( C , D ) Total lipids extracted from the indicated endolysosomal membrane samples ( 30 μg of proteins ) were either ( C ) subjected to a phosphate assay to quantify the bulk of phospholipids or ( D ) separated by TLC . Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining . CHOL , cholesterol ; LBPA lysobisphosphatidic acid ; PC , phosphatidylcoline ; PE phosphatidylethanolamine ; PI phosphatidyinositol ; SM sphingomyelin . ( A , C ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus MSD and WT versus MPS ‐ IIIA . Scale bar , 10 μm ( B ) .

O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O

(C, D) Total lipids extracted from the indicated endolysosomal membrane samples (30 μg of proteins) were either (C) subjected to a phosphate assay to quantify the bulk of phospholipids or (D) separated by TLC. Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining. CHOL, cholesterol; LBPA lysobisphosphatidic acid; PC, phosphatidylcoline; PE phosphatidylethanolamine; PI phosphatidyinositol; SM sphingomyelin. (A, C) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus MSD and WT versus MPS‐IIIA. Scale bar, 10 μm (B).


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

B-SMALL_MOLECULE O O O O O B-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O

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


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

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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


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 ) .

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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).


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( 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 .

O O O B-SUBCELLULAR I-SUBCELLULAR O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(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 .

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(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 .

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(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 O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O

(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).


( 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 .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-ORGANISM O B-ORGANISM O O B-CELL_LINE O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_LINE O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O

(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 .

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(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 .

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(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 ) .

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(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 .

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(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 O O O O B-GENEPROD O O O B-ORGANISM B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O

(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).


( 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 ) .

O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O B-SMALL_MOLECULE O O O B-ORGANISM B-CELL_LINE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O 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-ORGANISM O O O O O B-SMALL_MOLECULE O B-ORGANISM O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O B-ORGANISM O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O

(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 .

O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O

(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 .

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(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 O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE O

(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.


( 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 . ) .

O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O

(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 .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O

(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 O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O B-SMALL_MOLECULE O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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.


( A ) Blood ammonia in 8 - 9 - week - old C57BL / 6J wild - type ( WT ) mice ( n = 8 ) and Becn1F121A mice with constitutive activation of autophagy ( n = 8 ) at baseline and 30 min after i . p . injection of NH4Cl ( 10 mmol / kg ) . * * p < 0 . 01 ( Unpaired t - test ) . ns : not statistically significant difference .

O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Blood ammonia in 8-9-week-old C57BL/6J wild-type (WT) mice (n=8) and Becn1F121A mice with constitutive activation of autophagy (n=8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **p<0.01 (Unpaired t-test). ns: not statistically significant difference.


( B ) Urinary orotic acid of 12 - week - old spf - ash mice treated with TB - 1 ( 15 mg / kg , i . p ) or vehicle at various times as indicated by the arrows ( n = 5 mice / group ) . * p < 0 . 05 ( Two - way ANOVA ) .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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-ORGANISM O O O O O O O O O O O O O O O O O

(B) Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n=5 mice/group). *p<0.05 (Two-way ANOVA).


( C ; D ) Western blotting and densitometric quantifications of autophagy markers ( LC3II : autophagosomes ; p62 and NBR1 : cargo receptors ) in livers of spf - ash mice harvested after ten days of treatment with TB - 1 or vehicle . β - actin was used as loading control . n = 5 mice / group . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - test ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

(C; D) Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after ten days of treatment with TB-1 or vehicle. β-actin was used as loading control. n=5 mice/group. **p<0.01, *p<0.05 (Unpaired t-test).


( E ) Survival curves of spf - ash mice fed with a high protein diet ( HPD ) for ten days and treated with TB - 1 alone or combined with scavenger drug and L - Arginine , or treated with scavenger drug and L - Arginine , or left untreated . WT control were included ( n = 5 / group ) . * * * p < 0 . 001 ( Log - rank Mantel - Cox test ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) and L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were age - , gender - and strain ( C3H ) - matched . All values are shown as averages ± S . E . M .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Survival curves of spf-ash mice fed with a high protein diet (HPD) for ten days and treated with TB-1 alone or combined with scavenger drug and L-Arginine, or treated with scavenger drug and L-Arginine, or left untreated. WT control were included (n=5/group). ***p<0.001 (Log-rank Mantel-Cox test). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


( F ) Blood ammonia levels determined after 4 days under HPD ( n = 5 mice / group ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) and L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were age - , gender - and strain ( C3H ) - matched . All values are shown as averages ± S . E . M .

O O O B-TISSUE B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Blood ammonia levels determined after 4 days under HPD (n=5 mice/group); ***p<0.001, **p<0.01, *p<0.05 (One-way ANOVA). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


( A ) Survival curves of AslNeo / Neo mice and age - matched wild - type ( WT ) controls treated with TB - 1 ( 15 mg / kg , i . p . , every 48 hours starting at day 10 of age ) or vehicle . WT + Vehicle n = 8 ; WT + TB - 1 n = 8 ; AslNeo / Neo + Vehicle n = 20 ; AslNeo / Neo + TB - 1 n = 16 . * p < 0 . 05 ( Log - rank Mantel - Cox test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Survival curves of AslNeo/Neo mice and age-matched wild-type (WT) controls treated with TB-1 (15 mg/kg, i.p., every 48 hours starting at day 10 of age) or vehicle. WT + Vehicle n=8; WT + TB-1 n=8; AslNeo/Neo + Vehicle n=20; AslNeo/Neo + TB-1 n=16. *p<0.05 (Log-rank Mantel-Cox test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( B ) Isotopic enrichment of 15N - labeled urea in blood , 20 min after i . p . injection of 15NH4Cl tracer ( 4 mmol / kg ) in AslNeo / Neo mice treated with TB - 1 ( n = 15 ) or vehicle ( n = 9 ) . * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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) Isotopic enrichment of 15N-labeled urea in blood, 20 min after i.p. injection of 15NH4Cl tracer (4 mmol/kg) in AslNeo/Neo mice treated with TB-1 (n=15) or vehicle (n=9). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( C ) Representative Western blotting bands of LC3 , p62 and NBR1 in livers of AslNeo / Neo mice treated with TB - 1 or vehicle . GAPDH and β - actin were used as loading controls . ( D ) Densitometric quantifications . AslNeo / Neo + Vehicle n = 5 ; AslNeo / Neo + TB - 1 n = 9 . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative Western blotting bands of LC3, p62 and NBR1 in livers of AslNeo/Neo mice treated with TB-1 or vehicle. GAPDH and β-actin were used as loading controls. (D) Densitometric quantifications. AslNeo/Neo + Vehicle n=5; AslNeo/Neo + TB-1 n=9. **p<0.01, *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( E ) Argininosuccinate in dried blood spots ( DBS ) of WT and AslNeo / Neo mice injected with TB - 1 or vehicle ( n = 4 - 14 mice / group ) . * * * p < 0 . 001 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Argininosuccinate in dried blood spots (DBS) of WT and AslNeo/Neo mice injected with TB-1 or vehicle (n=4-14 mice/group). ***p<0.001, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( F ) Isotopic enrichment of 15N - labeled argininosuccinate in livers of AslNeo / Neo mice treated with TB - 1 ( n = 11 ) or vehicle ( n = 5 ) . * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-TISSUE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Isotopic enrichment of 15N-labeled argininosuccinate in livers of AslNeo/Neo mice treated with TB-1 (n=11) or vehicle (n=5). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( A ) Hematoxylin and eosin ( H & E , upper panels ) and periodic acid Schiff ( PAS , lower panels ) staining of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bars : 500 µm .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(A) Hematoxylin and eosin (H&E, upper panels) and periodic acid Schiff (PAS, lower panels) staining of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bars: 500 µm.


( B ) Computational analysis of PAS staining ( n ≥ 4 mice / group ) . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O 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) Computational analysis of PAS staining (n≥4 mice/group). **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.


( C ) Quantification of hepatic glycogen in vehicle ( n = 6 ) - and TB - 1 - treated AslNeo / Neo mice ( n = 12 ) compared to WT ( n = 5 ) controls . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M .

O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of hepatic glycogen in vehicle (n=6)- and TB-1-treated AslNeo/Neo mice (n=12) compared to WT (n=5) controls. **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.


( D ) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo / Neo mice treated with either with TB - 1 or vehicle ( n = 4 mice / group ) . * p < 0 . 05 ( One - way ANOVA ) . GAPDH was used as loading control . Data information : All values are shown as averages ± S . E . M .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(D) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo/Neo mice treated with either with TB-1 or vehicle (n=4 mice/group). *p<0.05 (One-way ANOVA). GAPDH was used as loading control. Data information: All values are shown as averages ± S.E.M.


( E ) Representative electron microscopy images of liver samples harvested from WT and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bar : 900 nm .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(E) Representative electron microscopy images of liver samples harvested from WT and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bar: 900 nm.


( A ) Representative electron microscopy images of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . False color on the images indicates glycogen within the nuclei . Scale bar : 1 μm . ( B ) Quantification of nuclei containing glycogen ( approx . 180 nuclei were analyzed in total per condition , n = 3 mice / group ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O B-ORGANISM O O O O

(A) Representative electron microscopy images of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. False color on the images indicates glycogen within the nuclei. Scale bar: 1 μm. (B) Quantification of nuclei containing glycogen (approx. 180 nuclei were analyzed in total per condition, n=3 mice/group).


( A ) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h . Then , WB was used to measure p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein expression . " Nor " = normal medium . Actin served as the loading control . The red text " - Cys " indicates that cystine deficiency most significantly activates AMPK and its substrates .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O

(A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h. Then, WB was used to measure p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein expression. "Nor" = normal medium. Actin served as the loading control. The red text "-Cys" indicates that cystine deficiency most significantly activates AMPK and its substrates.


( F ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein in 293T cells transfected with shRNAs targeting LKB1 , CaMKK2 , TAK1 or a non - targeting control ( NTC ) that were treated with cystine - deficient medium for 8 h . Actin served as the loading control .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O

(F) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells transfected with shRNAs targeting LKB1, CaMKK2, TAK1 or a non-targeting control (NTC) that were treated with cystine-deficient medium for 8 h. Actin served as the loading control.


( I ) 293T cells cultured with cystine - deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti - AMPKα , followed by WB analysis with anti - AMPKα and anti - CaMKK2 .

O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O

(I) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-AMPKα, followed by WB analysis with anti-AMPKα and anti-CaMKK2.


( E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK ACC , Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine - deficient medium for 8 h . Actin served as the loading control .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O

(E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine-deficient medium for 8 h. Actin served as the loading control.


( J ) WB analysis of p - AMPK and total AMPK protein expression in CARS - overexpressing 293T cells and RCC4 cells that were treated with 1 µg / ml STO - 609 or DMSO for 8 h . Actin served as the loading control .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O

(J) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO-609 or DMSO for 8 h. Actin served as the loading control.


( D , E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor , AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine - deficient medium for 8 h ( D ) or transfected with Flag - CARS for 48 h ( E ) . Actin served as the loading control .

O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O

(D, E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine-deficient medium for 8 h (D) or transfected with Flag-CARS for 48 h (E). Actin served as the loading control.


( F ) 293T cells were transfected with HA - AMPKγ2 alone or with Flag - CARS for 48 h and then cultured with cystine - deficient medium or complete medium for 8 h . Cell lysates were immunoprecipitated with anti - Flag , and a WB analysis was performed .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O

(F) 293T cells were transfected with HA-AMPKγ2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed.


( B ) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


( C , D ) The crystal violet assay ( C ) and apoptosis rate assay ( D ) were performed in 293T cells transfected with shRNAs targeting NTC , CARS , CaMKK2 or AMPKγ2 and further treated with cystine - deficient medium for 24 h with or without 1 mM AICAR . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) The crystal violet assay (C) and apoptosis rate assay (D) were performed in 293T cells transfected with shRNAs targeting NTC, CARS, CaMKK2 or AMPKγ2 and further treated with cystine-deficient medium for 24 h with or without 1 mM AICAR. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


( F WB analysis of p - AMPK , total AMPK , CARS , AMPKγ2 , and CaMKK2 protein expression with 786 - O cells transfected with Flag - EV , Flag - CARS , Flag - CaMKK2 or Flag - AMPKγ2 and further treated with cystine - deficient medium for 8 h . Actin served as the loading control . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F WB analysis of p-AMPK, total AMPK, CARS, AMPKγ2, and CaMKK2 protein expression with 786-O cells transfected with Flag-EV, Flag-CARS, Flag-CaMKK2 or Flag-AMPKγ2 and further treated with cystine-deficient medium for 8 h. Actin served as the loading control. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) produced with lopinavir ( LPV , 0 - 100 nM ) detecting p24 antibody .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O

(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24 antibody.


Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated THP - 1 shSAMHD1 cells . Mean ± SD , n = 3 .

O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O

Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 cells. Mean ± SD, n=3.


Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated U87 cells . Mean ± SD , n = 3 .

O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O

Titration of LPV-treated HIV-1 GFP viruses on PMA-treated U87 cells. Mean ± SD, n=3.


ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . G ) CXCL - 10 protein in cell supernatants from D - F ( ELISA ) .

B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O

PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). G) CXCL-10 protein in cell supernatants from D-F (ELISA).


( H ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 2 U RT / ml 30 nM LPV - treated HIV - 1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib . A control was stimulated with 1 ng / ml IFNβ .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O

(H) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ.


( I ) RT products from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 3 U RT / ml LPV - treated HIV - 1 GFP viruses .

O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O

(I) RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses.


( J ) Immunoblot of HIV - 1 R9 BaL virus particles ( 2 × 1011 genomes ) produced with LPV ( 0 - 100 nM ) detecting p24 .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O

(J) Immunoblot of HIV-1 R9 BaL virus particles (2×1011 genomes) produced with LPV (0-100 nM) detecting p24.


( K , L ) ISG qRT - PCR ( K ) and infection data ( L ) from primary monocyte - derived macrophages ( MDM ) infected for 24 h with LPV - treated HIV - 1 R9 BaL viruses ( 0 . 2 U RT / ml ) . Data are collated from two donors ( represented by circles and squares ) , n = 3 . Horizontal line represents the median . For experiments in which the virus dose used was normalised by RT activity , the number of genome copies was also measured by qPCR of virus . This gave dose equivalents of within 2 - 3 fold of RT equivalents .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O

(K, L) ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDM) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n=3. Horizontal line represents the median. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within 2-3 fold of RT equivalents.


( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) with varying proportions of ΔCA - SP1 protease cleavage mutation detecting p24 .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O

(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) with varying proportions of ΔCA-SP1 protease cleavage mutation detecting p24.


( B ) Titration of HIV - 1 GFP ΔCA - SP1 viruses on U87 cells . Mean ± SD , n = 3 .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O

(B) Titration of HIV-1 GFP ΔCA-SP1 viruses on U87 cells. Mean ± SD, n=3.


( C ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(C ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


D ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

D) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . E ) CXCL - 10 protein in supernatants from ( C , D ) ( ELISA ) .

B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O

PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). E) CXCL-10 protein in supernatants from (C, D) (ELISA).


( F ) RT products from THP - 1 cells transduced for 24 h with 6 × 109 genomes / ml ( approx . 0 . 5 U RT / ml ) HIV - 1 GFP ΔCA - SP1 viruses .

O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O

(F) RT products from THP-1 cells transduced for 24 h with 6×109 genomes/ml (approx. 0.5 U RT/ml) HIV-1 GFP ΔCA-SP1 viruses.


( G ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 016 - 0 . 2 U RT / ml ) . Data are shown as individual measurements , representative of 2 repeats .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.016 - 0.2 U RT/ml). Data are shown as individual measurements, representative of 2 repeats.


( H ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 mutant , or stimulated with 4 μg / ml cGAMP as a control , in the presence of DMSO vehicle or 2 μM ruxolitinib .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O

(H) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 mutant, or stimulated with 4 μg/ml cGAMP as a control, in the presence of DMSO vehicle or 2 μM ruxolitinib.


( I ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 3 × 108 genomes / ml or 1 . 5 × 109 genomes / ml , equivalent to 0 . 1 U RT / ml and 0 . 5 U RT / ml ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (3×108 genomes/ml or 1.5×109 genomes/ml, equivalent to 0.1 U RT/ml and 0.5 U RT/ml).


( J ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O

(J) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib.


primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib . K ) CXCL - 10 protein in supernatants from ( J ) ( ELISA ) .

O B-CELL_TYPE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O

primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib. K) CXCL-10 protein in supernatants from (J) (ELISA).


( A ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

(A) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.


THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . B ) CXCL - 10 protein in supernatants from ( A ) ( ELISA ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O

THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir. B) CXCL-10 protein in supernatants from (A) (ELISA).


ISG qRT - PCR from THP - 1 - IFIT - 1 cells transduced for 24 h with 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 109 and 3 × 109 genomes / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.


( E ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ΔCA - SP1 ( WT ) , 75 % ΔCA - SP1 , 75 % ΔCA - SP1 carrying a mutation in reverse transcriptase ( 75 % ΔCA - SP1 RT D185E ) or 75 % ΔCA - SP1 carrying a mutation in integrase ( 75 % ΔCA - SP1 INT D116N ) ( 3 . 75 × 109 , 7 . 5 × 109 and 1 . 5 × 1010 genomes / ml ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(E) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % ΔCA-SP1 (WT), 75 % ΔCA-SP1, 75 % ΔCA-SP1 carrying a mutation in reverse transcriptase (75 % ΔCA-SP1 RT D185E) or 75 % ΔCA-SP1 carrying a mutation in integrase (75 % ΔCA-SP1 INT D116N) (3.75×109, 7.5×109 and 1.5×1010 genomes/ml).


( F ) CXCL - 10 protein in supernatants from ( E ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

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(F) CXCL-10 protein in supernatants from (E) (ELISA). Triangles indicate CXCL-10 not detected.


( A ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING or MAVS , or a gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP 75 % ΔCA - SP1 ( 0 . 4 U RT / ml ) or stimulated by transfection with either HT - DNA ( 0 . 1 μg / ml ) or poly I : C ( 0 . 5 μg / ml ) .

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(A) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING or MAVS, or a gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP 75 % ΔCA-SP1 (0.4 U RT/ml) or stimulated by transfection with either HT-DNA (0.1 μg/ml) or poly I:C (0.5 μg/ml).


IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line stimulated for 24 h with poly I : C ( transfection , 0 . 5 μg / ml ) , HT - DNA ( transfection , 0 . 1 μg / ml ) , LPS ( 50 ng / ml ) or cGAMP ( transfection , 0 . 5 μg / ml )

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IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line stimulated for 24 h with poly I:C (transfection, 0.5 μg/ml), HT-DNA (transfection, 0.1 μg/ml), LPS (50 ng/ml) or cGAMP (transfection, 0.5 μg/ml)


IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml )

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IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml)


PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml ) ( C ) . D ) CXCL - 10 protein in supernatants from ( C ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

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PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml) (C). D) CXCL-10 protein in supernatants from (C) (ELISA). Triangles indicate CXCL-10 not detected.


( E ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) .

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(E) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).


( F ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) .

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(F) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).


PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . G ) CXCL - 10 protein in supernatants from ( E ) .

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PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). G) CXCL-10 protein in supernatants from (E).


PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . ( H ) CXCL - 10 protein in supernatants from ( F ) ( ELISA ) .

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PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). (H) CXCL-10 protein in supernatants from (F) (ELISA).


( I ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 control cells transduced for 48 h with WT , DRV - treated ( DRV , 12 . 5 nM ) or HIV - 1 GFP containing 90 % ΔCA - SP1 ( 1 × 1010 genomes / ml ) in the presence of DMSO vehicle , 2 μM ruxolitinib or 0 . 5 μg / ml H151 .

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(I) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 control cells transduced for 48 h with WT, DRV-treated (DRV, 12.5 nM) or HIV-1 GFP containing 90 % ΔCA-SP1 (1×1010 genomes/ml) in the presence of DMSO vehicle, 2 μM ruxolitinib or 0.5 μg/ml H151.


Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with a fixed dose of HIV - 1 GFP ( 5 × 107 genomes / ml ) and increasing doses of HIV - LUC ΔCA - SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O B-CELL_LINE O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with a fixed dose of HIV-1 GFP (5×107 genomes/ml) and increasing doses of HIV-LUC ΔCA-SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.


Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with LPV - treated HIV - LUC viruses Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O B-CELL_LINE O O O O B-GENEPROD O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with LPV-treated HIV-LUC viruses Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.


( A ) Infection data for THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(A) Infection data for THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).


THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . B ) IFIT - 1 reporter activity in supernatant from ( A ) .

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THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM). B) IFIT-1 reporter activity in supernatant from (A).


ISG qRT - PCR from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 25 - 1 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).


( E ) CXCL - 10 protein in supernatants of THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(E) CXCL-10 protein in supernatants of THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).


( F ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) and ruxolitinib ( Rux , 2 μM ) as indicated .

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(F) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM) and ruxolitinib (Rux, 2 μM) as indicated.


( G ) IRF reporter activity from THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP ( 3 U / ml and 6 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(G) IRF reporter activity from THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).


( H ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells lacking MAVS or a matching gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP ( 3 U / ml and 6 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(H) IFIT-1 reporter activity from THP-1-IFIT-1 cells lacking MAVS or a matching gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).


Depletion of RBM14 induces ectopic formation of centriolar protein complexesA - E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) ( A ) , Centrobin ( green ) and C - Nap1 ( magenta ) ( B ) , centrin - 2 ( green ) and CPAP ( magenta ) ( C ) , acetylated - tubulin ( green ) and CP110 ( magenta ) ( D ) , and centrin - 2 ( green ) and γ - tubulin ( magenta ) ( E ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition . Values are mean percentages ± standard error of mean ( SEM ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) . Note that we counted the number of C - Nap1 foci after anaphase in mitosis when C - Nap1 signals recover from the reduction in prometaphase .

O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O B-CELL_LINE O O B-CELL_LINE O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

Depletion of RBM14 induces ectopic formation of centriolar protein complexesA-E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta) (A), Centrobin (green) and C-Nap1 (magenta) (B), centrin-2 (green) and CPAP (magenta) (C), acetylated-tubulin (green) and CP110 (magenta) (D), and centrin-2 (green) and γ-tubulin (magenta) (E). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01, n.s., not significant (one-tailed t-test). Note that we counted the number of C-Nap1 foci after anaphase in mitosis when C-Nap1 signals recover from the reduction in prometaphase.


A U2OS cells or U2OS cells expressing FLAG - RBM14 full length ( FL , aa1 - 669 ) , N - terminal fragment ( RBM14 [ N ] , aa1 - 150 ) or C - terminal fragment ( RBM14 [ C ] , aa151 - 669 ) and treated with control siRNA or siRNA against 3 ′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin - 2 . Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition . Values are mean percentages ± SEM from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) .

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A U2OS cells or U2OS cells expressing FLAG-RBM14 full length (FL, aa1-669), N-terminal fragment (RBM14[N], aa1-150) or C-terminal fragment (RBM14[C], aa151-669) and treated with control siRNA or siRNA against 3′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin-2. Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).


C , D Cytoplasmic RBM14 could suppress centriole amplification in HU - treated cells . ( C ) U2OS cells or U2OS cells expressing FLAG - RBM14 FL or [ C ] and treated with or without HU were stained with antibodies against centrin - 2 ( green ) and FLAG ( magenta ) . ( D ) U2OS cells or U2OS cells expressing FLAG - RBM14 FL , FLAG - RBM14 - NES or GFP - RBM14 - PACT and treated with or without HU were stained with antibodies against FLAG ( magenta , left ) or GFP ( magenta , right ) as well as centrin - 2 ( green ) . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 10 μm . Histograms represent frequency of cells in interphase with excess centrin foci in each condition . The percentages of U2OS cells with centrosomal localization of the RBM14 full - length protein or mutants are shown below the immunofluorescence images . We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . Please note that cytoplasmic expression levels of GFP - RBM14 - PACT are less than those of FLAG - RBM14 FL ( ˜ 0 . 5 - fold ) or FLAG - RBM14 - NES ( ˜ 0 . 7 - fold ) .

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C, D Cytoplasmic RBM14 could suppress centriole amplification in HU-treated cells. (C) U2OS cells or U2OS cells expressing FLAG-RBM14 FL or [C] and treated with or without HU were stained with antibodies against centrin-2 (green) and FLAG (magenta). (D) U2OS cells or U2OS cells expressing FLAG-RBM14 FL, FLAG-RBM14-NES or GFP-RBM14-PACT and treated with or without HU were stained with antibodies against FLAG (magenta, left) or GFP (magenta, right) as well as centrin-2 (green). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 10 μm. Histograms represent frequency of cells in interphase with excess centrin foci in each condition. The percentages of U2OS cells with centrosomal localization of the RBM14 full-length protein or mutants are shown below the immunofluorescence images. We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Please note that cytoplasmic expression levels of GFP-RBM14-PACT are less than those of FLAG-RBM14 FL (˜0.5-fold) or FLAG-RBM14-NES (˜0.7-fold).


A HeLa cells immunoprecipitated with control IgG or STIL antibodies . Soluble cytosolic fractions ( input ) and immunoprecipitates ( IPs ) were analyzed by Western blotting using RBM14 , STIL or CPAP antibodies .

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A HeLa cells immunoprecipitated with control IgG or STIL antibodies. Soluble cytosolic fractions (input) and immunoprecipitates (IPs) were analyzed by Western blotting using RBM14, STIL or CPAP antibodies.


B GST pull - down assay testing interactions between purified STIL [ N ] ( ˜ 5 μg , aa1 - 1018 ) and GST - RBM14 [ N ] or [ C ] . The asterisks indicate non - specific bands .

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B GST pull-down assay testing interactions between purified STIL[N] (˜5 μg, aa1-1018) and GST-RBM14 [N] or [C]. The asterisks indicate non-specific bands.


D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and STIL ( magenta ) . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess STIL foci co - localized with centrin foci . Values are mean percentages ± SEM from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) .

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D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin-2 (green) and STIL (magenta). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess STIL foci co-localized with centrin foci. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01 (one-tailed t-test).


E In vitro competitive binding assay . GST pull - down experiment was performed as in ( B ) , with purified STIL [ N ] and GST - RBM14 [ C ] in the presence of the indicated amount of purified His - CPAP [ SBD ] , His - tagged STIL - Binding Domain of CPAP . The fraction of STIL [ N ] bound to GST - RBM14 [ C ] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N - terminal region of STIL . Input materials were analyzed by Western blotting using the STIL or CPAP antibodies . The precipitated GST - RBM14 [ C ] was analyzed by SDS - PAGE , stained with SimplyBlue ™ Safe ( Invitrogen ) .

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E In vitro competitive binding assay. GST pull-down experiment was performed as in (B), with purified STIL[N] and GST-RBM14[C] in the presence of the indicated amount of purified His-CPAP[SBD], His-tagged STIL-Binding Domain of CPAP. The fraction of STIL[N] bound to GST-RBM14[C] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N-terminal region of STIL. Input materials were analyzed by Western blotting using the STIL or CPAP antibodies. The precipitated GST-RBM14[C] was analyzed by SDS-PAGE, stained with SimplyBlue™ Safe (Invitrogen).


F Interaction between STIL and GFP - CPAP in the presence of FLAG - RBM14 FL or fragments . U2OS cells expressing GFP - CPAP were transfected with empty vector as a control , FLAG - RBM14 [ N ] , FLAG - RBM14 [ C ] or FLAG - RBM14 FL constructs , immunoprecipitated with STIL antibodies , and the resulting IPs were analyzed by Western blotting using STIL , CPAP or FLAG antibodies . Quantification of relative protein amounts of co - immunoprecipitated GFP - CPAP in each STIL - IP fraction , normalized with the amount of STIL precipitated , is shown below the panels . Means ± SEM were calculated from three independent experiments . * * P < 0 . 01 ( one - tailed t - test ) . Note that we did not find any significant effect of exogenous expression of RBM14 full - length protein or deletion mutants on the expression levels of GFP - CPAP or endogenous STIL proteins in this experiment .

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F Interaction between STIL and GFP-CPAP in the presence of FLAG-RBM14 FL or fragments. U2OS cells expressing GFP-CPAP were transfected with empty vector as a control, FLAG-RBM14[N], FLAG-RBM14[C] or FLAG-RBM14 FL constructs, immunoprecipitated with STIL antibodies, and the resulting IPs were analyzed by Western blotting using STIL, CPAP or FLAG antibodies. Quantification of relative protein amounts of co-immunoprecipitated GFP-CPAP in each STIL-IP fraction, normalized with the amount of STIL precipitated, is shown below the panels. Means ± SEM were calculated from three independent experiments. **P < 0.01 (one-tailed t-test). Note that we did not find any significant effect of exogenous expression of RBM14 full-length protein or deletion mutants on the expression levels of GFP-CPAP or endogenous STIL proteins in this experiment.


G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) . Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . Representative mitotic cells treated with siRNAs against RBM14 alone , RBM14 + STIL or RBM14 + CPAP are shown below . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm .

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G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta). Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Representative mitotic cells treated with siRNAs against RBM14 alone, RBM14 + STIL or RBM14 + CPAP are shown below. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm.