipitations from DLD1 parental, FTH1−/− or FTL−/− (clones 1 and 2 for each genotype) protein extracts. The presence of FTH1 and FTL was detected by western blot. Uncropped images of blots are shown in Supplementary Fig. 7.


figf7 ( a ) DLD1 cells were incubated in the presence or absence of DFX ( 30 μM ) and PIK - III ( 5 μM ) and the levels of FTH1 and FTL were analysed using western blotting . . ( b ) DLD parental or NCOA4 − / − cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined . ( c ) Ncoa4 − / − mouse embryonic stem cells were analysed for Ncoa4 and Fth1 by western blotting . Note the complete absence of the lower Fth1 band consistent with earlier findings . ( d ) Representative haemotoxylin and eosin staining of spleens from control or Ncoa4 − / − mice is shown and brown pigmented regions apparent in Ncoa4 − / − sections are indicated ( white arrows ) . ( e ) The presence of iron in spleens from control or Ncoa4 − / − mice was determined using Perls ' Prussian blue staining . Representative micrographs ( left panels ) or magnifications ( right panels ) are shown . Scale bars correspond to 100 μm and 20 μm in full and magnified panels respectively . ( f ) Quantitative image analysis of the data shown in e was performed as described in the Methods . Each data point represents relative iron levels in a single mouse spleen section and horizontal lines are means of each group ( n = 6 mice per group ; ∗ P 0 . 0006 for unpaired t - test ) . Uncropped images of blots are shown in Supplementary Fig . 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

figf7(a) DLD1 cells were incubated in the presence or absence of DFX (30 μM) and PIK-III (5 μM) and the levels of FTH1 and FTL were analysed using western blotting.. (b) DLD parental or NCOA4−/− cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined.(c) Ncoa4−/− mouse embryonic stem cells were analysed for Ncoa4 and Fth1 by western blotting. Note the complete absence of the lower Fth1 band consistent with earlier findings.(d) Representative haemotoxylin and eosin staining of spleens from control or Ncoa4−/− mice is shown and brown pigmented regions apparent in Ncoa4−/− sections are indicated (white arrows).(e) The presence of iron in spleens from control or Ncoa4−/− mice was determined using Perls' Prussian blue staining. Representative micrographs (left panels) or magnifications (right panels) are shown. Scale bars correspond to 100 μm and 20 μm in full and magnified panels respectively. (f) Quantitative image analysis of the data shown in e was performed as described in the Methods. Each data point represents relative iron levels in a single mouse spleen section and horizontal lines are means of each group (n = 6 mice per group; ∗P 0.0006 for unpaired t-test). Uncropped images of blots are shown in Supplementary Fig. 7.


Figure 1B , C Inhibitory effect of mycolactone on cell expression of CD38 ( Type II ) and CD138 , IL - 6R , and CD40 ( Type I ) transmembrane proteins ( B ) . Stimulatory effect of mycolactone on cell expression of BCMA , a Type III transmembrane protein ( C ) . Mean Fluorescence Intensities ( MFIs ) were measured in live cells 48 h after addition of mycolactone . Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments , relative to vehicle - treated controls ( Ctrl ) .

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Figure 1B, C Inhibitory effect of mycolactone on cell expression of CD38 (Type II) and CD138, IL-6R, and CD40 (Type I) transmembrane proteins (B). Stimulatory effect of mycolactone on cell expression of BCMA, a Type III transmembrane protein (C). Mean Fluorescence Intensities (MFIs) were measured in live cells 48 h after addition of mycolactone. Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments, relative to vehicle-treated controls (Ctrl).


Figure 2C Synergy between mycolactone and BZ in the MM . 1S , JIM3 and KMS - 11 cell lines , when treated as in ( B ) . Data are Mean Loewe scores ± SD shown as heatmaps . N = 6 ( cumulative data of 3 independent experiments with technical duplicates ) for MM . 1S and KMS - 11 , and N = 4 ( cumulative data of 2 independent experiments with technical duplicates ) for JIM3 . Statistical significance was assessed by Student ' s t - test : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . D , E MM . 1S cell were treated as in ( B ) but using carfilzomib instead of BZ ( D ) . Synergy scores ( E ) were calculated as in ( C ) . Data are Mean Loewe scores of technical duplicates , shown as heatmaps .

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Figure 2C Synergy between mycolactone and BZ in the MM.1S, JIM3 and KMS-11 cell lines, when treated as in (B). Data are Mean Loewe scores ± SD shown as heatmaps. N = 6 (cumulative data of 3 independent experiments with technical duplicates) for MM.1S and KMS-11, and N = 4 (cumulative data of 2 independent experiments with technical duplicates) for JIM3. Statistical significance was assessed by Student's t-test: *p<0.05; **p<0.01; ***p<0.001.D,E MM.1S cell were treated as in (B) but using carfilzomib instead of BZ (D). Synergy scores (E) were calculated as in (C). Data are Mean Loewe scores of technical duplicates, shown as heatmaps.


Figure 3In MM . 1S cells treated with mycolactone and / or BZ at the indicated concentrations , or vehicle as control for 6 h : ( D ) CHOP and GADD34 mRNA levels were quantified by qPCR Data information : Shown mRNA data are Mean fold changes ( 2 - ∆ ∆ CT ) ± SD , relative to untreated controls ( cumulative data of at least 2 independent experiments with technical duplicates or triplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison test , exact p - values indicated . Thapsigargin ( Tg , 2 µM , 6 h ) , Tunicamycin ( Tu , 2 µM , 6 h ) were used as positive controls . In MM . 1S cells treated with mycolactone and / or BZ at the indicated concentrations , or vehicle as control for 6 h : ( E ) ATF6 and GAPDH protein levels in cell lysates were assessed by Western blot with molecular weight markers ( kDa ) indicated on the right ; Bands corresponding to glycosylated ( G - ATF6 ) , non - glycosylated ( NG - ATF6 ) and cleaved ATF6 are indicated by arrows ; Data information : DTT ( 4 mM , 2 h ) were used as positive controls . shown data are representative of 2 independent experiments with similar results . In MM . 1S cells treated with mycolactone and / or BZ at the indicated concentrations , or vehicle as control for 6 h : ( F ) Total RNA was isolated and used as a template for RT - PCR of XBP - 1 ( upper panel ) which was then digested with Pst1 and separated on a 2 % agarose gel ( lower panel ) with molecular weight markers ( bp ) indicated on the left ; spliced XBP1 ( sXBP1 ) mRNA levels were also quantified by qPCR and normalized to total ( spliced + unspliced ) XBP1 mRNA levels , ( G ) BiP mRNA level were quantified by qPCR . Data information : Shown mRNA data are Mean fold changes ( 2 - ∆ ∆ CT ) ± SD , relative to untreated controls ( cumulative data of at least 2 independent experiments with technical duplicates or triplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison test , exact p - values indicated . Thapsigargin ( Tg , 2 µM , 6 h ) , Tunicamycin ( Tu , 2 µM , 6 h ) were used as positive controls . shown data are representative of 2 independent experiments with similar results .

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Figure 3In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (D) CHOP and GADD34 mRNA levels were quantified by qPCR Data information: Shown mRNA data are Mean fold changes (2-∆∆CT) ± SD, relative to untreated controls (cumulative data of at least 2 independent experiments with technical duplicates or triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6 h), Tunicamycin (Tu, 2 µM, 6 h) were used as positive controls.In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (E) ATF6 and GAPDH protein levels in cell lysates were assessed by Western blot with molecular weight markers (kDa) indicated on the right; Bands corresponding to glycosylated (G-ATF6), non-glycosylated (NG-ATF6) and cleaved ATF6 are indicated by arrows; Data information: DTT (4 mM, 2 h) were used as positive controls. shown data are representative of 2 independent experiments with similar results.In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (F) Total RNA was isolated and used as a template for RT-PCR of XBP-1 (upper panel) which was then digested with Pst1 and separated on a 2% agarose gel (lower panel) with molecular weight markers (bp) indicated on the left; spliced XBP1 (sXBP1) mRNA levels were also quantified by qPCR and normalized to total (spliced + unspliced) XBP1 mRNA levels, (G) BiP mRNA level were quantified by qPCR. Data information: Shown mRNA data are Mean fold changes (2-∆∆CT) ± SD, relative to untreated controls (cumulative data of at least 2 independent experiments with technical duplicates or triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6 h), Tunicamycin (Tu, 2 µM, 6 h) were used as positive controls. shown data are representative of 2 independent experiments with similar results.


Figure 4C MM . 1S BzR cells were treated for 24 h with mycolactone and / or BZ at the indicated concentrations , or vehicle as control . Data are Mean % ± SD of live , apoptotic and dead cells relative to total cells . N = 6 ( cumulative data of 3 independent experiments with technical duplicates ) . E ATF4 , CHOP , sXBP1 and BiP mRNA levels were quantified by qPCR in MM . 1s BzR cells treated as in ( C ) . sXBP1 mRNA levels were normalized to total ( spliced + unspliced ) XBP1 mRNA level . Data are Mean RNA fold changes ( 2 - ∆ ∆ CT ) ± SD , relative to untreated controls . N = 5 ( cumulative data of 2 independent experiments with technical duplicates and triplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison test , exact p - values indicated . Thapsigargin ( Tg , 2 µM , 6h ) was used as a positive control .

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Figure 4C MM.1S BzR cells were treated for 24 h with mycolactone and/or BZ at the indicated concentrations, or vehicle as control. Data are Mean % ± SD of live, apoptotic and dead cells relative to total cells. N = 6 (cumulative data of 3 independent experiments with technical duplicates).E ATF4, CHOP, sXBP1 and BiP mRNA levels were quantified by qPCR in MM.1s BzR cells treated as in (C). sXBP1 mRNA levels were normalized to total (spliced + unspliced) XBP1 mRNA level. Data are Mean RNA fold changes (2-∆∆CT) ± SD, relative to untreated controls. N = 5 (cumulative data of 2 independent experiments with technical duplicates and triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.


Figure 5C v - abl pro - B cells were treated with mycolactone and / or BZ at the indicated concentrations for 6 h . CHOP mRNA levels were quantified by qPCR . Data are Mean RNA fold changes ( 2 - ∆ ∆ CT ) ± SD relative to untreated controls . N = 6 ( cumulative data of 3 independent experiments with technical duplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison test , exact p - values indicated . Data information : Thapsigargin ( Tg , 2 µM , 6h ) was used as a positive control . D v - abl pro - B cells were treated for 6 h with mycolactone and / or BZ at the indicated concentrations . ATF4 protein levels were assessed in cell lysates by Western blot ( top panel ) and quantified relative to GADPH levels ( lower panel ) . Data information : Data shown are representative of experiments performed with two independent v - abl cell clones , with similar results . Thapsigargin ( Tg , 2 µM , 6h ) was used as a positive control .

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Figure 5C v-abl pro-B cells were treated with mycolactone and/or BZ at the indicated concentrations for 6 h. CHOP mRNA levels were quantified by qPCR. Data are Mean RNA fold changes (2-∆∆CT) ± SD relative to untreated controls. N = 6 (cumulative data of 3 independent experiments with technical duplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Data information: Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.D v-abl pro-B cells were treated for 6 h with mycolactone and/or BZ at the indicated concentrations. ATF4 protein levels were assessed in cell lysates by Western blot (top panel) and quantified relative to GADPH levels (lower panel). Data information: Data shown are representative of experiments performed with two independent v-abl cell clones, with similar results. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.


Figure 6Mononuclear cells from bone marrow aspirates of newly diagnosed ( # 2 , # 5 , # 9 and # 13 ) or relapsed ( # 7 and # 12 ) MM patients were treated with mycolactone and / or BZ at the indicated concentrations , or vehicle as control , for 18 h . Then , MM cells were identified by staining with anti - CD38 and anti - CD138 antibodies A Following treatment , induction of apoptosis / necrosis was measured by exposure of annexin V and PI incorporation . Data are Mean % of live cells from technical duplicates , relative to controls . Mononuclear cells from bone marrow aspirates of newly diagnosed ( # 2 , # 5 , # 9 and # 13 ) or relapsed ( # 7 and # 12 ) MM patients were treated with mycolactone and / or BZ at the indicated concentrations , or vehicle as control , for 18 h . B Synergy between mycolactone and BZ in treated tumors . Data are Mean Loewe scores from technical duplicates , shown as heatmaps .

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Figure 6Mononuclear cells from bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients were treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control, for 18 h. Then, MM cells were identified by staining with anti-CD38 and anti-CD138 antibodies A Following treatment, induction of apoptosis/necrosis was measured by exposure of annexin V and PI incorporation. Data are Mean % of live cells from technical duplicates, relative to controls.Mononuclear cells from bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients were treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control, for 18 h. B Synergy between mycolactone and BZ in treated tumors. Data are Mean Loewe scores from technical duplicates, shown as heatmaps.


Figure 7A C57BL / 6 mice ( N = 2 / group ) were injected intraperitoneally with BZ ( 0 . 5 mg . Kg - 1 ) , BZ ( 0 . 5 mg . Kg - 1 ) + mycolactone ( 0 . 3 mg . Kg - 1 ) , DMSO as vehicle controls every 3 . 5 days , or left untreated . Blood samples were analyzed 5 , 15 , 20 and 25 days after the first injection and percentages of CD3 + and CD19 + cells determined by flow cytometry . Results are expressed as % of positive cells among total mononuclear cells , each dot representing the Mean % from 2 mice . B NSG mice ( N = 9 / group ) were injected subcutaneously with 3 . 106 MM . 1S cells at day 0 . Seven days later , mice were injected with DMSO , BZ ( 0 . 5 mg . Kg - 1 ) and / or mycolactone ( 0 . 3 mg . Kg - 1 ) every 3 . 5 days intraperitoneally and tumor growth was followed by daily measurement of the tumor diameter . Data are Mean tumor diameters ± SD and represent cumulative data from 3 independent experiments with 3 mice / group . Difference between groups were analyzed with Tukey ' s multiple comparison test using Two - way Anova , with mixed effect model for each time points : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( exact p - values indicated ) . Only significant differences are shown .

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Figure 7A C57BL/6 mice (N=2/group) were injected intraperitoneally with BZ (0.5 mg.Kg-1), BZ (0.5 mg.Kg-1) + mycolactone (0.3 mg.Kg-1), DMSO as vehicle controls every 3.5 days, or left untreated. Blood samples were analyzed 5, 15, 20 and 25 days after the first injection and percentages of CD3+ and CD19+ cells determined by flow cytometry. Results are expressed as % of positive cells among total mononuclear cells, each dot representing the Mean % from 2 mice.B NSG mice (N=9/group) were injected subcutaneously with 3.106 MM.1S cells at day 0. Seven days later, mice were injected with DMSO, BZ (0.5 mg.Kg-1) and/or mycolactone (0.3 mg.Kg-1) every 3.5 days intraperitoneally and tumor growth was followed by daily measurement of the tumor diameter. Data are Mean tumor diameters ± SD and represent cumulative data from 3 independent experiments with 3 mice/group. Difference between groups were analyzed with Tukey's multiple comparison test using Two-way Anova, with mixed effect model for each time points: *p<0.05; **p<0.01; ***p<0.001 (exact p-values indicated). Only significant differences are shown.


Figure 1a , Seckel cells contain normal centrosome numbers and mitotic spindle poles . Interphase centrosomes of Seckel cells have no CPAP immunoreactivity but recruit similar levels of centrosomal proteins ( ii and iii ) . b , Seckel cells display overly long cilia . Arl13b labels cilium . Scale bar 1 μm . Quantification of cilia length , * * * p < 0 . 001 ; t - test ; Scatter plot showing mean with ± SD ( iii ) . Cells displaying normal ( 0 - 5 μm ) , long ( 5 - 10 μm ) and extra long ( > 10 μm ) cilia . * * * p < 0 . 001 ; Ordinary Two - way ANOVA ( iv ) . Average cilia length of WT and Seckel cells ( v ) . n = 3 independent experiments , 228 cells for WT and 297 cells for Seckel . Error bars are s . e . m . * * * p < 0 . 001 , t - test . c , Electron microscopy ( EM ) of Seckel and WT fibroblasts . In contrast to WT , Seckel basal body has disorganized microtubule of doublet and triplets ( red arrows and red circles in schematics ) . d , Longitudinal serial - sectioning EM of WT ( top ) Seckel fibroblasts ( bottom ) . In contrast to WT basal bodies that have structurally normal distal and sub - distal appendages ( yellow arrows and red triangles of DA and SDA in schematics ) , Seckel basal body displays either lack or misplaced appendages . n > 15 basal bodies . e , Seckel cells show reduced EdU incorporation after 72 hrs . Error bars are s . e . m . * * p < 0 . 01 , ttest ; n = 4 independent experiments , 727 cells for WT and 844 cells for Seckel .

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Figure 1a, Seckel cells contain normal centrosome numbers and mitotic spindle poles. Interphase centrosomes of Seckel cells have no CPAP immunoreactivity but recruit similar levels of centrosomal proteins (ii and iii).b, Seckel cells display overly long cilia. Arl13b labels cilium. Scale bar 1 μm. Quantification of cilia length, *** p<0.001; t-test; Scatter plot showing mean with ±SD (iii). Cells displaying normal (0-5 μm), long (5-10 μm) and extra long (>10 μm) cilia. *** p<0.001; Ordinary Two-way ANOVA (iv). Average cilia length of WT and Seckel cells (v). n = 3 independent experiments, 228 cells for WT and 297 cells for Seckel. Error bars are s. e. m. *** p<0.001, t-test.c, Electron microscopy (EM) of Seckel and WT fibroblasts. In contrast to WT, Seckel basal body has disorganized microtubule of doublet and triplets (red arrows and red circles in schematics).d, Longitudinal serial-sectioning EM of WT (top) Seckel fibroblasts (bottom). In contrast to WT basal bodies that have structurally normal distal and sub-distal appendages (yellow arrows and red triangles of DA and SDA in schematics), Seckel basal body displays either lack or misplaced appendages. n> 15 basal bodies.e, Seckel cells show reduced EdU incorporation after 72 hrs. Error bars are s. e. m. ** p<0.01, ttest; n = 4 independent experiments, 727 cells for WT and 844 cells for Seckel.


Figure 2a . Experimental plan . Both Seckel and WT cells were synchronized at G0 by serum starvation for 96 hrs . This treatment causes Seckel and WT cells to have equivalent cilia length allowing us to estimate the rate of cilia disassembly ( see below and Appendix Fig . S2a ) . b , Dynamic CPAP localization during cilium disassembly and cell cycle re - entry . Cep152 levels remain unchanged . Arl13b ( green ) or acetylated α - tubulin ( magenta ) marks the cilium . n > 100 cells . Bar diagrams at right shows semi quantitative intensity measurements . Error bars are ± SD . c , Seckel cells show neither CPAP dynamics nor cilium disassembly upon serum stimulation . Cep152 levels remain constant . n > 100 cells . Bar diagrams at right shows semi quantitative intensity measurements . Error bars are ± SD . d , Cilium disassembly dynamics in WT and Seckel cells . Relative cilium disassembly rate shows retarded cilium disassembly in Seckel cells ( Error bars are s . e . m . ) ( ii ) . Quantification and distribution of cilia length in WT and Seckel fibroblasts . Scatter plot shows mean with ± SD ( i ) . e , Seckel cells with retarded cilium disassembly show delayed G1 - S transition assessed by nuclearCyclin A immunoreactivity . n = 3 independent experiments , n > 200 cells for WT and Seckel . Error bars are s . e . m . f , Seckel cells show delayed mitotic entry . Note that an unresorbed Arl13b - positive ciliary remnant ( ciliary membrane , green ) is present with one of the centrosomes ( Paridaen et al , 2013 ) . Bar diagram shows mitotic index of WT and Seckel cells 24 hrs after serum stimulation . n = 3 independent experiments , n > 300 cells for WT and Seckel . Error bars are s . e . m . Scale bar 1 μm .

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Figure 2a. Experimental plan. Both Seckel and WT cells were synchronized at G0 by serum starvation for 96 hrs. This treatment causes Seckel and WT cells to have equivalent cilia length allowing us to estimate the rate of cilia disassembly (see below and Appendix Fig. S2a).b, Dynamic CPAP localization during cilium disassembly and cell cycle re-entry. Cep152 levels remain unchanged. Arl13b (green) or acetylated α-tubulin (magenta) marks the cilium. n> 100 cells. Bar diagrams at right shows semi quantitative intensity measurements. Error bars are ± SD.c, Seckel cells show neither CPAP dynamics nor cilium disassembly upon serum stimulation. Cep152 levels remain constant. n> 100 cells. Bar diagrams at right shows semi quantitative intensity measurements. Error bars are ± SD.d, Cilium disassembly dynamics in WT and Seckel cells. Relative cilium disassembly rate shows retarded cilium disassembly in Seckel cells (Error bars are s. e. m.) (ii). Quantification and distribution of cilia length in WT and Seckel fibroblasts. Scatter plot shows mean with ± SD (i).e, Seckel cells with retarded cilium disassembly show delayed G1-S transition assessed by nuclearCyclin A immunoreactivity. n = 3 independent experiments, n> 200 cells for WT and Seckel. Error bars are s.e.m.f, Seckel cells show delayed mitotic entry. Note that an unresorbed Arl13b-positive ciliary remnant (ciliary membrane, green) is present with one of the centrosomes(Paridaen et al, 2013). Bar diagram shows mitotic index of WT and Seckel cells 24 hrs after serum stimulation. n = 3 independent experiments, n> 300 cells for WT and Seckel. Error bars are s.e.m. Scale bar 1 μm.


Figure 3a , CPAP co - immunopurifies with CDC components from HeLa extracts . Centrosomal proteins Cap350 and ODF2 do not co - purify with CPAP . b , Levels of CDC components ( CPAP , Aurora A , OFD1 , and Nde - 1 ) are differentially regulated in response to serum starvation and stimulation in WT and Seckel fibroblasts . Specifically , they are elevated 1h after serum stimulation , thus enabling cilium disassembly . Elevated CDC levels in WT cells correlate with an elevated level of CPAP as shown in Fig . 2 as well as with the expression of cyclin A , a G1 - S marker . GAPDH was used as a loading control indicating an equivalent amount of WT and Seckel fibroblast total proteins were loaded . c . WT CPAP ( WT - cells IP ) but not Seckel variant CPAP ( Seckel cells IP ) co - immunopurifies with CDC components from asynchronously grown WT and Seckelfibroblastscell extracts . The IP samples were adjusted so that equivalent amounts of immunoprecipitatedCPAP were used in samples . Seckel ( 5x ) and WT ( 1x ) fibroblast cell extracts were used as input controls to show the efficacy of the various antibodies . Note that CPAP abundance in Seckel cells is ~ 5 times lower than that of WT cells . CentrosomalproteinsCap350 and ODF2 do not co - purify with CPAP indicating the specificity of CPAP interaction with CDC components . d , GST pull down ( i ) and immunopurification assays ( ii ) of CPAP ' s CC5 domain demonstrate its sufficiency in mediating CPAP - CDC protein interactions . The CC5 fragment is expressed as a GFP fusion protein in HeLa cells and immunopurified using anti - GFP antibodies . HeLa extracts were used as input controls . CAP350 does not co - purify with CPAP . Both Cap350 and ODF2 are not pulled down by CPAP ' s CC5 domain .

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Figure 3a, CPAP co-immunopurifies with CDC components from HeLa extracts. Centrosomal proteins Cap350 and ODF2 do not co-purify with CPAP.b, Levels of CDC components (CPAP, Aurora A, OFD1, and Nde-1) are differentially regulated in response to serum starvation and stimulation in WT and Seckel fibroblasts. Specifically, they are elevated 1h after serum stimulation, thus enabling cilium disassembly. Elevated CDC levels in WT cells correlate with an elevated level of CPAP as shown in Fig. 2 as well as with the expression of cyclin A, a G1-S marker. GAPDH was used as a loading control indicating an equivalent amount of WT and Seckel fibroblast total proteins were loaded.c. WT CPAP (WT-cells IP) but not Seckel variant CPAP (Seckel cells IP) co-immunopurifies with CDC components from asynchronously grown WT and Seckelfibroblastscell extracts. The IP samples were adjusted so that equivalent amounts of immunoprecipitatedCPAP were used in samples. Seckel (5x) and WT (1x) fibroblast cell extracts were used as input controls to show the efficacy of the various antibodies. Note that CPAP abundance in Seckel cells is ~5 times lower than that of WT cells. CentrosomalproteinsCap350 and ODF2 do not co-purify with CPAP indicating the specificity of CPAP interaction with CDC components.d, GST pull down (i) and immunopurification assays (ii) of CPAP's CC5 domain demonstrate its sufficiency in mediating CPAP-CDC protein interactions. The CC5 fragment is expressed as a GFP fusion protein in HeLa cells and immunopurified using anti-GFP antibodies. HeLa extracts were used as input controls. CAP350 does not co-purify with CPAP. Both Cap350 and ODF2 are not pulled down by CPAP's CC5 domain.


Figure 4a , Seckel NPCs fail to efficiently recruit CDC components . Fraction of non - ciliated and ciliated WT and Seckel NPCs . Ciliated cells are further quantified and grouped based on whether they are positive or negative for Aurora A and CPAP at their basal bodies . Scale bar 0 . 5 μm ( i - ii ) . Proportion of ciliated cells is higher in Seckel NPCs ( * * p < 0 . 01 ) . Compared to WT , a higher proportion of Seckel NPCs lack Aurora A and CPAP in their basal bodies ( * p < 0 . 05 for Auroa A and * * * p < 0 . 001 for CPAP ) . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons test . n = 3 independent experiments . 127 cells for WT and 97 cells for Seckel . Error bars are s . e . m . ( iii ) Seckel cells consistently contain Nde - 1 and Ofd1 with a significantly lesser intensity . t test ; error bars are s . e . m . ; n = 3 independent experiments . 110 cells for WT and 100 cells for Seckel . b , Proportion of ciliated cells is higher in Seckel NPCs . Error bars are s . e . m . * * p < 0 . 01 , t - test . n = 3 independent experiments . 127 cells for WT and 97 cells for Seckel . c , Seckel NPCs exhibit longer cilia . Error bars are ± s . e . m . * * * p < 0 . 001 , t - test . NPCs were derived from two independent iPS clones . n = 3 independent experiments . 146 cells for WT , 135 cells for Seckel ( clone 1 ) and 202 ( clone 2 ) . d , SeckelNPCs show reduced proliferation and delayed G1 - S transition . Error bars are ± s . e . m . * * * p < 0 . 001 ( EdU ) , * p < 0 . 05 ( Cyclin - A ) , t - test . For cyclin A , n = 3 independent experiments . 270 cells for WT and 232 cells for Seckel cells . For EdU , 700 cells for WT and 1630 cells for Seckel . e , Olomoucine triggers differentiation of WT NPCs into TUJ1 - positive neurons under non - differentiating conditions . Error bars are s . e . m . * * * p < 0 . 001 , Ordinary Two - way ANOVA followed by Tukey ' s multiple comparisons test . n = 3 independent experiments . 298 cells for untreated , 151 cells for DMSO and 146 cells for olomoucine , 25 μM . f , Seckel NPCs differentiated into TUJ1 - positive neurons under non - differentiating conditions . Error bars show ± s . e . m . * * * p < 0 . 001 , t - test . n = 3 independent experiments . 633 cells for WT and 275 cells for Seckel . Scale bar 5 μm . g , Expression of CPAP - GFP or CPAP - CC5 - GFP in Seckel NPCs slightly increases nestin - positive cells ( i ) , decreases neuronal differentiation ( for CC5 * p < 0 . 05 ) ( ii ) , decreases ciliary length ( for CPAP * * * p < 0 . 001 and for CC5 * * p < 0 . 01 ) ( iii ) , elevates mitotic index ( for CC5 * p < 0 . 05 ) ( iv ) and decreases the number of ciliated cells ( for CPAP * p < 0 . 05 and for CC5 * * p < 0 . 01 ) ( v ) compared to control ( mock electroporation ) . Error bars show s . e . m . One - way ANOVA followed by Tukey ' s multiple comparisons test . n = 3 independent experiments with > 350 ( nestinstaining , differentiation and mitotic index ) , and > 70 cells ( cilium length ) cells .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Seckel NPCs fail to efficiently recruit CDC components. Fraction of non-ciliated and ciliated WT and Seckel NPCs. Ciliated cells are further quantified and grouped based on whether they are positive or negative for Aurora A and CPAP at their basal bodies. Scale bar 0.5 μm (i-ii). Proportion of ciliated cells is higher in Seckel NPCs (**p<0.01). Compared to WT, a higher proportion of Seckel NPCs lack Aurora A and CPAP in their basal bodies (*p<0.05 for Auroa A and ***p<0.001 for CPAP). Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 127 cells for WT and 97 cells for Seckel. Error bars are s. e. m. (iii) Seckel cells consistently contain Nde-1 and Ofd1 with a significantly lesser intensity. t test; error bars are s.e.m.; n = 3 independent experiments. 110 cells for WT and 100 cells for Seckel.b, Proportion of ciliated cells is higher in Seckel NPCs. Error bars are s. e. m. ** p<0.01, t-test. n = 3 independent experiments. 127 cells for WT and 97 cells for Seckel.c, Seckel NPCs exhibit longer cilia. Error bars are ± s. e. m. *** p<0.001, t-test. NPCs were derived from two independent iPS clones. n = 3 independent experiments. 146 cells for WT, 135 cells for Seckel (clone 1) and 202 (clone 2).d, SeckelNPCs show reduced proliferation and delayed G1-S transition. Error bars are ± s. e. m. *** p<0.001 (EdU), * p<0.05 (Cyclin-A), t-test. For cyclin A, n = 3 independent experiments. 270 cells for WT and 232 cells for Seckel cells. For EdU, 700 cells for WT and 1630 cells for Seckel.e, Olomoucine triggers differentiation of WT NPCs into TUJ1-positive neurons under non-differentiating conditions. Error bars are s. e. m. *** p<0.001, Ordinary Two-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments. 298 cells for untreated, 151 cells for DMSO and 146 cells for olomoucine, 25 μM.f, Seckel NPCs differentiated into TUJ1-positive neurons under non-differentiating conditions. Error bars show ±s. e. m. *** p<0.001, t-test. n = 3 independent experiments. 633 cells for WT and 275 cells for Seckel. Scale bar 5 μm.g, Expression of CPAP-GFP or CPAP-CC5-GFP in Seckel NPCs slightly increases nestin-positive cells (i), decreases neuronal differentiation (for CC5 *p<0.05) (ii), decreases ciliary length (for CPAP ***p<0.001 and for CC5 **p<0.01) (iii), elevates mitotic index (for CC5 *p<0.05) (iv) and decreases the number of ciliated cells (for CPAP *p<0.05 and for CC5 **p<0.01) (v) compared to control (mock electroporation). Error bars show s. e. m. One-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments with >350 (nestinstaining, differentiation and mitotic index), and >70 cells (cilium length) cells.


Figure 5a - b , siRNA - mediated down regulation of CDC components in WT NPCs causes longer cilia ( Ofd1 * * * p < 0 . 001 , Nde1 * * p < 0 . 01 ) , retarded cilia disassembly as shown by increased numbers of ciliated cells ( Ofd1 * * p < 0 . 01 , Nde1 * p < 0 . 05 ) and differentiation of NPCs into TUJ1 positive cells ( Ofd1 * * p < 0 . 001 , Nde1 * p < 0 . 01 ) . In controls , a non - targeting siRNA ( scrambled ) is used . Western blots at right validate siRNAs - mediated Nde1 and OFD1 depletion . Error bars are s . e . m . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons test . n = 3 independent experiments . 275 cells for scrambled , 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA . a - b , siRNA - mediated down regulation of CDC components in WT NPCs causes longer cilia ( Ofd1 * * * p < 0 . 001 , Nde1 * * p < 0 . 01 ) , retarded cilia disassembly as shown by increased numbers of ciliated cells ( Ofd1 * * p < 0 . 01 , Nde1 * p < 0 . 05 ) and differentiation of NPCs into TUJ1 positive cells ( Ofd1 * * p < 0 . 001 , Nde1 * p < 0 . 01 ) . In controls , a non - targeting siRNA ( scrambled ) is used . Western blots at right validate siRNAs - mediated Nde1 and OFD1 depletion . Error bars are s . e . m . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons test . n = 3 independent experiments . 275 cells for scrambled , 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA . c , VX680 ( 0 . 5 μM , a concentration that will specifically allow neuronal differentiation ) was used to inhibit Aurora A activity . Bar diagrams below show quantifications . Compared to DMSO - treated control NPCs , a higher proportion of VX680 treated NPCs are ciliated ( * * * p < 0 . 001 ) , exhibiting longer cilia ( * * * p < 0 . 001 ) and show Tuj1 positive neurons ( * * * p < 0 . 001 ) . Error bars are s . e . m . For ciliated cells and cilium length : n = 3 independent experiments . 164 cells for DMSO - treated and 77 cells for VX680 - treated . For NPC differentiation : n = 4 independent experiments . 286 cells for DMSO - treated and 118 cells for VX680 - treated . Scale bar 1 μm . d , Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours . Inhibition of Ofd1 ( * p < 0 . 05 ) , Nde1 ( * * p < 0 . 01 ) and Aurora A ( * * * p < 0 . 001 ) caused significantly reduced incorporation of EdU . e - g , Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP . Both low - and high - exposures are given ( e ) . Increased cilia length and NPC differentiation upon expression of RNAi - resistant CPAP E1235V compared to WT CPAP ( f - g ) . Error bars are s . e . m . * * * p < 0 . 001 ( cilia length ) , * p < 0 . 05 ( differentiation ) , t - test . For cilia length : n = 4 independent experiments . 25 cells for CPAP , 47 cells for CPAP E1235V . For differentiation : n = 4 independent experiments . 145 for CPAP and 81 for CPAP E1235V . Scale bar 1 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5a-b, siRNA-mediated down regulation of CDC components in WT NPCs causes longer cilia (Ofd1 ***p<0.001, Nde1 **p<0.01), retarded cilia disassembly as shown by increased numbers of ciliated cells (Ofd1 **p<0.01, Nde1 *p<0.05) and differentiation of NPCs into TUJ1 positive cells (Ofd1 **p<0.001, Nde1 *p<0.01). In controls, a non-targeting siRNA (scrambled) is used. Western blots at right validate siRNAs-mediated Nde1 and OFD1 depletion. Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 275 cells for scrambled, 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA.a-b, siRNA-mediated down regulation of CDC components in WT NPCs causes longer cilia (Ofd1 ***p<0.001, Nde1 **p<0.01), retarded cilia disassembly as shown by increased numbers of ciliated cells (Ofd1 **p<0.01, Nde1 *p<0.05) and differentiation of NPCs into TUJ1 positive cells (Ofd1 **p<0.001, Nde1 *p<0.01). In controls, a non-targeting siRNA (scrambled) is used. Western blots at right validate siRNAs-mediated Nde1 and OFD1 depletion. Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 275 cells for scrambled, 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA.c, VX680 (0.5 μM, a concentration that will specifically allow neuronal differentiation) was used to inhibit Aurora A activity. Bar diagrams below show quantifications. Compared to DMSO-treated control NPCs, a higher proportion of VX680 treated NPCs are ciliated (***p<0.001), exhibiting longer cilia (***p<0.001) and show Tuj1 positive neurons (***p<0.001). Error bars are s. e. m. For ciliated cells and cilium length: n = 3 independent experiments. 164 cells for DMSO-treated and 77 cells for VX680-treated. For NPC differentiation: n = 4 independent experiments. 286 cells for DMSO-treated and 118 cells for VX680-treated. Scale bar 1 μm.d, Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours. Inhibition of Ofd1 (*p<0.05), Nde1 (**p<0.01) and Aurora A (***p<0.001) caused significantly reduced incorporation of EdU.e-g, Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP. Both low -and high -exposures are given (e). Increased cilia length and NPC differentiation upon expression of RNAi-resistant CPAP E1235V compared to WT CPAP (f-g). Error bars are s. e. m. *** p<0.001 (cilia length), * p<0.05 (differentiation), t-test. For cilia length: n = 4 independent experiments. 25 cells for CPAP, 47 cells for CPAP E1235V. For differentiation: n = 4 independent experiments. 145 for CPAP and 81 for CPAP E1235V. Scale bar 1 μm.


Figure 6a , iPS - derived 14 - day old organoid displaying complex morphological structures of brain - like tissues , parenthesis . Scale bar 500 μm . The ventricular zone ( VZ ) with radial glial cells stained with nestin ( magenta ) and neurons stained with TUJ1 is shown at higher magnification . Scale bar 20 μm . b , Reduced Nestin / Pax - 6 - positive progenitors and increased TUJ1 - positive neurons in Seckel organoids . In ventricular zones of WT organoids , there are significantly more Pax6 positive cells than TUJ1 positive cells , which is not the case in Seckel organoids ( * p < 0 . 05 ) . TUJ1 / Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs . Pax6 cells in Seckel organoid ventricular zones ( * p < 0 . 05 ) . Error bars are s . e . m . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons test . n = 10 WT organoids ( 2520 cells ) . Seckeln = 10 organoids ( 2024 cells ) . Scale bar 20 μm . c , ( i ) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen ( * p < 0 . 05 ) . Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids . ( * p < 0 . 05 ) . Error bars are s . e . m . t - test . n = 6 for WT organoid and 6 for Seckel organoid . Scale bar 20 μm . ( ii ) Seckel organoid cells display longer cilia . Error bars are s . e . m . * * * p < 0 . 001 , t - test . n = 3 for WT organoids and 4 for Seckel organoids . Scale bar 1 μm . d , Seckel organoids electroporated with constructs expressing GFP alone ( i ) , GFP with CC5 - GFP and ( ii ) GFP with CPAP - GFP ( iii ) . Middle panel shows staining with anti - GFP antibodies ( red ) to show the transfected area . Note the presence of increased Pax6 - positive ( magenta ) NPCs in CC5 and CPAP electroporated tissues compared to GFP control . Likewise , there is a larger neural epithelium with multiple RGs ( arrowheads ) . Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone ( iv ) lower TUJ1 / Pax6 ratio ( * * p < 0 . 01 and * * * p < 0 . 001 respectively ) ( v ) smaller lumen diameter ( * * p < 0 . 01 ) and ( vi ) shorter cilia length ( * * p < 0 . 01 and * * * p < 0 . 001 respectively ) . Error bars are s . e . m . One - way ANOVA followed by Dunnett ' s multiple comparisons test . n = 3 organoids with 225 cells for GFP , 537 cells for CPAP and 569 for CC5 . At least 10 ventricles from 3 independent organoids were used to measure the CPAP diameter . t test . Error bars are s . e . m . Scale bar 20 μm . e , Rescue effect of CC5 and CPAPelectroporation on cleavage planes of phospho - vimentin - positive RGs in Seckel organoids . Compared to GFP control , electroporation of CC5 or GFP increases the proportion of horizontally orientated SeckelRGs ( for CC5 * * * p < 0 . 001 ) and decreases the portion of obliquely ( for CC5 * p < 0 . 05 ) or vertically oriented SeckelGFP ( for CPAP and CC5 * * p < 0 . 01 ) . Error bars are s . e . m . Two - way ANOVA followed by Tukey ' s multiple comparisons test . n = 4 organoids . 100 cells for GFP control , 112 cells for CPAP and 99 cells for CC5 .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, iPS-derived 14-day old organoid displaying complex morphological structures of brain-like tissues, parenthesis. Scale bar 500 μm. The ventricular zone (VZ) with radial glial cells stained with nestin (magenta) and neurons stained with TUJ1 is shown at higher magnification. Scale bar 20 μm.b, Reduced Nestin/Pax-6-positive progenitors and increased TUJ1-positive neurons in Seckel organoids. In ventricular zones of WT organoids, there are significantly more Pax6 positive cells than TUJ1 positive cells, which is not the case in Seckel organoids (*p<0.05). TUJ1/Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs. Pax6 cells in Seckel organoid ventricular zones (* p<0.05). Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 10 WT organoids (2520 cells). Seckeln = 10 organoids (2024 cells). Scale bar 20 μm.c, (i) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen (* p<0.05). Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids. (*p<0.05). Error bars are s. e. m. t-test. n=6 for WT organoid and 6 for Seckel organoid. Scale bar 20 μm. (ii) Seckel organoid cells display longer cilia. Error bars are s. e. m. *** p<0.001, t-test. n=3 for WT organoids and 4 for Seckel organoids. Scale bar 1 μm.d, Seckel organoids electroporated with constructs expressing GFP alone (i), GFP with CC5-GFP and (ii) GFP with CPAP-GFP (iii). Middle panel shows staining with anti-GFP antibodies (red) to show the transfected area. Note the presence of increased Pax6-positive (magenta) NPCs in CC5 and CPAP electroporated tissues compared to GFP control. Likewise, there is a larger neural epithelium with multiple RGs (arrowheads). Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone (iv) lower TUJ1/Pax6 ratio (**p<0.01 and ***p<0.001 respectively) (v) smaller lumen diameter (**p<0.01) and (vi) shorter cilia length (**p<0.01 and ***p<0.001 respectively). Error bars are s. e. m. One-way ANOVA followed by Dunnett's multiple comparisons test. n = 3 organoids with 225 cells for GFP, 537 cells for CPAP and 569 for CC5. At least 10 ventricles from 3 independent organoids were used to measure the CPAP diameter. t test. Error bars are s.e.m. Scale bar 20 μm.e, Rescue effect of CC5 and CPAPelectroporation on cleavage planes of phospho-vimentin-positive RGs in Seckel organoids. Compared to GFP control, electroporation of CC5 or GFP increases the proportion of horizontally orientated SeckelRGs (for CC5 ***p<0.001) and decreases the portion of obliquely (for CC5 *p<0.05) or vertically oriented SeckelGFP (for CPAP and CC5 **p<0.01). Error bars are s.e.m. Two-way ANOVA followed by Tukey's multiple comparisons test. n = 4 organoids. 100 cells for GFP control, 112 cells for CPAP and 99 cells for CC5.


Figure 1B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths . Yeast cells expressing clogger proteins ( cytochrome b2 - DHFR and cytochrome b2 ∆ 19 - DHFR ) or cytosolic DHFR for comparison under control of the galactose - inducible promoter were grown to mid - log phase on lactate medium . Ten - fold serial dilutions were dropped on lactate ( no induction ) or lactate with 0 . 5 % galactose medium ( induction ) . MTS , matrix - targeting signal . E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates . Cells were grown in lactate medium and clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before samples were harvested and lysed . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

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Figure 1B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths. Yeast cells expressing clogger proteins (cytochrome b2-DHFR and cytochrome b2∆19-DHFR) or cytosolic DHFR for comparison under control of the galactose-inducible promoter were grown to mid-log phase on lactate medium. Ten-fold serial dilutions were dropped on lactate (no induction) or lactate with 0.5% galactose medium (induction). MTS, matrix-targeting signal.E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates. Cells were grown in lactate medium and clogger expression was induced for 4.5 h with 0.5% galactose before samples were harvested and lysed. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


Figure 2A The clogger was expressed in wild type ( WT ) and ∆ rpn4 ( ∆ ) cells for 4 . 5 h with medium containing 0 . 5 % galactose . Medium was exchanged for a non - inducing lactate medium . Precursor ( pre ) and mature ( m ) forms of the b2 ∆ - DHFR clogger were visualized by Western blotting using a DHFR - specific antibody . G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆ rpn4 cells . The cytosolic soluble DHFR protein was expressed for control . The strains were grown in lactate medium and clogger expression was induced with 0 . 5 % galactose for 6 h .

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Figure 2A The clogger was expressed in wild type (WT) and ∆rpn4 (∆) cells for 4.5 h with medium containing 0.5% galactose. Medium was exchanged for a non-inducing lactate medium. Precursor (pre) and mature (m) forms of the b2∆-DHFR clogger were visualized by Western blotting using a DHFR-specific antibody.G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆rpn4 cells. The cytosolic soluble DHFR protein was expressed for control. The strains were grown in lactate medium and clogger expression was induced with 0.5% galactose for 6 h.


Figure 3A Cytosolic DHFR ( control ) or the clogger protein b2 - DHFR were expressed in wild type and ∆ rpn4 cells for 4 . 5 h . The distribution of constitutively expressed Hsp104 - GFP was visualized by fluorescence microscopy . Diameter of Hsp104 - GFP - bound aggregates were quantified . . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates . Significance was assessed using two - sided , paired Student ' s t - test . P values are indicated as asterisks * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . See also Fig . EV4A for quantification of the number of aggregates per cell . B Hsp104 - GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures . SN , supernatant representing the non - bound fraction . The signals for Ssa1 and Sod1 are shown for control .

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Figure 3A Cytosolic DHFR (control) or the clogger protein b2-DHFR were expressed in wild type and ∆rpn4 cells for 4.5 h. The distribution of constitutively expressed Hsp104-GFP was visualized by fluorescence microscopy. Diameter of Hsp104-GFP-bound aggregates were quantified. . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates. Significance was assessed using two-sided, paired Student's t-test. P values are indicated as asterisks ** p ≤ 0.01, **** p ≤ 0.0001. See also Fig. EV4A for quantification of the number of aggregates per cell.B Hsp104-GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures. SN, supernatant representing the non-bound fraction. The signals for Ssa1 and Sod1 are shown for control.


Figure 5A The mitochondrial proteins Pdb1 , Mam33 and Aim17 were co - expressed for 4 . 5 h with clogger as fusions with the red fluorescent protein RFP ( mCherry ) . Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4 . 5 h after induction of the clogger or cytosolic DHFR for control . For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17 - expressing plasmids and grown to mid - log phase in lactate medium . Clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before microscopy was perfomed . Please note that in the absence of Hsp42 and Hsp104 , no Aim17 - RFP - containing granules are formed . C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N - terminally truncated version of Mam33 - RFP ( both under control of GAL promoter ) together with constitutively expressed Hsp104 - GFP ( TPI promoter ) . The cells were grown on lactate plus 0 . 5 % galactose - containing medium for 4 . 5 h . The distribution of the fluorescent proteins was visualized by microscopy . D Wild type cells expressing Hsp104 - GFP were grown on glucose or lactate medium to log phase . Cells were diluted and visualized by life cell imaging . Please note , that in glucose media , Hsp104 - GFP is initially dispersed in cells and only aggregates when cells become stationary . In contrast , Hsp104 - GFP - containing granules are frequent on lactate medium even at log phase . See movie EV1 for life cell imaging of glucose - grown cells .

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Figure 5A The mitochondrial proteins Pdb1, Mam33 and Aim17 were co-expressed for 4.5 h with clogger as fusions with the red fluorescent protein RFP (mCherry). Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4.5 h after induction of the clogger or cytosolic DHFR for control. For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17-expressing plasmids and grown to mid-log phase in lactate medium. Clogger expression was induced for 4.5 h with 0.5 % galactose before microscopy was perfomed. Please note that in the absence of Hsp42 and Hsp104, no Aim17-RFP-containing granules are formed. C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N-terminally truncated version of Mam33-RFP (both under control of GAL promoter) together with constitutively expressed Hsp104-GFP (TPI promoter) . The cells were grown on lactate plus 0.5% galactose-containing medium for 4.5 h. The distribution of the fluorescent proteins was visualized by microscopy.D Wild type cells expressing Hsp104-GFP were grown on glucose or lactate medium to log phase. Cells were diluted and visualized by life cell imaging. Please note, that in glucose media, Hsp104-GFP is initially dispersed in cells and only aggregates when cells become stationary. In contrast, Hsp104-GFP-containing granules are frequent on lactate medium even at log phase. See movie EV1 for life cell imaging of glucose-grown cells.


Figure 6E Clogger and Mam33 - HA were co - expressed for 4 . 5 h in wild type and ∆ hsp42 cells . Mitochondria were isolated and subjected to Western blotting to detect mature ( m ) and precursor ( pre ) forms of Mam33 - HA . The matrix protein Tim16 served as loading control . F The mitochondria used for E were treated with proteinase K ( PK ) to remove surface - exposed proteins . Mitochondrial membranes were lysed with NP - 40 and soluble ( S ) and aggregated ( P ) proteins were separated by centrifugation . T , total . G To measure the toxicity of clogger expression , clogger proteins were induced for 24 h with lactate medium containing 0 . 5 % galactose in the mutants indicated . Aliquots were removed and the number of living cells were assessed by a plating assay on glucose - containing plates . The resistance of ∆ rpn4 to clogger expression depends on Hsp104 and Hsp42 . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

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

Figure 6E Clogger and Mam33-HA were co-expressed for 4.5 h in wild type and ∆hsp42 cells. Mitochondria were isolated and subjected to Western blotting to detect mature (m) and precursor (pre) forms of Mam33-HA. The matrix protein Tim16 served as loading control.F The mitochondria used for E were treated with proteinase K (PK) to remove surface-exposed proteins. Mitochondrial membranes were lysed with NP-40 and soluble (S) and aggregated (P) proteins were separated by centrifugation. T, total.G To measure the toxicity of clogger expression, clogger proteins were induced for 24 h with lactate medium containing 0.5 % galactose in the mutants indicated. Aliquots were removed and the number of living cells were assessed by a plating assay on glucose-containing plates. The resistance of ∆rpn4 to clogger expression depends on Hsp104 and Hsp42. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


Figure 1A . 3D image of an EGFP - CaaX - expressing Ramos B cell embedded in Matrigel ( left ) and segmentation of the inner dome ( center ) using our own script based on Watershed segmentation and segmentation of the cell surface ( right ) using an nnU - Net model that we trained based on our own - generated ground truth .

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Figure 1A. 3D image of an EGFP-CaaX-expressing Ramos B cell embedded in Matrigel (left) and segmentation of the inner dome (center) using our own script based on Watershed segmentation and segmentation of the cell surface (right) using an nnU-Net model that we trained based on our own-generated ground truth.


Figure 3C . Cluster density at surface features . IgM - BCR cluster density within different topographical features quantified as cluster count per surface feature volume . F , G , H . Continuous analysis of IgM - BCR cluster density using geodesic distance maps initiated from ( F ) microvilli tips towards microvilli roots , ( G ) centerline of ridges towards cell body surface , and ( H ) nodes towards cell body surface .

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Figure 3C. Cluster density at surface features. IgM-BCR cluster density within different topographical features quantified as cluster count per surface feature volume.F, G, H. Continuous analysis of IgM-BCR cluster density using geodesic distance maps initiated from (F) microvilli tips towards microvilli roots, (G) centerline of ridges towards cell body surface, and (H) nodes towards cell body surface.


Figure 4A , B . Sum projections of the skeletonized ridge network and microvilli ( A ) and segmented IgM - BCR clusters ( B ) in an untreated and 100 µM CK - 666 - treated cell for 15 consecutive time frames amounting to 37 . 5 s . G . Continuous analysis of IgM - BCR cluster density in 15 - 30 min 100 µM CK - 666 - treated cells using geodesic distance maps initiating from distinct surface features ( centerline of ridges , nodes and microvilli tips ) ( to compare to untreated in Fig 3F ) . Data points and the dotted lines represent mean ± SD . H . Time - course images of EGFP - CaaX and IgM - BCR intensity in an elongating microvillus ( purple , MV mask ) .

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Figure 4A, B. Sum projections of the skeletonized ridge network and microvilli (A) and segmented IgM-BCR clusters (B) in an untreated and 100 µM CK-666-treated cell for 15 consecutive time frames amounting to 37.5 s.G. Continuous analysis of IgM-BCR cluster density in 15-30 min 100 µM CK-666-treated cells using geodesic distance maps initiating from distinct surface features (centerline of ridges, nodes and microvilli tips) (to compare to untreated in Fig 3F). Data points and the dotted lines represent mean ± SD. H. Time-course images of EGFP-CaaX and IgM-BCR intensity in an elongating microvillus (purple, MV mask).


Figure 5A . IgM - BCR cluster distribution upon Ag stimulation . Ramos B cells in the Matrigel were stimulated with 50 ng / ml NIP - 15 - BSA Ag for at least 15 minutes . Ag - MC on the B cell surface were segmented and overlaid with the ridge network ( including microvilli ) with a 1 . 25x and 3 . 0x local intensity cut - off for cluster detection . G , H . Staticity scores for different surface features ( R + N , MV ) ( G ) and for segmented IgM - BCR clusters assigned to different surface features ( H ) in unstimulated and Ag - stimulated cells . Violin plot solid lines represent the median and dotted lines represent the quartiles .

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Figure 5A. IgM-BCR cluster distribution upon Ag stimulation. Ramos B cells in the Matrigel were stimulated with 50 ng/ml NIP-15-BSA Ag for at least 15 minutes. Ag-MC on the B cell surface were segmented and overlaid with the ridge network (including microvilli) with a 1.25x and 3.0x local intensity cut-off for cluster detection.G, H. Staticity scores for different surface features (R+N, MV) (G) and for segmented IgM-BCR clusters assigned to different surface features (H) in unstimulated and Ag-stimulated cells. Violin plot solid lines represent the median and dotted lines represent the quartiles.


Figure 6A . Phalloidin - Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP - CaaX and IgM - BCR with or without 30 min 2 µM LatA treatment . D . Surface - masked EGFP - CaaX and IgM - BCR signal and overlay of segmented elevations ( yellow ) with the surface - masked IgM - BCR signal in cells treated with LatA and LatA + Ag ( 50 ng / ml NIP - 15 - BSA ) . E . Time frames from a LatA + Ag cell showing directed movement of Ag - MC towards the elevations ( yellow ) . Each Ag - MC shown with a different colored arrow . F . IgM - BCR intensity enrichment within all the segmented clusters in LatA and Lat + Ag cells . G . Cluster density at surface features . Quantification of the IgM - BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM - BCR clusters ( left panel , local contrast cutoff = 1x ) and for Ag - MC ( right panel , local contrast cutoff = 3x ) . HI . Surface - masked EGFP - CaaX and IgM - BCR signal for a LatA and LatA + Ag cell and the sum projections of their segmented IgM - BCR clusters ( duration : 37 . 5 s ) . IgM - BCR clusters are color - coded ( green and yellow ) according to their assigned surface region .

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Figure 6A. Phalloidin-Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP-CaaX and IgM-BCR with or without 30 min 2 µM LatA treatment.D. Surface-masked EGFP-CaaX and IgM-BCR signal and overlay of segmented elevations (yellow) with the surface-masked IgM-BCR signal in cells treated with LatA and LatA+Ag (50 ng/ml NIP-15-BSA). E. Time frames from a LatA+Ag cell showing directed movement of Ag-MC towards the elevations (yellow). Each Ag-MC shown with a different colored arrow.F. IgM-BCR intensity enrichment within all the segmented clusters in LatA and Lat+Ag cells. G. Cluster density at surface features. Quantification of the IgM-BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM-BCR clusters (left panel, local contrast cutoff = 1x) and for Ag-MC (right panel, local contrast cutoff = 3x). HI. Surface-masked EGFP-CaaX and IgM-BCR signal for a LatA and LatA+Ag cell and the sum projections of their segmented IgM-BCR clusters (duration: 37.5 s). IgM-BCR clusters are color-coded (green and yellow) according to their assigned surface region.


Figure 2D ) Cells expressing empty vector ( EV ) , or PRKAA1 - myc , PRKAB2 and PRKAG2 - FLAG ( AMPK ) alone or with myc - Ulk1 WT ( ULK ) were cultured in full medium ( F ) , EBSS ( St ) , or serum - free medium ( SFM ) alone or in the presence of 991 ( 1µM ) or MRT68921 ( 1μM ) for 90 minutes as indicated . 2 % Loading control and pull down samples were analysed by Western blot .

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Figure 2D) Cells expressing empty vector (EV), or PRKAA1-myc, PRKAB2 and PRKAG2-FLAG (AMPK) alone or with myc-Ulk1 WT (ULK) were cultured in full medium (F), EBSS (St), or serum-free medium (SFM) alone or in the presence of 991 (1µM) or MRT68921 (1μM) for 90 minutes as indicated. 2% Loading control and pull down samples were analysed by Western blot.


Figure 3A ) VPS15 - ZZ was co - expressed with Ulk1 and Ulk2 wild type ( WT ) or kinase inactive ( KI ) before analysis by Western blot . An electrophoretic mobility shift in VPS15 - ZZ was noted with WT only .

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Figure 3A) VPS15-ZZ was co-expressed with Ulk1 and Ulk2 wild type (WT) or kinase inactive (KI) before analysis by Western blot. An electrophoretic mobility shift in VPS15-ZZ was noted with WT only.


Figure 4A ) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot . LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA - derived clones . B ) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr , fixed and imaged for WIPI2 ( green ) . Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles ( red asterisks ) . Quantification of WIPI2 spots , mean + / - SEM , n = 3 . D ) Wild type ( WT ) , 6SA or 6SE VPS15 - HA , or empty vector ( EV ) , were expressed in VPS15 KO or HEK293A cells . Cells were starved for 1 hr and the distributions of WIPI2 ( green ) , HA ( red ) and LC3 ( blue ) were assessed , mean + / - SEM , n = 4 .

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 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 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

Figure 4A) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot. LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA-derived clones.B) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr, fixed and imaged for WIPI2 (green). Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles (red asterisks). Quantification of WIPI2 spots, mean +/- SEM, n=3.D) Wild type (WT), 6SA or 6SE VPS15-HA, or empty vector (EV), were expressed in VPS15 KO or HEK293A cells. Cells were starved for 1 hr and the distributions of WIPI2 (green), HA (red) and LC3 (blue) were assessed, mean +/- SEM, n=4.