Figure 3Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSDCholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale barCholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale bar : 10 μm ( A , C , D , F ) . Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale bar : 10 μm ( A , C , D , F ) . Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale bar : 10 μm ( A , C , D , F ) .

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 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 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 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 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

Figure 3Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSDCholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale barCholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).


Figure 4 ( A ) Endolysosomal membranes from MSD , MPS ‐ IIIA and WT MEFs were treated with 1 % Triton X ‐ 114 and loaded on a sucrose gradient . Immunoblots with Flotillin ‐ 1 identified DRMs in fractions 2 , 3 and 4 ( arrows ) . The fractions at the bottom of the gradient ( 12 and 13 ) correspond to high ‐ density detergent soluble fractions , whereas the remaining ones were defined as intermediate fractions ( intermediate ‐ I : 5 , 6 , 7 8 ; intermediate ‐ II : 9 , 10 and 11 ) . The percentage of Flotillin ‐ 1 in DRMs was calculated from the densitometric quantification of immunoblots . ( B ) Immuno ‐ EM of GM1 lipid was carried out in WT , MSD and MPS ‐ IIIA MEFs by staining cells with anti ‐ cholera toxin B antibodies ( see Materials and methods section ) . The number of GM1 ‐ positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT . ( C ) Endolysosomal membranes from MSD , MPS ‐ IIIA and WT MEFs were stained with C ‐ laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value ( see Materials and methods section for details ) . Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft ( DRMs ) and soluble fractions . ( D ) Equal aliquots from either DRMs or soluble fractions were pooled , the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions . ( E ) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient . Values represent the mean ± s . e . m . values of three experiments ( A - D ) . * P0 . 05 , Student ' s t ‐ test : ( A - C ) : WT versus MSD and WT versus MPS ‐ IIIA ; ( D ) : WT versus MSD and WT versus MPS ‐ IIIA for each fraction . Scale bar : 0 . 3 μm ( B ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were treated with 1% Triton X‐114 and loaded on a sucrose gradient. Immunoblots with Flotillin‐1 identified DRMs in fractions 2, 3 and 4 (arrows). The fractions at the bottom of the gradient (12 and 13) correspond to high‐density detergent soluble fractions, whereas the remaining ones were defined as intermediate fractions (intermediate‐I: 5, 6, 7 8; intermediate‐II: 9, 10 and 11). The percentage of Flotillin‐1 in DRMs was calculated from the densitometric quantification of immunoblots.(B) Immuno‐EM of GM1 lipid was carried out in WT, MSD and MPS‐IIIA MEFs by staining cells with anti‐cholera toxin B antibodies (see Materials and methods section). The number of GM1‐positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT.(C) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were stained with C‐laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value (see Materials and methods section for details). Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft (DRMs) and soluble fractions.(D) Equal aliquots from either DRMs or soluble fractions were pooled, the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions.(E) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient. Values represent the mean±s.e.m. values of three experiments (A-D). *P0.05, Student's t‐test: (A-C): WT versus MSD and WT versus MPS‐IIIA; (D): WT versus MSD and WT versus MPS‐IIIA for each fraction. Scale bar: 0.3 μm (B).


Figure 5 ( A ) VAMP7 , Vti1b and syntaxin 7 distributions in endo ‐ lysosomal membranes from MSD , MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions . To simplify the analysis the DRM , the intermediate ‐ I , the intermediate ‐ II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting . SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment . In MSD and MPS ‐ IIIA MEFs , all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes . Cholesterol modulation results in a change of SNARE distribution . ( B ) The percentage of each analysed SNARE observed in DRM fractions was quantified from blots ( ImageJ densitometry analysis ) and displayed as relative amount versus WT . ( C , E ) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in ( C ) endolysosomal membranes and ( E ) total cell lysates from WT ( untreated and cholesterol treated ) and MSD MEFs ( untreated and MβCD treated ) . In the graphs , the protein levels were displayed as relative amount versus WT . ( D ) WT and MSD MEFs transfected with GFP - VAMP7 were stained with anti ‐ GFP for immuno ‐ EM . The enlarged image shows internalization of GFP - VAMP7 particles ( arrow ) . ( F ) Syntaxin 5 , Sec22 and SNAP23 distribution in total membrane derived from control WT and MSD MEFs . Syntaxin 5 immunoblot shows two bands ( * , 35 kDa and * * , 42 kDa ) corresponding to the two isoforms of the protein . ( G ) Distribution profile of Rab7 in WT and MSD lysosomal membranes . Values represent the mean ± s . e . m . values of three independent experiments ( B , C , E ) . * P0 . 05 , Student ' s t ‐ test : ( B ) : WT versus MSD , WT versus MPS ‐ IIIA , WT versus WT + cholesterol , MSD versus MSD + MβCD , and MPS ‐ IIIA versus MβCD for each analysed SNARE ; ( C , E ) : WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD for each analysed protein . Scale bar : 0 . 3 μm ( D ) .

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

Figure 5(A) VAMP7, Vti1b and syntaxin 7 distributions in endo‐lysosomal membranes from MSD, MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions. To simplify the analysis the DRM, the intermediate‐I, the intermediate‐II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting. SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment. In MSD and MPS‐IIIA MEFs, all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes. Cholesterol modulation results in a change of SNARE distribution.(B) The percentage of each analysed SNARE observed in DRM fractions was quantified from blots (ImageJ densitometry analysis) and displayed as relative amount versus WT.(C, E) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in (C) endolysosomal membranes and (E) total cell lysates from WT (untreated and cholesterol treated) and MSD MEFs (untreated and MβCD treated). In the graphs, the protein levels were displayed as relative amount versus WT.(D) WT and MSD MEFs transfected with GFP-VAMP7 were stained with anti‐GFP for immuno‐EM. The enlarged image shows internalization of GFP-VAMP7 particles (arrow).(F) Syntaxin 5, Sec22 and SNAP23 distribution in total membrane derived from control WT and MSD MEFs. Syntaxin 5 immunoblot shows two bands (*, 35 kDa and **, 42 kDa) corresponding to the two isoforms of the protein.(G) Distribution profile of Rab7 in WT and MSD lysosomal membranes. Values represent the mean±s.e.m. values of three independent experiments (B, C, E). *P0.05, Student's t‐test: (B): WT versus MSD, WT versus MPS‐IIIA, WT versus WT+cholesterol, MSD versus MSD+MβCD, and MPS‐IIIA versus MβCD for each analysed SNARE; (C, E): WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each analysed protein. Scale bar: 0.3 μm (D).


Figure 6 ( A ) SDS ‐ resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total , detergent insoluble ( DRM ) and detergent soluble ( Sol . ) endo ‐ lysosomal membrane fractions derived from MSD and WT MEFs . The SDS ‐ resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD . Immunoblots revealed the presence of low molecular weight complexes ( * , 50 - 60 kDa ) and high molecular weight complexes ( * * , > 80 kDa ) . The percentage of Vti1b in SDS ‐ resistant complexes in total endolysosomal membranes ( bottom ‐ left chart ) and the amount of Vti1b ‐ containing SDS ‐ resistant complexes in DRM and soluble fractions ( bottom ‐ right chart ) were calculated by the densitometric quantification of the correspondent immunoblots ( ImageJ ) . Values represent the mean ± s . e . m . values of three independent measurements . * P0 . 05 , Student ' s t ‐ test : WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD ( bottom ‐ left chart ) ; WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD for each fraction ( bottom ‐ right chart ) . ( B ) Syntaxin 7 and VAMP7 were co ‐ immunoprecipitated with Vti1b using anti ‐ Vti1b antibodies in WT ( untreated or cholesterol treated ) and in MSD ( not treated or MβCD treated ) MEFs . The amount of Vti1b precipitated in each cell line is also shown . ( C ) SDS ‐ resistant complexes are decorated by anti ‐ syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs . ( D ) Membrane ‐ associated α ‐ SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates ( total ) , intracellular membranes recovered after centrifugation from a post ‐ nuclear supernatant fraction ( membrane associated ) and cell lysates devoid of membranes ( cytosolic released ) derived from MSD ( untreated or MβCD treated ) and WT ( untreated or cholesterol treated ) MEFs .

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

Figure 6(A) SDS‐resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total, detergent insoluble (DRM) and detergent soluble (Sol.) endo‐lysosomal membrane fractions derived from MSD and WT MEFs. The SDS‐resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD. Immunoblots revealed the presence of low molecular weight complexes (*, 50-60 kDa) and high molecular weight complexes (**, >80 kDa). The percentage of Vti1b in SDS‐resistant complexes in total endolysosomal membranes (bottom‐left chart) and the amount of Vti1b‐containing SDS‐resistant complexes in DRM and soluble fractions (bottom‐right chart) were calculated by the densitometric quantification of the correspondent immunoblots (ImageJ). Values represent the mean±s.e.m. values of three independent measurements. *P0.05, Student's t‐test: WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD (bottom‐left chart); WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each fraction (bottom‐right chart).(B) Syntaxin 7 and VAMP7 were co‐immunoprecipitated with Vti1b using anti‐Vti1b antibodies in WT (untreated or cholesterol treated) and in MSD (not treated or MβCD treated) MEFs. The amount of Vti1b precipitated in each cell line is also shown.(C) SDS‐resistant complexes are decorated by anti‐syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs.(D) Membrane‐associated α‐SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates (total), intracellular membranes recovered after centrifugation from a post‐nuclear supernatant fraction (membrane associated) and cell lysates devoid of membranes (cytosolic released) derived from MSD (untreated or MβCD treated) and WT (untreated or cholesterol treated) MEFs.


Figure 7 ( A ) MSD , MPS ‐ IIIA and WT MEFs were subjected to a triple labelling with anti ‐ VAMP7 , anti ‐ Vti1b and anti ‐ LAMP1 antibodies . The merges between VAMP7 and Vti1b ( double merges in yellow ) and between VAMP7 , Vti1b and LAMP1 ( triple merges in white ) are shown ( see also enlarged images showing the extent of co ‐ localization in different regions of the cells ) . The VAMP7 - Vti1b co ‐ localization was quantified in 15 different cells and displayed as % of Vti1b co ‐ localizing with VAMP7 ( means ± s . e . m . ) . ( B ) Co ‐ localization of Vti1b with epsinR was quantified by double ‐ labelling experiments in MSD and MPS ‐ IIIA ( untreated and MβCD treated ) and in control WT ( untreated and cholesterol treated ) MEFs . The chart displays merge values ( means ± s . e . m . ) that represent the percentage of Vti1b co ‐ localizing with epsinR measured in 15 different cells . ( C ) Vti1b trafficking was monitored by FRAP analysis in WT ( untreated and cholesterol treated ) and MSD ( untreated and MβCD treated ) MEFs transfected with GFP - Vti1b ( see Materials and methods section for details ) . FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach ( 100 % ) and are representative of 10 recordings from different cells .

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

Figure 7(A) MSD, MPS‐IIIA and WT MEFs were subjected to a triple labelling with anti‐VAMP7, anti‐Vti1b and anti‐LAMP1 antibodies. The merges between VAMP7 and Vti1b (double merges in yellow) and between VAMP7, Vti1b and LAMP1 (triple merges in white) are shown (see also enlarged images showing the extent of co‐localization in different regions of the cells). The VAMP7-Vti1b co‐localization was quantified in 15 different cells and displayed as % of Vti1b co‐localizing with VAMP7 (means±s.e.m.).(B) Co‐localization of Vti1b with epsinR was quantified by double‐labelling experiments in MSD and MPS‐IIIA (untreated and MβCD treated) and in control WT (untreated and cholesterol treated) MEFs. The chart displays merge values (means±s.e.m.) that represent the percentage of Vti1b co‐localizing with epsinR measured in 15 different cells.(C) Vti1b trafficking was monitored by FRAP analysis in WT (untreated and cholesterol treated) and MSD (untreated and MβCD treated) MEFs transfected with GFP-Vti1b (see Materials and methods section for details). FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells.


Figure 1b , Representative phase images of the morphology of WT and Apc - / - organoids before mRNA isolation . c , Heat map illustrating differentially expressed genes ( DEGs ) encoding heat shock proteins between WT and Apc - / - organoids . d , Volcano plot of DEGs between normal mouse intestinal tissue and adenomas ( GSE65461 ) . e , f , Relative gene expression of Hspb1 ( * * * P = 0 . 0004 , n = 6 ) ( e ) and protein expression of HSP25 ( f ) in unrecombined versus recombined murine organoids . g , Gene expression of Hspb1 in normal ( N ) versus adenoma ( A ) tissue ( * * * P = 0 . 0002 , n = 4 ) . H , RNA - ISH for Hspb1 in murine intestinal adenoma . Scalebar , 100 μm . Zoom panel , 25 μmi , Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue , ( * * * * P < 0 . 0001 , paired t - test , n = 59 pairs ) .

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

Figure 1b, Representative phase images of the morphology of WT and Apc-/- organoids before mRNA isolation.c, Heat map illustrating differentially expressed genes (DEGs) encoding heat shock proteins between WT and Apc-/- organoids.d, Volcano plot of DEGs between normal mouse intestinal tissue and adenomas (GSE65461).e, f, Relative gene expression of Hspb1 (***P=0.0004, n=6)(e) and protein expression of HSP25 (f) in unrecombined versus recombined murine organoids.g, Gene expression of Hspb1 in normal (N) versus adenoma (A) tissue (***P=0.0002, n=4).H, RNA-ISH for Hspb1 in murine intestinal adenoma. Scalebar, 100 μm. Zoom panel, 25 μmi, Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue, (****P<0.0001, paired t-test, n=59 pairs).


Figure 2Relative gene expression of AXIN2 ( * P = 0 . 0129 ) , P21 ( * P = 0 . 0103 ) , and HSPB1 ( * P = 0 . 0236 ) ( b ) in Ls174t - dnTCF4 cells the presence or absence of doxycycline . protein expression of HSP27 ( c ) in Ls174t - dnTCF4 cells the presence or absence of doxycycline . Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR - 99021 ( * * P = 0 . 0016 , one way ANOVA , n = 4 ) ( d ) protein expression of HSP25 in WT and Apc - / - organoids ( e ) . g , h , Representative images of single cell Hspb1 KO clones passaged after loss of Apc ( g ) , and quantification of their clonogenic potential ( * P = 0 . 0147 ( # 1 ) , * * P = 0 . 0035 ( # 2 ) , * * P = 0 . 0032 ( # 3 ) ) ( h ) . i , j , Representative images of Apc - / - organoids cultured in the absence or presence of 60 μM BVDU ( i ) , and quantification of their clonogenic potential ( * * P = 0 . 0039 ) ( j ) . k , l , Relative gene expression of Wnt target genes Axin2 ( * * P = 0 . 0080 ) and Lgr5 ( * P = 0 . 0142 ) in Apc - / - organoids cultured in the absence or presence of 60 μM BVDU . m , RNA - ISH for Lgr5 in control or BVDU - treated Apc - / - organoids . Scale bar , 50 μm , zoom panel 20 μm .

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Figure 2Relative gene expression of AXIN2 (*P=0.0129), P21(*P=0.0103), and HSPB1(*P=0.0236) (b) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.protein expression of HSP27 (c) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR-99021 (**P=0.0016, one way ANOVA, n=4) (d)protein expression of HSP25 in WT and Apc-/- organoids (e).g, h, Representative images of single cell Hspb1 KO clones passaged after loss of Apc (g), and quantification of their clonogenic potential (*P=0.0147(#1), **P=0.0035(#2), **P=0.0032(#3)) (h).i, j, Representative images of Apc-/- organoids cultured in the absence or presence of 60 μM BVDU (i), and quantification of their clonogenic potential (**P=0.0039) (j).k, l, Relative gene expression of Wnt target genes Axin2 (**P=0.0080) and Lgr5 (*P=0.0142) in Apc-/- organoids cultured in the absence or presence of 60 μM BVDU.m, RNA-ISH for Lgr5 in control or BVDU-treated Apc-/- organoids. Scale bar, 50 μm, zoom panel 20 μm.


Figure 3b , c , Visualization of proliferating cells in tissues incubated with EdU for 2 - hours ( b ) , and quantification of the hyperproliferative zone ( * * P = 0 . 0057 , n = 3 mice ) ( c ) . e , f , RNA - ISH for Notum reveals Apc - mutant clones in crypt bottoms of control or BVDU - treated mice ( e ) , and quantification of the abundance of mutant crypts ( n = 2 mice per condition , n = 5 technical replicates per mouse ) ( f ) . g , h , Clone size distributions of Notum + crypts in control ( n = 265 crypts ) or BVDU - treated ( n = 260 crypts ) mice , the average clone size and number of fixed clones are included in the figure . Survival curves for control and BVDU - treated mice ( P = 0 . 0351 , Mantel - Cox test ) ( j ) macroscopic images of adenoma development in the distal small intestine ( k ) boxplots for the number of adenomas per intestinal region ( * * P = 0 . 0013 ) ( l ) , n = 8 control , n = 11 treated .

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Figure 3b, c, Visualization of proliferating cells in tissues incubated with EdU for 2-hours (b), and quantification of the hyperproliferative zone (**P=0.0057, n=3 mice) (c).e, f, RNA-ISH for Notum reveals Apc-mutant clones in crypt bottoms of control or BVDU-treated mice (e), and quantification of the abundance of mutant crypts (n=2 mice per condition, n=5 technical replicates per mouse) (f).g, h, Clone size distributions of Notum+ crypts in control (n=265 crypts) or BVDU-treated (n=260 crypts) mice, the average clone size and number of fixed clones are included in the figure.Survival curves for control and BVDU-treated mice (P=0.0351, Mantel-Cox test) (j)macroscopic images of adenoma development in the distal small intestine (k)boxplots for the number of adenomas per intestinal region(**P=0.0013) (l), n=8 control, n=11 treated.


figf1 ( a ) [ 3H ] thymidine incorporation at 72 h by wild - type ( WT ) and Irgm1 - / - CD4 + T cells activated with soluble mAb to CD3 and irradiated syngenic wild - type splenocytes . ( b ) [ 3H ] thymidine incorporation at 72 h by OT - II and Irgm1 - / - OT - II CD4 + T cells stimulated with OVA protein ( OVA ) or OVA peptide of amino acids 323 - 339 ( OVA ( 323 - 339 ) ) in the presence of syngenic splenic DCs . ( c , d ) Kinetic analysis of [ 3H ] thymidine incorporation ( c ) and cell numbers ( d ) in cultures of OT - II and Irgm1 − / − OT - II spleen cells stimulated with OVA peptide . ( e ) Flow cytometric analysis of the activation and proliferation of OVA peptide - stimulated OT - II and Irgm1 - / - OT - II CD4 + T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU . Numbers in quadrants indicate percent viable CD4 + cells . ( f ) Enzyme - linked immunosorbent assay of IL - 2 produced by OT - II and Irgm1 - / - OT - II CD4 + T cells stimulated with OVA peptide in the presence of splenic DCs , measured at 72 h . Data are representative of more than five ( a , b ) or three ( c - f ) independent experiments with similar results ( mean and s . d . of triplicate cultures , a - d , f ) .

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figf1(a) [3H]thymidine incorporation at 72 h by wild-type (WT) and Irgm1-/- CD4+ T cells activated with soluble mAb to CD3 and irradiated syngenic wild-type splenocytes.(b) [3H]thymidine incorporation at 72 h by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA protein (OVA) or OVA peptide of amino acids 323-339 (OVA(323-339)) in the presence of syngenic splenic DCs.(c,d) Kinetic analysis of [3H]thymidine incorporation (c) and cell numbers (d) in cultures of OT-II and Irgm1−/− OT-II spleen cells stimulated with OVA peptide.(e) Flow cytometric analysis of the activation and proliferation of OVA peptide-stimulated OT-II and Irgm1-/- OT-II CD4+ T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU. Numbers in quadrants indicate percent viable CD4+ cells.(f) Enzyme-linked immunosorbent assay of IL-2 produced by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DCs, measured at 72 h. Data are representative of more than five (a,b) or three (c-f) independent experiments with similar results (mean and s.d. of triplicate cultures, a-d,f).


figf2 ( a ) Immunoblot analysis of Irgm1 in lysates of flow cytometry - sorted naive wild - type CD4 + lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 ( 10 μg / ml ) alone or with mAb to CD28 ( 2 μg / ml ) in the presence or absence of IFN - γ - neutralizing mAb . Actin , loading control . ( b ) Enzyme - linked immunosorbent assay of IFN - γ produced by OT - II and Irgm1 − / − OT - II CD4 + T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions ( TH1 or TH2 ) or nonpolarized conditions ( N ) , assessed in 72 - hour cultures . ND , not detected . ( c ) Accumulation of polarized and nonpolarized OT - II ( open symbols ) or Irgm1 − / − OT - II ( filled symbols ) CD4 + T cells after peptide stimulation , quantified by counting of viable cells . ( d ) Viable OT - II and Irgm1 − / − OT - II CD4 + T cells 72 h after restimulation with OVA peptide in the presence of T cell - depleted APC samples . ( e ) Flow cytometry of the viability and division of OT - II and Irgm1 − / − OT - II CD4 + T cells stimulated with peptide , allowed to ' rest ' for 7 d in IL - 2 , then labeled with CFSE and restimulated with OVA peptide in the presence of T cell - depleted APC samples and assessed 72 h later . ( f ) Flow cytometric analysis of the population expansion of CFSE - labeled OT - II and Irgm1 − / − OT - II CD4 + T cells activated with OVA peptide in the presence or absence of IFN - γ - neutralizing mAb . Results in e , f obtained after gating on CD4 + T cells ; numbers in outlined areas ( e ) and in top left corners ( f ) indicate percent CD4 + cells . PI , propidium iodide . ( g ) [ 3H ] thymidine incorporation by wild - type , Irgm1 − / − , Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng − / − splenocytes . Data are representative of two independent experiments with similar results ( mean and s . d . of triplicate cultures , b - d , g ) .

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 O O O O O O O O O O O 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 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 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 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

figf2(a) Immunoblot analysis of Irgm1 in lysates of flow cytometry-sorted naive wild-type CD4+ lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 (10 μg/ml) alone or with mAb to CD28 (2 μg/ml) in the presence or absence of IFN-γ-neutralizing mAb. Actin, loading control.(b) Enzyme-linked immunosorbent assay of IFN-γ produced by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions (TH1 or TH2) or nonpolarized conditions (N), assessed in 72-hour cultures. ND, not detected.(c) Accumulation of polarized and nonpolarized OT-II (open symbols) or Irgm1−/− OT-II (filled symbols) CD4+ T cells after peptide stimulation, quantified by counting of viable cells.(d) Viable OT-II and Irgm1−/− OT-II CD4+ T cells 72 h after restimulation with OVA peptide in the presence of T cell-depleted APC samples.(e) Flow cytometry of the viability and division of OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with peptide, allowed to 'rest' for 7 d in IL-2, then labeled with CFSE and restimulated with OVA peptide in the presence of T cell-depleted APC samples and assessed 72 h later.(f) Flow cytometric analysis of the population expansion of CFSE-labeled OT-II and Irgm1−/− OT-II CD4+ T cells activated with OVA peptide in the presence or absence of IFN-γ-neutralizing mAb. Results in e,f obtained after gating on CD4+ T cells; numbers in outlined areas (e) and in top left corners (f) indicate percent CD4+ cells. PI, propidium iodide.(g) [3H]thymidine incorporation by wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng−/− splenocytes. Data are representative of two independent experiments with similar results (mean and s.d. of triplicate cultures, b-d,g).


figf3 ( a ) Flow cytometric analysis of the survival of CD45 . 2 + Ifng − / − ( open symbols ) or Irgm1 − / − Ifng − / − ( filled symbols ) CD4 + T lymphocytes preactivated with agonistic mAb to CD3 in vitro , then transferred intraperitoneally into congenic CD45 . 1 + C57BL / 6 . SJL recipients left untreated or inoculated intraperitoneally with T . gondii or M . avium 3 d earlier , assessed in peritoneal exudate cell samples collected at day 4 after transfer . ( b ) Survival of M . avium - infected wild - type , Irgm1 − / − , Ifng − / − and Irgm1 − / − Ifng − / − mice ( n = 5 mice per genotype ) . ( c - e ) Splenic bacterial loads ( c ) , circulating lymphocytes ( d ) and splenic lymphocytes ( e ) , assessed at week 4 after M . avium infection ( n = 5 mice per genotype ) . Each symbol in a , c - e represents an individual mouse . Data are representative of two independent experiments .

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figf3(a) Flow cytometric analysis of the survival of CD45.2+ Ifng−/− (open symbols) or Irgm1−/−Ifng−/− (filled symbols) CD4+ T lymphocytes preactivated with agonistic mAb to CD3 in vitro, then transferred intraperitoneally into congenic CD45.1+ C57BL/6.SJL recipients left untreated or inoculated intraperitoneally with T. gondii or M. avium 3 d earlier, assessed in peritoneal exudate cell samples collected at day 4 after transfer.(b) Survival of M. avium-infected wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− mice (n = 5 mice per genotype).(c-e) Splenic bacterial loads (c), circulating lymphocytes (d) and splenic lymphocytes (e), assessed at week 4 after M. avium infection (n = 5 mice per genotype). Each symbol in a,c-e represents an individual mouse. Data are representative of two independent experiments.


figf4 ( a ) [ 3H ] thymidine incorporation by naive CD4 + lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN - γ - deficient irradiated APCs , with IL - 2 ( 10 U / ml ) added 24 h after the initiation of culture . ( b ) Accumulation of viable CD4 + T cells at 72 h in the presence of medium alone ( Med ) or IFN - γ ( 5 U / ml ) . ( c ) Flow cytometric analysis of the viability and division of CD4 + T cells labeled with CFSE and activated as described in a with T cell - depleted Ifng − / − APC samples in the presence or absence of IFN - γ ( 5 U / ml ) . Numbers in plots indicate percent CFSE + CD4 + T cell populations . ( d - f ) Death of Ifng − / − and Irgm1 − / − Ifng − / − T cell populations activated with mAb to CD3 and expanded in IL - 2 , then treated with IFN - γ ( d ) , agonistic mAb to CD3 ( e ) or Fas ligand ( FasL ; f ) , assessed by propidium iodide staining at 48 h after treatment . Open bars , Ifng − / − ; filled bars , Irgm1 − / − Ifng − / − ( a , b , d - f ) . Data are representative of three independent experiments with similar results ( mean and s . d . of triplicate cultures a , b , d - f ) .

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figf4(a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture.(b) Accumulation of viable CD4+ T cells at 72 h in the presence of medium alone (Med) or IFN-γ (5 U/ml).(c) Flow cytometric analysis of the viability and division of CD4+ T cells labeled with CFSE and activated as described in a with T cell-depleted Ifng−/− APC samples in the presence or absence of IFN-γ (5 U/ml). Numbers in plots indicate percent CFSE+CD4+ T cell populations.(d-f) Death of Ifng−/− and Irgm1−/−Ifng−/− T cell populations activated with mAb to CD3 and expanded in IL-2, then treated with IFN-γ (d), agonistic mAb to CD3 (e) or Fas ligand (FasL; f), assessed by propidium iodide staining at 48 h after treatment. Open bars, Ifng−/−; filled bars, Irgm1−/−Ifng−/− (a,b,d-f). Data are representative of three independent experiments with similar results (mean and s.d. of triplicate cultures a,b,d-f).


figf5 ( a - f ) Electron microscopy analysis of Ifng − / − CD4 + lymphocytes ( a ) and Irgm1 − / − Ifng − / − CD4 + lymphocytes ( b - f ) exposed for 30 h to IFN - γ ( 5 U / ml ) . ( c ) Enlargement of the area outlined in b ; arrowheads indicate two autophagic vacuoles . ( d ) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells . Each symbol represents one section . ( e ) Irgm1 − / − CD4 + T cell with many vacuoles containing dense materials . ( f ) Two Irgm1 − / − CD4 + cells with vacuolated cytoplasma . Original magnification ( a - c , e , f ) , × 1 , 000 . Data are representative of two experiments . ( g , h ) Death of Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells in the presence or absence of wortmannin or Ly294002 ( g ) , assessed by flow cytometry with propidium iodide staining at 48 h after IFN - γ exposure . Data are representative of three independent experiments ( mean and s . d . of triplicate cultures ) . ( g , h ) Death of Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells transfected with control or beclin1 - specific small interfering RNA , assessed by flow cytometry with propidium iodide staining at 48 h after IFN - γ exposure . Right ( h ) , immunoblot analysis of beclin1 expression . C , control small interfering RNA ; B , beclin1 - specific small interfering RNA . Data are representative of three independent experiments ( mean and s . d . of triplicate cultures ) .

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figf5(a-f) Electron microscopy analysis of Ifng−/− CD4+ lymphocytes (a) and Irgm1−/−Ifng−/− CD4+ lymphocytes (b-f) exposed for 30 h to IFN-γ (5 U/ml). (c) Enlargement of the area outlined in b; arrowheads indicate two autophagic vacuoles. (d) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells. Each symbol represents one section. (e) Irgm1−/− CD4+ T cell with many vacuoles containing dense materials. (f) Two Irgm1−/− CD4+ cells with vacuolated cytoplasma. Original magnification (a-c,e,f), × 1,000. Data are representative of two experiments.(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells in the presence or absence of wortmannin or Ly294002 (g), assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells transfected with control or beclin1-specific small interfering RNA, assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Right (h), immunoblot analysis of beclin1 expression. C, control small interfering RNA; B, beclin1-specific small interfering RNA. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).


Figure 1B Vero E6 cells were inoculated with SARS - CoV - 2 , fixed with paraformaldehyde after two days , stained with 30 nM of the indicated Alexa Fluor 488 ( AF488 ) - labeled anti - RBD VHH antibodies , and imaged by confocal laser scanning microscopy ( CLSM ) . C Immunofluorescence ( IF ) staining as in B , but with the indicated AF568 - labeled anti - S1ΔRBD VHH antibodies .

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Figure 1B Vero E6 cells were inoculated with SARS-CoV-2, fixed with paraformaldehyde after two days, stained with 30 nM of the indicated Alexa Fluor 488 (AF488)-labeled anti-RBD VHH antibodies, and imaged by confocal laser scanning microscopy (CLSM). C Immunofluorescence (IF) staining as in B, but with the indicated AF568-labeled anti-S1ΔRBD VHH antibodies.


Figure 2BLI sensorgrams of intermediate VHHs A SARS - CoV - 2 RBD - loaded sensors were incubated with nanobodies at the indicated concentrations for 300 s , followed by dipping in assay buffer for 600 s . The response curves ( black ) were fitted using either a 1 : 1 ( for VHH - 72 ) or 2 : 1 ( for Re6B06 and Re9F06 ) binding models to obtain KDs . Fitted curves are shown in red . For Re6B06 and Re9F06 , some secondary binding with weaker affinity at high VHH concentrations was observed ( denoted as KD2 ) .

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Figure 2BLI sensorgrams of intermediate VHHs A SARS-CoV-2 RBD-loaded sensors were incubated with nanobodies at the indicated concentrations for 300 s, followed by dipping in assay buffer for 600 s. The response curves (black) were fitted using either a 1:1 (for VHH-72) or 2:1 (for Re6B06 and Re9F06) binding models to obtain KDs. Fitted curves are shown in red. For Re6B06 and Re9F06, some secondary binding with weaker affinity at high VHH concentrations was observed (denoted as KD2).


Figure 3A Vero E6 cells were infected with SARS - CoV - 2 , pre - incubated with indicated concentrations of the neutralizing VHH - 72 Cells were fixed two days after inoculation , stained with sets of anti - RBD ( green ) and anti - S1ΔRBD ( red ) nanobodies and analyzed by CLSM . We used a cocktail of such fluorophore - labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody . B Vero E6 cells were infected as in A . Viral RNA in the culture supernatants was quantified by reverse transcription ( RT ) - qPCR . RNA signals are normalized to the respective minus - VHH controls ( 100 % ) . Note the log10 scale of the y - axis . " Inoculum " ( red dotted line ) marks the RNA level detected at the time of infection .

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Figure 3A Vero E6 cells were infected with SARS-CoV-2, pre-incubated with indicated concentrations of the neutralizing VHH-72 Cells were fixed two days after inoculation, stained with sets of anti-RBD (green) and anti-S1ΔRBD (red) nanobodies and analyzed by CLSM. We used a cocktail of such fluorophore-labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody.B Vero E6 cells were infected as in A. Viral RNA in the culture supernatants was quantified by reverse transcription (RT)-qPCR. RNA signals are normalized to the respective minus-VHH controls (100%). Note the log10 scale of the y-axis. "Inoculum" (red dotted line) marks the RNA level detected at the time of infection.


Figure 4Neutralization of SARS - CoV - 2 by the indicated VHH antibodies at nanomolar ( A and B ) or picomolar ( C and D ) concentrations . The neutralization experiment was performed ; n " indicates the number of independent biological

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Figure 4Neutralization of SARS-CoV-2 by the indicated VHH antibodies at nanomolar (A and B) or picomolar (C and D) concentrations. The neutralization experiment was performed ;n" indicates the number of independent biological replicates


Figure 5A HeLa cells were transiently transfected to express the SARS - CoV - 2 Spike protein . Following fixation , cells were stained for 1 hour with fluorophore - labeled Re10B10 ( 5 nM , green ) and Re7E02 ( 15 nM , red ) in the presence of the indicated unlabeled VHH competitors . Competitor ( 150 nM ) was added 20 min prior to the labeled nanobodies . The weakly binding competitors Re5A08 , Re9F06 , Re6B06 , and , Re6D06 were added as trimers ( see below ) . Cells were imaged by CLSM . For each competitor , the sequence class and the binding site on the RBD ( epitope 1 or 2 ) are indicated .

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Figure 5A HeLa cells were transiently transfected to express the SARS-CoV-2 Spike protein. Following fixation, cells were stained for 1 hour with fluorophore-labeled Re10B10 (5 nM, green) and Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled nanobodies. The weakly binding competitors Re5A08, Re9F06, Re6B06, and, Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated.


Figure 7F Neutralization of SARS - CoV - 2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations . The neutralization experiment was

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Figure 7F Neutralization of SARS-CoV-2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations. The neutralization experiment was performed


Figure 8B Re6B06 monomer and trimer variants ( 20 nM ) were analyzed by BLI for RBD - binding , with 900 s association and 1500 s dissociation steps . Note the avidity effect of Re6B06 trimerization . C Neutralization of SARS - CoV - 2 by the indicated VHH antibodies . The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized . Re6B06 , for example , is 30 000 - fold more potent when symmetry - matched with the virus Spike .

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Figure 8B Re6B06 monomer and trimer variants (20 nM) were analyzed by BLI for RBD-binding, with 900 s association and 1500 s dissociation steps. Note the avidity effect of Re6B06 trimerization.C Neutralization of SARS-CoV-2 by the indicated VHH antibodies. The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized. Re6B06, for example, is 30 000-fold more potent when symmetry-matched with the virus Spike.


Figure 9C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI . Immobilized RBD was incubated with 100 nM of nanobodies for 450 s , followed by assay buffer for 900 s .

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Figure 9C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI. Immobilized RBD was incubated with 100 nM of nanobodies for 450 s, followed by assay buffer for 900 s.


Figure 10A and B Neutralization of SARS - CoV - 2 B . 1 . 351 / Beta by the indicated VHH antibody constructs . The neutralization experiment was performed as described in Fig 3A , with the difference being that a mutant - optimized anti - RBD / S1 VHH cocktail was used for immunofluorescence staining Two titrations for the Re9F06 trimer are shown , corresponding to the standard ( upper row ) or a five - fold higher virus inoculum ( lower row ) that was used for even more stringent neutralization conditions .

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Figure 10A and B Neutralization of SARS-CoV-2 B.1.351/Beta by the indicated VHH antibody constructs. The neutralization experiment was performed as described in Fig 3A, with the difference being that a mutant-optimized anti-RBD/S1 VHH cocktail was used for immunofluorescence staining Two titrations for the Re9F06 trimer are shown, corresponding to the standard (upper row) or a five-fold higher virus inoculum (lower row) that was used for even more stringent neutralization conditions.


Figure 1 ( B ) A fragment of genomic DNA ( 2 . 2 kb ) was amplified by PCR and in vitro sgRNA - guided Cas9 - mediated cleavage assay was performed with each of the sgRNAs . sgRNA - targeting sites are indicated by arrows , genomic DNA size is indicated by asterisk .

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Figure 1(B) A fragment of genomic DNA (2.2 kb) was amplified by PCR and in vitro sgRNA-guided Cas9-mediated cleavage assay was performed with each of the sgRNAs. sgRNA-targeting sites are indicated by arrows, genomic DNA size is indicated by asterisk.


Figure 4 ( C ) Immunohistochemistry of day 8 spleen sections for host mouse receiving 5x105 isolated CD45 . 2 B cells from CLK09 ( left ) and CLK19 ( right ) KI mouse . Green , B220 ; Blue , CD3 ; White , CD45 . 2 ; Red , GL7 . White arrows indicate the CD45 . 2 B cells .

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Figure 4(C) Immunohistochemistry of day 8 spleen sections for host mouse receiving 5x105 isolated CD45.2 B cells from CLK09 (left) and CLK19 (right) KI mouse. Green, B220; Blue, CD3; White, CD45.2; Red, GL7.White arrows indicate the CD45.2 B cells.


Figure 6 ( A ) CD45 . 2 frequency among GCs induced by eOD - GT8 60 mer at different time points . Adoptively transferred mice with 1 in 104 of precursor frequency were immunized with eOD - GT8 60mer . On days 8 , 15 and 36 , the splenic B cells were isolated to measure the CD45 . 2 cell frequency in GC B cells by FACS using the markers mentioned above by FACS . Gated plots represent the CD45 . 2 frequency among GC B cells . ( B ) Quantification of CD45 . 2 frequency among GCs at three time points . X - axis represents adoptively transferred mice groups with different precursor frequency ( 1 in 104 , 2 in 105 , 3 in 106 ) at three time point , y axis represents CD45 . 2 frequency among GC . Each circle represents one mouse . For CLK21 and CLK09 panel , n = 5 mice in each group . For CLK19 panel , n = 3 mice in each group . Bars indicate mean ± SD from mice in each group . ( C ) IgG titers detected by ELISA . eOD - GT8 or eOD - GT8 KO soluble proteins were used to coat ELISA plates . Sera from adoptively transferred mice at different time points were used to detect by ELISA . Bars indicate geometric mean and geometric SD from mice in each group . For CLK21 and CLK09 panel , n = 5 mice in each group . For CLK19 panel , n = 3 mice in each group . X - axis represents precursor frequency , and Y - axis represents the change of area under curve ( AUC coated eOD - GT8 - AUC coated eOD - GT8 KO ) . Each circle represents one mouse .

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Figure 6(A) CD45.2 frequency among GCs induced by eOD-GT8 60 mer at different time points. Adoptively transferred mice with 1 in 104 of precursor frequency were immunized with eOD-GT8 60mer. On days 8, 15 and 36, the splenic B cells were isolated to measure the CD45.2 cell frequency in GC B cells by FACS using the markers mentioned above by FACS. Gated plots represent the CD45.2 frequency among GC B cells. (B) Quantification of CD45.2 frequency among GCs at three time points. X-axis represents adoptively transferred mice groups with different precursor frequency (1 in 104, 2 in 105, 3 in 106) at three time point, y axis represents CD45.2 frequency among GC. Each circle represents one mouse. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. Bars indicate mean ± SD from mice in each group.(C) IgG titers detected by ELISA. eOD-GT8 or eOD-GT8 KO soluble proteins were used to coat ELISA plates. Sera from adoptively transferred mice at different time points were used to detect by ELISA. Bars indicate geometric mean and geometric SD from mice in each group. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. X-axis represents precursor frequency, and Y-axis represents the change of area under curve (AUC coated eOD-GT8 -AUC coated eOD-GT8 KO). Each circle represents one mouse.


Figure 1B . BrdU - sensitized U - 2 - OS cells stably expressing GFP - NLS - CDKL5 were pre - incubated with DMSO ( mock ) , olaparib ( 5 μM ) , talazoparib ( 50 nM ) or PDD00017273 ( 0 . 3 μM ) for 1 h prior to micro - irradiation and live - imaging at the indicated times post - irradiation . One of three independent experiments is shown . Scale bar is 10 μmE . BrdU - sensitized parental or PARP1Δ / Δ , PARP2Δ / Δ , PARP1 / 2Δ / Δ U - 2 - OS cells transiently expressing GFP NLS - CDKL5 were subjected to 355 nm line micro - irradiation followed by time lapse imaging . One of two independent experiments is shown . Scale bar is 10 μmG . Cells subjected to the workflow in F . were detergent - extracted and fixed before staining with anti - GFP or fibrillarin ( nucleoli ) . Scale bar is 10 μm H . Quantification of the detergent - insoluble GFP - NLS - CDKL5 signal ( minus nucleolar signal ) . The mean ± SD from three biological experiments is shown . Statistical significance was assessed by one - way - ANOVA - test . Asterisks * * indicate P - value of < 0 . 01 ; ns - not significant .

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Figure 1B. BrdU-sensitized U-2-OS cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM), talazoparib (50 nM) or PDD00017273 (0.3 μM) for 1 h prior to micro-irradiation and live-imaging at the indicated times post-irradiation. One of three independent experiments is shown. Scale bar is 10 μmE. BrdU-sensitized parental or PARP1Δ/Δ, PARP2Δ/Δ, PARP1/2Δ/Δ U-2-OS cells transiently expressing GFP NLS-CDKL5 were subjected to 355 nm line micro-irradiation followed by time lapse imaging. One of two independent experiments is shown. Scale bar is 10 μmG. Cells subjected to the workflow in F. were detergent-extracted and fixed before staining with anti-GFP or fibrillarin (nucleoli). Scale bar is 10 μm H. Quantification of the detergent-insoluble GFP-NLS-CDKL5 signal (minus nucleolar signal). The mean ± SD from three biological experiments is shown. Statistical significance was assessed by one-way-ANOVA-test. Asterisks ** indicate P-value of <0.01; ns - not significant.


Figure 2B . BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS , the GFP - NLS - CDKL5 deletion mutants shown in A , or full length ( FL ) GFP - NLS - CDKL5 were subjected to line micro - irradiation ( 355 nm ) and time lapse imaging . Three independent experiments were performed , and one representative experiment is shown . Scale bar is 10 μmD . Same as in B except that the GFP - NLS tagged CDKL5 fragments indicated were examined . Scale bar is 10 μmF . Recombinant fragments of CDKL5 fused to GST ( 1 . 2 , 2 . 5 , 5 , 10 µg ) , or GST , were dot - blotted on nitrocellulose membrane and then incubated with synthetic PAR . PAR binding was detected by far western blotting . APLF was used as positive control . One of three independent experiments is shown ( G - H ) . U - 2 - OS ( Flp - In T - Rex ) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min . Extracts were subjected to immunoprecipitation with antibodies against CDKL5 ( G ) or PAR ( H ) ( or non - specific IgG as control ) . Precipitates , and input lysates , were analyzed by western blotting using the indicated antibodies . One of two independent experiments is

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Figure 2B. BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS, the GFP-NLS-CDKL5 deletion mutants shown in A, or full length (FL) GFP-NLS-CDKL5 were subjected to line micro-irradiation (355 nm) and time lapse imaging. Three independent experiments were performed, and one representative experiment is shown. Scale bar is 10 μmD. Same as in B except that the GFP-NLS tagged CDKL5 fragments indicated were examined. Scale bar is 10 μmF. Recombinant fragments of CDKL5 fused to GST (1.2, 2.5, 5, 10 µg), or GST, were dot-blotted on nitrocellulose membrane and then incubated with synthetic PAR. PAR binding was detected by far western blotting. APLF was used as positive control. One of three independent experiments is shown(G-H) .U-2-OS (Flp-In T-Rex) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min. Extracts were subjected to immunoprecipitation with antibodies against CDKL5 (G) or PAR (H) (or non-specific IgG as control). Precipitates, and input lysates, were analyzed by western blotting using the indicated antibodies. One of two independent experiments is shown


Figure 3A . ( Top ) BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS - CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro - irradiation ( 405 nm ) . ( Bottom ) Quantitation of spot intensities . Data represent the mean ± SEM of two independent experiments ; > 50 micro - irradiated cells per point . Scale bar is 10 μmBrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS - CDKL5 , mCherry - XRCC1 or mCherry - FAN1 were pre - incubated with indicated transcription inhibitors prior to line micro - irradiation ( 355 nm ) and time lapse imaging . One of three independent experiments is shown . Scale bar is 10 μmE . Same as B - D . except that BrdU - sensitized cells stably expressing GFP - NLS - CDKL5 were also pre - incubated with olaparib as control . Cells were subjected to line micro - irradiation , fixed and then subjected to indirect immunofluorescence using antibodies against GFP , PAR and γH2AX . Scale bar is 10 μmF . Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging . Scale bar is 10

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Figure 3A. (Top) BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro-irradiation (405 nm). (Bottom) Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point. Scale bar is 10 μmBrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μmE. Same as B-D. except that BrdU-sensitized cells stably expressing GFP-NLS-CDKL5 were also pre-incubated with olaparib as control. Cells were subjected to line micro-irradiation, fixed and then subjected to indirect immunofluorescence using antibodies against GFP, PAR and γH2AX. Scale bar is 10 μmF. Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging. Scale bar is 10 μm


Figure 5B . Boxplots showing VSN - normalised intensity of phospho - peptides corresponding to EP400 pSer729 , ELOA pSer311and TTDN1 pSer40 from the experiment in A . The central band of the boxplot indicates the median value , while the hinges represent the first and third quartile ( bottom and top of boxplot , respectively ) . The whiskers extend to the largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times the interquartile range from their respective hinge . In all cases the data was derived from 3 biological replicates . C . Boxplots of the VSN - adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG - EP400 , FLAG - ELOA and FLAG - TTDN1 from the experiment in A . The central band of the boxplot indicates the median value , while the hinges represent the first and third quartile ( bottom and top of boxplot , respectively ) . The whiskers extend to the largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times the interquartile range from their respective hinge . Datapoints further removed than the whiskers are plotted individually . The experiment was conducted using three biological replicates within each respective group , each TMT channel represents a single biological replicate .

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 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

Figure 5B. Boxplots showing VSN-normalised intensity of phospho-peptides corresponding to EP400 pSer729, ELOA pSer311and TTDN1 pSer40 from the experiment in A. The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. In all cases the data was derived from 3 biological replicates.C. Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG-EP400, FLAG-ELOA and FLAG-TTDN1 from the experiment in A.The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. Datapoints further removed than the whiskers are plotted individually. The experiment was conducted using three biological replicates within each respective group, each TMT channel represents a single biological replicate.


Figure 6A . HEK293 cells were co - transfected with CDKL5 ( wild type " WT " or kinase - dead " KD " K42R mutant ) fused to an NLS , and FLAG - ELOA ( wild type " WT " or a S311A mutant " SA " ) . Anti - FLAG precipitates or cell extracts were probed with the antibodies indicated . One of three independent experiments is shown . B . Same as A . showing a range of pathogenic ( red ) and benign ( blue ) CDKL5 variants . ( C - E ) . Wild type ( WT ) , CDKL5 - disrupted ( CDKL5Δ / Δ ) or siRNA - transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks . Quantification of ELOA - pSer311 signal at the laser tracks is shown . Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated ( n ) . For simplicity , only intensities greater than zero are shown . Statistical significance was assessed by one - way - ANOVA - test or unpaired t test with Welch ' s correction . Asterisks * * * * indicate P - values of < 0 . 0001 . Scale bar is 10

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 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

Figure 6A. HEK293 cells were co-transfected with CDKL5 (wild type "WT" or kinase-dead "KD" K42R mutant) fused to an NLS, and FLAG-ELOA (wild type "WT" or a S311A mutant "SA"). Anti-FLAG precipitates or cell extracts were probed with the antibodies indicated. One of three independent experiments is shown. B. Same as A. showing a range of pathogenic (red) and benign (blue) CDKL5 variants. (C-E). Wild type (WT), CDKL5-disrupted (CDKL5Δ/Δ) or siRNA-transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks. Quantification of ELOA-pSer311 signal at the laser tracks is shown. Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated (n). For simplicity, only intensities greater than zero are shown. Statistical significance was assessed by one-way-ANOVA-test or unpaired t test with Welch's correction. Asterisks **** indicate P-values of <0.0001. Scale bar is 10 μm


Figure 7D . ( Left ) Quantification of transcription in U - 2 - OS 263 IFII reporter cells from experiment in B . > 150 cells were analysed per condition per experiment . The mean ± SD from four independent experiments is shown . ( Right ) Quantitative RT - PCR analysis of YFP - MS2 mRNA in U - 2 - OS 263 reporter cells . Data represent mean ± SD in different biological replicates as indicated ( n ) . Statistical significance for all the data was assessed by two - way - ANOVA - test , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; ns - not significant .

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

Figure 7D. (Left) Quantification of transcription in U-2-OS 263 IFII reporter cells from experiment in B. >150 cells were analysed per condition per experiment. The mean ± SD from four independent experiments is shown. (Right) Quantitative RT-PCR analysis of YFP-MS2 mRNA in U-2-OS 263 reporter cells. Data represent mean ± SD in different biological replicates as indicated (n). Statistical significance for all the data was assessed by two-way-ANOVA-test, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant.


Figure 8B , C . Quantitative PCR with reverse transcription ( qRT - PCR ) analysis of SLCO5a1 ( B ) and RYR2 expression levels ( C ) ( left panels ) U - 2 - OS HA - ER - I - PpoI cells depleted of CDKL5 transiently transfected with FLAG - tagged CDKL5 wild type ( WT ) or a K42R - mutated kinase dead ( KD ) mutant , or empty vector , at the times indicated after inducing I - PpoI . The mean ± SD from two qPCR replicates of two independent experiments is shown . Statistical significance for all the data was assessed by two - way - ANOVA - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; ns - not significant . I - PpoI - mediated cutting efficiency in the relevant gene is shown in the right - hand

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

Figure 8B, C. Quantitative PCR with reverse transcription (qRT-PCR) analysis of SLCO5a1 (B) and RYR2 expression levels (C) (left panels) U-2-OS HA-ER-I-PpoI cells depleted of CDKL5 transiently transfected with FLAG-tagged CDKL5 wild type (WT) or a K42R-mutated kinase dead (KD) mutant, or empty vector, at the times indicated after inducing I-PpoI. The mean ± SD from two qPCR replicates of two independent experiments is shown. Statistical significance for all the data was assessed by two-way-ANOVA-test *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant. I-PpoI-mediated cutting efficiency in the relevant gene is shown in the right-hand panel


Figure 1A ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles . Intracellular bacteria were enumerated by their ability to form colonies on agar plates following cell lysis at the indicated time points . Statistical analysis comparing Tbk1 - / - MEFs and cells expressing TBK1 alleles . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates . B ) Replication of S . Typhimurium in Atg5 - / - MEFs cells complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 ( 10 nM ) or DMSO . Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post inoculation ( p . i . ) following cell lysis . C ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium . Percentage of S . Typhimurium coated with ubiquitin ( detected by FK2 antibody ) at 1 or 4 h p . i . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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 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

Figure 1A) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs stably expressing the indicated TBK1 alleles. Intracellular bacteria were enumerated by their ability to form colonies on agar plates following cell lysis at the indicated time points. Statistical analysis comparing Tbk1-/- MEFs and cells expressing TBK1 alleles. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.B) Replication of S. Typhimurium in Atg5-/- MEFs cells complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 (10 nM) or DMSO. Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post inoculation (p.i.) following cell lysis.C) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium. Percentage of S. Typhimurium coated with ubiquitin (detected by FK2 antibody) at 1 or 4 h p.i. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


Figure 2A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : LC3B ( A ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM . A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with the indicated GFP : WIPI alleles ( B ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM . A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S . Typhimurium ( C - H ) . A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : DFCP1 ( FYVE * denotes a PI ( 3 ) P - binding mutant of DFCP1 ) ( D - E ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM . A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S . Typhimurium ( C - H ) . A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : ML1N * 2 , a probe for PI ( 3 , 5 ) P2 ( G ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM . A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S . Typhimurium ( C - H ) .

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 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 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 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

Figure 2A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:LC3B (A). Where indicated, Wortmannin (Wort) was added at 100 nM.A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with the indicated GFP:WIPI alleles (B). Where indicated, Wortmannin (Wort) was added at 100 nM.A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S. Typhimurium (C-H).A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:DFCP1 (FYVE* denotes a PI(3)P-binding mutant of DFCP1) (D-E). Where indicated, Wortmannin (Wort) was added at 100 nM.A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S. Typhimurium (C-H).A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:ML1N*2, a probe for PI(3,5)P2 (G). Where indicated, Wortmannin (Wort) was added at 100 nM.A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S. Typhimurium (C-H).


Figure 3A - B ) Kinetics of S . Typhimurium replication in MEFs depleted of WIPI1 ( A ) . Intracellular bacteria were enumerated by their ability to form colonies on agar plates . Western blot for GFP : WIPI1 upon the indicated siRNA treatments . A - B ) Kinetics of S . Typhimurium replication in MEFs depleted of WIPI2 ( B ) . Intracellular bacteria were enumerated by their ability to form colonies on agar plates . quantitative RT - PCR for WIPI2 upon the indicated siRNA treatments .

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

Figure 3A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI1 (A). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. Western blot for GFP:WIPI1 upon the indicated siRNA treatments.A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI2 (B). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. quantitative RT-PCR for WIPI2 upon the indicated siRNA treatments.


Figure 4A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . S . Typhimurium replication kinetics ( A , D ) . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical significance to Tbk1 - / - MEFs expressing TBK1ΔC is indicated . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates . A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . ( B ) Percentage of GFP : WIPI1 positive S . Typhimurium at 1 h p . i . in the indicated complemented MEFs . Mean and s . e . m . of four independent experiments . > 200 bacteria counted per coverslip . * p < 0 . 05 , * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test . A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . S . Typhimurium replication kinetics ( A , D ) . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical significance to Tbk1 - / - MEFs expressing TBK1ΔC is indicated . Western blot for Flag - tagged TBK1 variants in post nuclearcell lysates .

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 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 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

Figure 4A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of four independent experiments. >200 bacteria counted per coverslip. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test.A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclearcell lysates.


Figure 5E ) Lumier binding assay . Binding of the indicated luciferase - tagged NDP52 variants to beads coated with GST , GST : galectin - 8 , or GST : ubiquitin . Coomassie stain of purified GST proteins . Western blot for luciferase - tagged NDP52 alleles in total cell lysates . F ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - NDP52 fusion proteins . Infected cells were lysed at the indicated times post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical comparison with Tbk1 - / - MEFs expressing TBK1ΔC is indicated . * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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 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

Figure 5E) Lumier binding assay. Binding of the indicated luciferase-tagged NDP52 variants to beads coated with GST, GST:galectin-8, or GST:ubiquitin. Coomassie stain of purified GST proteins. Western blot for luciferase-tagged NDP52 alleles in total cell lysates.F) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-NDP52 fusion proteins. Infected cells were lysed at the indicated times post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical comparison with Tbk1-/- MEFs expressing TBK1ΔC is indicated. ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


Figure 6A - B ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - galectin fusion proteins . S . Typhimurium replication kinetics ( A ) . Infected cells were lysed at the indicated times post inoculation ( p . i . ) . and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 05 , * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates . A - B ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - galectin fusion proteins . ( B ) Percentage of GFP : WIPI1 positive S . Typhimurium at 1 h p . i . in the indicated complemented MEFs . Mean and s . e . m . of three independent experiments . > 200 bacteria counted per coverslip . * p < 0 . 05 , one - way ANOVA with Dunnett ' s multiple comparisons 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 O O O O O O O O O O O O O O O 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

Figure 6A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. S. Typhimurium replication kinetics (A). Infected cells were lysed at the indicated times post inoculation (p.i.). and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of three independent experiments. >200 bacteria counted per coverslip. *p<0.05, one-way ANOVA with Dunnett's multiple comparisons test.