B , C Cell death measured by lactase dehydrogenase ( LDH ) ( B ) and cell viability measured by 3 - ( 4 , 5 - dimethyl - thiazol - . 2 - yl ) - 2 , 5 - diphenyl tetrazolium bromide ( MTT ) ( C ) in T98 cells 42 hrs post - transfection of pure and interrupted SCA8 repeat tracts ; LDH n = 8 , MTT n = 12 , n = independent experiments , * p < 0 . 05 , NT not transfected , EV : empty vector ; unpaired t test ; mean ± SEM .

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B, C Cell death measured by lactase dehydrogenase (LDH) (B) and cell viability measured by 3-(4,5- dimethyl-thiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (C) in T98 cells 42 hrs post-transfection of pure and interrupted SCA8 repeat tracts; LDH n=8, MTT n=12, n=independent experiments, * p < 0.05, NT not transfected, EV: empty vector; unpaired t test; mean ± SEM.


D , E qRT - PCR showing transcript levels of Pure 96 and Int . 95 ( D , n = 6 ; p = 0 . 0187 ) , and Pure 104 and Int . 102 ( E , n = 6 ; * * * * p < 0 . 0001 ) repeats ; n = independent experiments ; unpaired t test ; mean ± SEM .

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D, E qRT-PCR showing transcript levels of Pure 96 and Int.95 (D, n=6; p=0.0187), and Pure 104 and Int.102 (E, n=6; **** p<0.0001) repeats; n=independent experiments; unpaired t test; mean ± SEM.


B , C Cell death ( B ; LDH , n = 8 ) and viability ( C ; MTT , n = 6 ) assays in T98 cells 42 - hrs post - transfection . NT : not transfected ; EV : empty vector ; n = independent experiments ; * p < 0 . 05 ; unpaired t test ; mean ± SEM .

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B, C Cell death (B; LDH, n=8) and viability (C; MTT, n=6) assays in T98 cells 42-hrs post-transfection. NT: not transfected; EV: empty vector; n=independent experiments; * p < 0.05; unpaired t test; mean ± SEM.


Protein blots of polyGln proteins expressed in transfected HEK293T cells with interrupted or pure polyGln repeats ; EV : empty vector ; n = 3

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Protein blots of polyGln proteins expressed in transfected HEK293T cells with interrupted or pure polyGln repeats; EV: empty vector; n=3 transfections


Immunofluorescence of intranuclear inclusions for polyGln expressed from Alt . polyGln and Alt . Int . polyGln constructs in HEK293T cells ; scale bar : 50μm ; green arrow heads indicate cells positive for Alt . Int . polyGln intranuclear inclusions ; dashed white box in merge panels indicates the region show in the orthogonal ( Ortho ) zoomed images which show cross section and localization of diffuse polyGln staining and nuclear polyGln ( Arg ) inclusions with alpha tubulin as a cytoplasmic marker ; n = 4 experiments with n > 55 cells per

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Immunofluorescence of intranuclear inclusions for polyGln expressed from Alt. polyGln and Alt. Int. polyGln constructs in HEK293T cells; scale bar: 50μm; green arrow heads indicate cells positive for Alt. Int. polyGln intranuclear inclusions; dashed white box in merge panels indicates the region show in the orthogonal (Ortho) zoomed images which show cross section and localization of diffuse polyGln staining and nuclear polyGln(Arg) inclusions with alpha tubulin as a cytoplasmic marker; n=4 experiments with n>55 cells per experiment


K PolyGln ( Arg ) proteins but not pure polyGln proteins colocalize with nucleophosmin in HEK293T cells ; scale bars : 20µm ; dashed white box in merge panels indicates the region shown as an orthogonal projection in the ortho zoom panels which show the co - localization of polyGln inclusions and nucleophosmin in three dimensions .

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K PolyGln(Arg) proteins but not pure polyGln proteins colocalize with nucleophosmin in HEK293T cells; scale bars: 20µm; dashed white box in merge panels indicates the region shown as an orthogonal projection in the ortho zoom panels which show the co-localization of polyGln inclusions and nucleophosmin in three dimensions.


A - C Protein blotting ( A ) and quantification ( B , C ) of polySer RAN proteins in HEK293T cells from interrupted ( Int . 95 ; Int . 102 ) or pure ( Pure 96 ; Pure 104 ) CAG repeats . EV : empty vector , n = 3 transfections , * * p < 0 . 01 , * p < 0 . 05 , unpaired t test , mean ± SEM .

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A-C Protein blotting (A) and quantification (B, C) of polySer RAN proteins in HEK293T cells from interrupted (Int.95; Int.102) or pure (Pure 96; Pure 104) CAG repeats. EV: empty vector, n=3 transfections, ** p<0.01, *p<0.05, unpaired t test, mean ± SEM.


D , Immunofluorescence of RAN polySer protein aggregates from CGG interrupted and pure CAG repeat tracts in HEK293T cells ; scale bar : 10μm . n > 20 cells per construct for each experiment ( Exp . )

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D, Immunofluorescence of RAN polySer protein aggregates from CGG interrupted and pure CAG repeat tracts in HEK293T cells; scale bar: 10μm. n>20 cells per construct for each experiment (Exp.)


F , Protein blot of polyAla RAN proteins expressed from pure or interrupted constructs ; n = 3

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F, Protein blot of polyAla RAN proteins expressed from pure or interrupted constructs; n=3 transfections


I , J Protein blotting ( I ) and quantification ( J ) of phospho eIF2α ( p - eIF2α ) relative to total eIF2α ; n = 4 transfections , * p < 0 . 05 , unpaired t test , mean ± SD .

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I, J Protein blotting (I) and quantification (J) of phospho eIF2α (p-eIF2α) relative to total eIF2α; n=4 transfections, * p<0.05, unpaired t test, mean ± SD.


Representative Southern blots showing DSB repair products in WT , rad59 ∆ , rad52 - R70A and rad59 ∆ rad52 - R70A cells . DNA was digested with Bsp1286I and probed with a MAT - distal sequence ( yellow box in panel B ) .

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Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel B).


Viability of indicated strains ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - test was used to determine the p -

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Viability of indicated strains (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value


Representative Southern blots showing DSB repair products in WT , rad59 ∆ , and rad52 - R70A . DNA was digested with XhoI and EcoRI and probed with a Z sequence ( yellow box ) .

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Representative Southern blots showing DSB repair products in WT, rad59∆, and rad52-R70A. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box).


Percentage of chromosome loss of indicated mutants . Data information : χ2 test is used to determine the p - value and number of colonies tested per mutant is indicated in Appendix Table S1 .

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Percentage of chromosome loss of indicated mutants. Data information: χ2 test is used to determine the p-value and number of colonies tested per mutant is indicated in Appendix Table S1.


Schematic of mutation analysis during allelic recombination ( left ) . Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site . Mutation rate ( right ) of WT , rad59 ∆ , rad52 - R70A was calculated as previously described ( Saini et . al . , 2013 ) . Data information : χ Mutation rates are reported as the median value , and statistical comparisons between median mutation rates were performed using the Mann - Whitney U - test .

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Schematic of mutation analysis during allelic recombination (left). Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site. Mutation rate (right) of WT, rad59∆, rad52-R70A was calculated as previously described (Saini et. al., 2013). Data information: χ Mutation rates are reported as the median value, and statistical comparisons between median mutation rates were performed using the Mann-Whitney U-test.


Representative Southern blots showing DSB repair products of indicated mutants in regular mating type switching ( H - 1400 assay ) .

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Representative Southern blots showing DSB repair products of indicated mutants in regular mating type switching (H-1400 assay).


Viability of indicated mutants are shown . ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - test was used to determine the p -

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

Viability of indicated mutants are shown. (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value


( A - C ) Representative Southern blots showing DSB repair products ( top ) , percentage of BIR among repair products ( bottom left ) and viability ( bottom right ) of WT and sir2 ∆ in the H - 150 assay ( A ) , H - - 150 no - gap assay ( B ) or native mating type switching H - 1400 assay ( C ) . ( mean ± SD ; n = 3 ) . Welch ' s unpaired t - test was used to determine the p - value in panels A - C .

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(A-C) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of WT and sir2∆ in the H-150 assay (A), H--150 no-gap assay (B) or native mating type switching H-1400 assay (C). (mean ± SD; n = 3). Welch's unpaired t-test was used to determine the p-value in panels A-C.


( A - B ) Representative Southern blots showing DSB repair products ( top ) , percentage of BIR among repair products ( bottom left ) and viability ( bottom right ) of indicated mutants in the H - 150 no - gap assay ( A ) or H - 150 assay ( B ) . ( mean ± SD ; n ≥ 3 ) . Welch ' s unpaired t - test was used to determine the p - value in panels A and B .

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(A-B) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of indicated mutants in the H-150 no-gap assay (A) or H-150 assay (B). (mean ± SD; n ≥3). Welch's unpaired t-test was used to determine the p-value in panels A and B.


Representative fluorescence microscopy images of total , CD142 - and CD142 + ASPCs isolated with the use of the respective anti - CD142 antibodies : SinoBiological ( SB ) , SinoBiological - LL ( SB - LL ) , BiOrbyt ( BO ) and R & D Systems ( RnD ) ( Appendix Fig . S2B , Appendix Fig . S5A , Methods ) , after in vitro adipogenic differentiation ;

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Representative fluorescence microscopy images of total, CD142- and CD142+ ASPCs isolated with the use of the respective anti-CD142 antibodies: SinoBiological (SB), SinoBiological-LL (SB-LL), BiOrbyt (BO) and R&D Systems (RnD) (Appendix Fig. S2B, Appendix Fig. S5A, Methods), after in vitro adipogenic differentiation;


Fraction of differentiated cells per ASPC type shown in E , as quantified by the " adiposcore " ; marker shapes correspond to different anti - CD142 antibodies used for isolation as indicated , n = 9 - 15 , 3 - 4 biological replicates , 3 - 5 independent wells for each ;

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Fraction of differentiated cells per ASPC type shown in E, as quantified by the "adiposcore"; marker shapes correspond to different anti-CD142 antibodies used for isolation as indicated, n=9-15, 3-4 biological replicates, 3-5 independent wells for each;


Representative fluorescence microscopy images of CD142 - , 20 % CD142 + and ( 5 - 7 % ) CD142 + ASPCs isolated following the sorting strategy shown in Appendix Fig . S7A , after in vitro adipogenic differentiation with the indicated white adipogenic differentiation cocktails ( Methods ) ;

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Representative fluorescence microscopy images of CD142-, 20% CD142+ and (5-7%) CD142+ ASPCs isolated following the sorting strategy shown in Appendix Fig. S7A, after in vitro adipogenic differentiation with the indicated white adipogenic differentiation cocktails (Methods);


Fraction of differentiated cells per ASPC type and differentiation cocktail shown in J , as quantified by the " adipo - score " ; marker shapes correspond to different biological replicates , n = 8 - 17 , 3 - 5 biological replicates , 2 - 5 independent wells for each ;

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Fraction of differentiated cells per ASPC type and differentiation cocktail shown in J, as quantified by the "adipo- score"; marker shapes correspond to different biological replicates, n=8-17, 3-5 biological replicates, 2-5 independent wells for each;


Representative fluorescence microscopy images of male - and female - derived total , CD142 - and CD142 + ASPCs , isolated following the sorting strategy shown in Appendix Fig . S8A after in vitro adipogenic differentiation ; Fraction of differentiated cells per ASPC type and per sex shown in L , as quantified by the " adiposcore " ; bar colour shading corresponds to male - and female - derived cells as indicated ; marker shapes correspond to different biological replicates , n = 9 - 10 , 4 biological replicates , 2 - 3 independent wells for each ;

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Representative fluorescence microscopy images of male- and female-derived total, CD142- and CD142+ ASPCs, isolated following the sorting strategy shown in Appendix Fig. S8A after in vitro adipogenic differentiation;   Fraction of differentiated cells per ASPC type and per sex shown in L, as quantified by the "adiposcore"; bar colour shading corresponds to male- and female-derived cells as indicated; marker shapes correspond to different biological replicates, n=9-10, 4 biological replicates, 2-3 independent wells for each;


Representative fluorescence microscopy images of P0 - , P12 - 17 - , 4wo - , 7wo - and 11wo - derived total , CD142 - and CD142 + APSCs after in vitro adipogenic differentiation ; Fraction of differentiated cells per ASPC type shown in D , as quantified by the " adiposcore " ; bar colour shading corresponds to pre - and post - weaning - derived cells as indicated ; marker shapes correspond to different biological replicates , n = 14 - 65 , 12 - 13 biological replicates , 2 - 6 independent wells for each

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 B-GENEPROD O O B-GENEPROD O O 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 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 O O O O O O O O O O O

Representative fluorescence microscopy images of P0-, P12-17-, 4wo-, 7wo- and 11wo-derived total, CD142- and CD142+ APSCs after in vitro adipogenic differentiation;   Fraction of differentiated cells per ASPC type shown in D, as quantified by the "adiposcore"; bar colour shading corresponds to pre- and post-weaning-derived cells as indicated; marker shapes correspond to different biological replicates, n=14-65, 12-13 biological replicates, 2-6 independent wells for each


Representative fluorescence microscopy images of CD142 - ASPCs co - cultured with an empty transwell ( T - well ) or with wild - type ( WT ) CD142 + ASPCs after in vitro adipogenic differentiation ; Fraction of differentiated CD142 - ASPCs co - cultured with an empty transwell ( T - well ) or with wild - type CD142 + ASPCs ( non - treated or treated with control scrambled siRNA ) after in vitro adipogenic differentiation and as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 10 , 4 biological replicates , 2 - 3 independent wells for each

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Representative fluorescence microscopy images of CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type (WT) CD142+ ASPCs after in vitro adipogenic differentiation;   Fraction of differentiated CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type CD142+ ASPCs (non-treated or treated with control scrambled siRNA) after in vitro adipogenic differentiation and as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each


Representative fluorescence microscopy imaged of human - derived stromal vascular fraction ( SVF ) , containing the ASPCs , after in vitro adipogenic differentiation when co - cultured with total , CD142 - or CD142 + mouse ASPCs ; Fraction of differentiated human - derived SVF , containing the ASPCs co - cultured with total , CD142 - or CD142 + mouse ASPCs after in vitro adipogenic differentiation ( Methods ) shown in E ; marker shapes correspond to different biological replicates , n = 9 , 3 biological replicates , 3 independent wells for each

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Representative fluorescence microscopy imaged of human-derived stromal vascular fraction (SVF), containing the ASPCs, after in vitro adipogenic differentiation when co-cultured with total, CD142- or CD142+ mouse ASPCs;   Fraction of differentiated human-derived SVF, containing the ASPCs co-cultured with total, CD142- or CD142+ mouse ASPCs after in vitro adipogenic differentiation (Methods) shown in E; marker shapes correspond to different biological replicates, n=9, 3 biological replicates, 3 independent wells for each


Fraction of differentiated adult - derived CD142 - ASPCs , as quantified by the " adiposcore " , treated with the indicated recombinant proteins shown in J ; marker shapes correspond to different biological replicates , n = 11 - 59 , 2 - 10 biological replicates , 3 - 9 independent wells for

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Fraction of differentiated adult-derived CD142- ASPCs, as quantified by the "adiposcore", treated with the indicated recombinant proteins shown in J; marker shapes correspond to different biological replicates, n=11-59, 2-10 biological replicates, 3-9 independent wells for each


Representative fluorescence microscopy images of adult - derived CD142 - ASPCs after in vitro adipogenic differentiation ; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg - specific candidates : CD142 , MGP , GDF10 , CLEC11A , CPE and BGN at 100 ng /

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Representative fluorescence microscopy images of adult-derived CD142- ASPCs after in vitro adipogenic differentiation; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg-specific candidates: CD142, MGP, GDF10, CLEC11A, CPE and BGN at 100 ng/m


Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) carrying control ( scr ) or siRNA - mediated knockdowns of selected CD142 + ASPC ( Areg ) - specific candidate genes : F3 , Mgp , and Gdf10 after adipogenic differentiation ( i . e . , after exposure to an adipogenic cocktail , Methods )

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Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) carrying control (scr) or siRNA-mediated knockdowns of selected CD142+ ASPC (Areg)-specific candidate genes: F3, Mgp, and Gdf10 after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods)


Fraction of differentiated CD142 - ASPCs , as quantified by the " adiposcore " , co - cultured with CD142 + ASPCs ( Aregs ) subjected to specific knockdowns ( or scrambled ( scr ) siRNA control ) , as indicated in A ; marker shapes correspond to different biological replicates , n = 10 , 4 biological replicates , 2 - 3 independent wells for each ;

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Fraction of differentiated CD142- ASPCs, as quantified by the "adiposcore", co-cultured with CD142+ ASPCs (Aregs) subjected to specific knockdowns (or scrambled (scr) siRNA control), as indicated in A; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each;


Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) ( in the top well ) subjected to the indicated compounds at the indicated concentration ; AGN and BMS are antagonists of RAR , DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA , and HX is an antagonist of RXR ; Fraction of differentiated CD142 - ASPCs indicated in G , as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 4 - 15 , 2 - 5 biological replicates , 2 - 3 independent wells for each ;

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Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) (in the top well) subjected to the indicated compounds at the indicated concentration; AGN and BMS are antagonists of RAR, DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA, and HX is an antagonist of RXR;   Fraction of differentiated CD142- ASPCs indicated in G, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=4-15, 2-5 biological replicates, 2-3 independent wells for each;


Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) subjected to control ( scr ) or siRNA - mediated knockdowns of selected RA signalling pathway genes : Rara , Rarb , Rarg , Rbp1 , and Rxrg after adipogenic differentiation ( i . e . , after exposure to an adipogenic cocktail , Methods ) ; Fraction of differentiated CD142 - ASPCs indicated in I , as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 24 - 29 , 4 biological replicates , 5 - 8 independent wells for each .

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Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) subjected to control (scr) or siRNA-mediated knockdowns of selected RA signalling pathway genes: Rara, Rarb, Rarg, Rbp1, and Rxrg after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods);   Fraction of differentiated CD142- ASPCs indicated in I, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=24-29, 4 biological replicates, 5-8 independent wells for each.


A Representative image of tumor - bearing mice and tumors after subcutaneous injection of Hepa1 - 6 hepatoma cells into WT and Tet2 - / - ( KO ) mice .

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A Representative image of tumor-bearing mice and tumors after subcutaneous injection of Hepa1-6 hepatoma cells into WT and Tet2-/- (KO) mice.


B Mean tumor volumes in WT and Tet2 - / - mice ( n = 6 ) .

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B Mean tumor volumes in WT and Tet2-/- mice (n=6).


C Tumor weights 18 days after tumor cell injection into WT and KO mice ( n = 6 ) .

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C Tumor weights 18 days after tumor cell injection into WT and KO mice (n=6).


D Kaplan - Meier survival curves of tumor - bearing and tumor - free WT and KO mice ( n = 15 ) .

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D Kaplan-Meier survival curves of tumor-bearing and tumor-free WT and KO mice (n=15).


E Representative images of lungs with metastasis from WT and KO mice injected with Hepa1 - 6 cells via tail vein , as well as quantification of the number of lung metastasis ( n = 4 ) .

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E Representative images of lungs with metastasis from WT and KO mice injected with Hepa1-6 cells via tail vein, as well as quantification of the number of lung metastasis (n=4).


F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells , as well as quantification of tumor weights ( n = 5 ) .

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F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells, as well as quantification of tumor weights (n=5).


A , B Immunohistochemistry analysis of CD8a , CD4 ( A ) , PCNA , and Foxp3 ( B ) in tumors from WT and Tet2 - / - ( KO ) mice subcutaneously injected with Hepa1 - 6 hepatoma cells . Corresponding bar charts display positive staining , quantified using Image Pro Plus 6 . 0 software ( n = 3 ) . Pictures were captured at 400X magnification . Scale bar = 50 μm .

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A, B Immunohistochemistry analysis of CD8a, CD4 (A), PCNA, and Foxp3 (B) in tumors from WT and Tet2-/- (KO) mice subcutaneously injected with Hepa1-6 hepatoma cells. Corresponding bar charts display positive staining, quantified using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.


D Tumor growth curves in WT and KO mice treated with PBS or with anti - PD - 1 Ab ( n = 3 ) .

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D Tumor growth curves in WT and KO mice treated with PBS or with anti-PD-1 Ab (n =3).


E Representatives images of day - 19 tumors from WT and KO from panel D .

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E Representatives images of day-19 tumors from WT and KO from panel D.


F Tumor weights from WT and KO mice at day 19 .

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F Tumor weights from WT and KO mice at day 19.


G Flow cytometry analysis of splenic CD8 + T cells in WT and KO mice with or without anti - PD - 1 treatment after tumor challenge .

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G Flow cytometry analysis of splenic CD8+ T cells in WT and KO mice with or without anti-PD-1 treatment after tumor challenge.


H - K GSEA analysis showing the significant down - regulation of genes in GO biological processes : chemokine signaling pathway ( H ) , T cell receptor signaling pathway ( I ) , innate immune response ( J ) and cytokine receptor interaction ( K ) . The x - axis represents that the mouse genes are ranked from the most up - regulated ( left end ) to the most down - regulated ( right end ) in comparison of syngeneic tumor from Hepa1 - 6 tumor - bearing KO to WT mice ( n = 4 ) . The y - axis is an enrichment score ( ES ) , which goes up ( > 0 ) and down ( < 0 ) representing the enrichment of up - regulated genes and down - regulated genes , respectively . NES is the normalized enrichment score . p - value was calculated by permutation .

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H-K GSEA analysis showing the significant down-regulation of genes in GO biological processes: chemokine signaling pathway (H), T cell receptor signaling pathway (I), innate immune response (J) and cytokine receptor interaction (K). The x-axis represents that the mouse genes are ranked from the most up-regulated (left end) to the most down-regulated (right end) in comparison of syngeneic tumor from Hepa1-6 tumor-bearing KO to WT mice (n=4). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of up-regulated genes and down-regulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.


A Percentages of MDSCs ( CD11b + Gr1 + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 11 , tumor - free ( - ) WT and Tet2 - / - （ KO ） mice ; n = 12 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 10 , tumor - free ( - ) WT and KO mice ; n = 13 , tumor - bearing ( + ) WT and KO mice , spleen ] . B Percentages of G - MDSCs ( CD11b + Ly6G + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 14 , tumor - free ( - ) WT and KO mice ; n = 15 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 15 , tumor - free ( - ) WT and KO mice ; n = 15 , tumor - bearing ( + ) WT and KO mice , spleen ] . C Percentages of M - MDSCs ( CD11b + Ly6C + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 5 , tumor - free ( - ) WT and KO mice ; n = 6 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 6 , tumor - free ( - ) WT and KO mice ; n = 6 , tumor - bearing ( + ) WT and KO mice , spleen ] .

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A Percentages of MDSCs (CD11b+Gr1+) gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and Tet2-/-（KO） mice; n=12, tumor-bearing (+) WT and KO mice, peripheral blood; n=10, tumor-free (-) WT and KO mice; n=13, tumor-bearing (+) WT and KO mice, spleen]. B Percentages of G-MDSCs (CD11b+Ly6G+) gated from the CD45+ cell population in peripheral blood and spleen [n=14, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, peripheral blood; n=15, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, spleen]. C Percentages of M-MDSCs (CD11b+Ly6C+) gated from the CD45+ cell population in peripheral blood and spleen [n=5, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, peripheral blood; n=6, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, spleen].


D Representative images of Ly6g and Ly6c immunohistochemistry in Hepa1 - 6 cell tumors from WT and KO mice . Corresponding bar charts display quantification of positive staining using Image Pro Plus 6 . 0 software ( n = 3 ) . Pictures were captured at 400X magnification . Scale bar = 50 μm .

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D Representative images of Ly6g and Ly6c immunohistochemistry in Hepa1-6 cell tumors from WT and KO mice. Corresponding bar charts display quantification of positive staining using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.


E Percentages of CD8 + T cells gated from the CD45 + cell population in peripheral blood and spleen [ n = 11 , tumor - free ( - ) WT and KO mice ; n = 5 , tumor - bearing ( + ) WT and Tet2 - / - KO mice , peripheral blood ; n = 16 , tumor - free ( - ) WT and KO mice ; n = 12 , tumor - bearing ( + ) WT and KO mice , spleen ] . F Percentages of CD4 + T cells gated from the CD45 + cell population in peripheral blood and spleen [ n = 8 , tumor - free ( - ) WT and KO mice ; n = 5 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 16 , tumor - free ( - ) WT and KO mice ; n = 11 , tumor - bearing ( + ) WT and KO mice , spleen ] .

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E Percentages of CD8+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and Tet2-/- KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=12, tumor-bearing (+) WT and KO mice, spleen]. F Percentages of CD4+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=8, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=11, tumor-bearing (+) WT and KO mice, spleen].


A Sorting efficiency of G - MDSCs ( CD11b + Ly6G + ) from spleens of tumor - bearing Tet2 - / - （ KO ） and WT mice .

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A Sorting efficiency of G-MDSCs (CD11b+Ly6G+) from spleens of tumor-bearing Tet2-/- （KO） and WT mice.


B GSEA analysis showing the significant upregulation of genes in GO biological processes : DNA replication ( left ) , cell cycle G1 - S phase transition ( middle ) , and ribosome biogenesis ( right ) . The x - axis represents that the mouse genes are ranked from the most upregulated ( left end ) to the most downregulated ( right end ) in comparison of tumor - bearing KO to WT mice from G - MDSCs of spleen ( n = 2 ) . The y - axis is an enrichment score ( ES ) , which goes up ( > 0 ) and down ( < 0 ) representing the enrichment of upregulated genes and downregulated genes , respectively . NES is the normalized enrichment score . p - value was calculated by permutation .

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B GSEA analysis showing the significant upregulation of genes in GO biological processes: DNA replication (left), cell cycle G1-S phase transition (middle), and ribosome biogenesis (right). The x-axis represents that the mouse genes are ranked from the most upregulated (left end) to the most downregulated (right end) in comparison of tumor-bearing KO to WT mice from G-MDSCs of spleen (n=2). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of upregulated genes and downregulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.


C G - MDSCs purified from spleen of tumor - bearing WT or Tet2 - / - mice were co - cultured with CD8 + T cells at the indicated cellular ratios for T cell activation assays , quantified by CFSE - labeling and dye dilution for 3 days in vitro . Representative histograms show CFSE signals in the in vitro T cell activation assays . D Quantification of CD8 + T cell proliferation in panel C . Each point represents a co - culture experiment ( n = 3 ) . ns , not significant . Graphs show mean ± SEM and statistical analysis by Student ' s t - test .

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C G-MDSCs purified from spleen of tumor-bearing WT or Tet2-/- mice were co-cultured with CD8+ T cells at the indicated cellular ratios for T cell activation assays, quantified by CFSE-labeling and dye dilution for 3 days in vitro. Representative histograms show CFSE signals in the in vitro T cell activation assays. D Quantification of CD8+ T cell proliferation in panel C. Each point represents a co-culture experiment (n=3). ns, not significant. Graphs show mean ± SEM and statistical analysis by Student's t-test.


B Representative flow cytometry plots of splenic G - MDSCs measured in tumor - free untreated Tet2 - / - ( KO ) and WT mice and after anti - Ly6g or PBS injections into tumor - bearing mice . Flow cytometric analysis of T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - Ly6g treatment after tumor challenge .

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B Representative flow cytometry plots of splenic G-MDSCs measured in tumor-free untreated Tet2-/- (KO) and WT mice and after anti-Ly6g or PBS injections into tumor-bearing mice. Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge.


C Tumor growth curves in WT and KO mice treated with either PBS or anti - mouse Ly6g ( n = 5 ) .

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C Tumor growth curves in WT and KO mice treated with either PBS or anti-mouse Ly6g (n=5).


D Day - 18 tumor weights at the endpoint of ( C ) [ n = 11 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 12 , anti - Ly6g antibody ( + ) injected tumor - bearing WT and KO mice ] .

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D Day-18 tumor weights at the endpoint of (C) [n=11, PBS (-) injected tumor-bearing WT and KO mice; n=12, anti-Ly6g antibody (+) injected tumor-bearing WT and KO mice].


E Percentages of CD8 + T cells from the spleens of tumor - bearing mice with or without anti - mouse Ly6g treatment [ n = 17 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 6 , anti - Ly6g ( + ) antibody injected tumor - bearing WT and KO mice ] . F Percentages of CD4 + T cells from the spleens of tumor - bearing mice with or without anti - mouse Ly6g treatment [ n = 16 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 6 , anti - Ly6g ( + ) antibody injected tumor - bearing WT and KO mice Flow cytometric analysis of T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - Ly6g treatment after tumor challenge .

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E Percentages of CD8+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=17, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice]. F Percentages of CD4+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=16, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge.


A Gene set enrichment analysis of the IL - 6 / JAK / STAT signaling pathway in G - MDSCs from tumor - bearing Tet2 - / - ( KO ) versus WT mice ( n = 2 ) . p - value was calculated by permutation .

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A Gene set enrichment analysis of the IL-6/JAK/STAT signaling pathway in G-MDSCs from tumor-bearing Tet2-/- (KO) versus WT mice (n=2). p-value was calculated by permutation.


B ELISA analysis of IL - 6 , IFN - γ , and IL - 2 levels in blood from WT and KO mice upon tumor challenge ( n = 4 , tumor - bearing WT mice ; n = 3 , tumor - bearing KO mice ) .

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B ELISA analysis of IL-6, IFN-γ, and IL-2 levels in blood from WT and KO mice upon tumor challenge (n=4, tumor-bearing WT mice; n=3, tumor-bearing KO mice).


C Tumor growth curves in tumor - bearing WT and KO mice treated with PBS or anti - mouse IL - 6 antibody ( n = 5 ) .

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C Tumor growth curves in tumor-bearing WT and KO mice treated with PBS or anti-mouse IL-6 antibody (n=5).


D Day - 18 tumor weights at the endpoint of ( C ) [ n = 10 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 7 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice ] .

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D Day-18 tumor weights at the endpoint of (C) [n=10, PBS (-) injected tumor-bearing WT and KO mice; n=7, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice].


E , F Percentages of CD11b + Ly6G + G - MDSCs gated from total cells in ( E ) spleen and ( F ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 antibody [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 4 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 4 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 4 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . G , H Percentages of CD8 + T cells gated from total cells in ( G ) spleen and ( H ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . I , J Percentages of CD4 + T cells gated from total cells in ( I ) spleen and ( J ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 4 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . Flow cytometric analysis of G - MDSCs and T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - IL - 6 treatment after tumor challenge .

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

E, F Percentages of CD11b+ Ly6G+ G-MDSCs gated from total cells in (E) spleen and (F) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 antibody [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. G, H Percentages of CD8+ T cells gated from total cells in (G) spleen and (H) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. I, J Percentages of CD4+ T cells gated from total cells in (I) spleen and (J) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. Flow cytometric analysis of G-MDSCs and T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-IL-6 treatment after tumor challenge.


A . Pull - down experiments using as a bait the biotinylated peptides mimicking the N - terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates ( top blot ) or purified His - tagged GOLPH3 protein ( bottom blot ) . The N - terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot ( LxxR motif in red , biotin at the C - terminus ) . Immunoblotting shows affinity - captured GOLPH3 ( endogenous and recombinant ) .

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

A. Pull-down experiments using as a bait the biotinylated peptides mimicking the N-terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot (LxxR motif in red, biotin at the C-terminus). Immunoblotting shows affinity-captured GOLPH3 (endogenous and recombinant).


B . Control or GOLPH3 - KD HeLa cells expressing luminal SI - GFP or the chimeras of sucrose - isomaltase containing the N - terminal cytosolic tails of LCS , Gb3S , GM3S and GD3S were fixed and labelled for surface localized GFP ( red ) . Scale bar , 20 μm .

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B. Control or GOLPH3-KD HeLa cells expressing luminal SI-GFP or the chimeras of sucrose-isomaltase containing the N-terminal cytosolic tails of LCS, Gb3S, GM3S and GD3S were fixed and labelled for surface localized GFP (red). Scale bar, 20 μm.


C . Pull - down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates ( top blot ) or purified His - tagged GOLPH3 protein ( bottom blot ) . The N - terminal cytosolic tail of LCS WT and LCS mutants ( mut . 1 , 2 and 3 ) is shown on the top of the blot ( mutations are in blue , LxxR motif in red , biotin is at the C - terminus ) .

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

C. Pull-down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail of LCS WT and LCS mutants (mut. 1, 2 and 3) is shown on the top of the blot (mutations are in blue, LxxR motif in red, biotin is at the C-terminus).


D . HeLa cells expressing luminal SI - GFP chimeras of sucrose - isomaltase ( SI - GFP , green ) containing the WT and mutant N - terminal cytosolic tails of LCS , were fixed and labelled for surface localized GFP ( red ) . Cells expressing LCS - SI - RxR and LCS - SI - LxxR chimeras shown PM staining . Scale bar , 20 μm , ( left ) . PM ( surface ) fluorescence intensity ( in Table S10 are listed the software used in this study ) of each chimera is normalised on the LCS - SI - GFP surface intensity ( data are means ± SEM derived from two biological replicates ; n is indicated in the graph , * * * p < 0 . 001 [ Student ' s t - test ] ) ( right ) .

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

D. HeLa cells expressing luminal SI-GFP chimeras of sucrose-isomaltase (SI-GFP, green) containing the WT and mutant N-terminal cytosolic tails of LCS, were fixed and labelled for surface localized GFP (red). Cells expressing LCS-SI-RxR and LCS-SI-LxxR chimeras shown PM staining. Scale bar, 20 μm, (left). PM (surface) fluorescence intensity (in Table S10 are listed the software used in this study) of each chimera is normalised on the LCS-SI-GFP surface intensity (data are means ± SEM derived from two biological replicates; n is indicated in the graph, ***p <0.001 [Student's t-test]) (right).


E . Quantitative flow cytometry - based analysis of the experiment reported in D ; n is indicated in the graph ( left ) . Percentage of cells with plasma membrane staining ( surface ) is reported for each chimera ( right ) .

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E. Quantitative flow cytometry-based analysis of the experiment reported in D; n is indicated in the graph (left). Percentage of cells with plasma membrane staining (surface) is reported for each chimera (right).


F . GOLPH3 - KD or OE HeLa cells were processed for ShTxB ( upper panels ) or ChTxB ( lower panels ) staining followed by flow cytometry analysis . Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition .

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F. GOLPH3-KD or OE HeLa cells were processed for ShTxB (upper panels) or ChTxB (lower panels) staining followed by flow cytometry analysis. Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition.


A . HeLa cells mock - treated or GOLPH3 - KD were transiently transfected with LCS _ GFP _ RUSH ( for simplicity , referred in the text as LCS - GR ) and subjected to the synchronisation protocol described in Materials and Methods . Cells were then subjected to IF at each indicated time point : LCS - GR ( green ) , cis Golgi marker GM130 ( red ) , and TGN marker TGN46 ( blue ) . Representative individual Golgi mini - stacks are shown . Scale bar , 1 µm . LCS - GR intra - Golgi trafficking in mock or GOLPH3 - KD conditions it has been quantified ( right graph ) by computational coalescence of line scans , and was performed by normalized line scan analysis ( normalisation of the distances was performed considering the GM130 peak as 0 , and the TGN46 peak as 1 ) . The n indicated in the graph refers to the number of Golgi stacks analysed in each condition .

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

A. HeLa cells mock-treated or GOLPH3-KD were transiently transfected with LCS_GFP_RUSH (for simplicity, referred in the text as LCS-GR) and subjected to the synchronisation protocol described in Materials and Methods. Cells were then subjected to IF at each indicated time point: LCS-GR (green), cis Golgi marker GM130 (red), and TGN marker TGN46 (blue). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. LCS-GR intra-Golgi trafficking in mock or GOLPH3-KD conditions it has been quantified (right graph) by computational coalescence of line scans, and was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1). The n indicated in the graph refers to the number of Golgi stacks analysed in each condition.


B . Mock and GOLPH3 - KD HeLa cells co - expressing LCS - GR and VSV - G were subjected to the co - synchronisation protocol described in Materials and Methods . Cells were then subjected to IF : LCS - GR or VSV - G ( green ) , GM130 ( blue ) , and TGN46 ( red ) . Representative individual Golgi mini - stacks are shown . Scale bar , 1 µm . C . Quantification of the experiment in ( B ) was performed by normalized line scan analysis ( normalisation of the distances was performed considering the GM130 peak as 0 , and the TGN46 peak as 1 ) ; n indicated in the graph refers to the number of Golgi stacks analysed in each condition , individual data points and means ± SEM are shown , * * * p < 0 . 001 ( Student ' s t test ) ; ns , not significant . D

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

B. Mock and GOLPH3-KD HeLa cells co-expressing LCS-GR and VSV-G were subjected to the co-synchronisation protocol described in Materials and Methods. Cells were then subjected to IF: LCS-GR or VSV-G (green), GM130 (blue), and TGN46 (red). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. C. Quantification of the experiment in (B) was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1); n indicated in the graph refers to the number of Golgi stacks analysed in each condition, individual data points and means ± SEM are shown, ***p < 0.001 (Student's t test); ns, not significant. D


D . HeLa cells were transfected with LCS - GR , subjected to the synchronisation protocol ( see Materials and Methods ) , fixed and processed for cryoimmunolabeling with an anti - GFP antibody ( 15 nm gold particles ) and anti - GM130 antibody ( 10 nm gold particles , black arrowheads ) peri - Golgi vesicles containing LCS - GR are marked by red arrowheads . Scale bar , 150 nm .

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D. HeLa cells were transfected with LCS-GR, subjected to the synchronisation protocol (see Materials and Methods), fixed and processed for cryoimmunolabeling with an anti-GFP antibody (15 nm gold particles) and anti-GM130 antibody (10 nm gold particles, black arrowheads) peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. Scale bar, 150 nm.


E , F . Quantification of the distribution of LCS - GR as measured by Frequency Distribution Analysis ( E ) and linear density ( LD , in F ) ( data are means ± SEM , number of Golgi stacks is n = 15 at 0 min , n = 14 at 10 min , n = 17 at 20 min . ( E ) The red arrows indicate the fraction of LCS - GR gold particles ( % ) in vesicles at each indicated time - point .

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 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 B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O

E, F. Quantification of the distribution of LCS-GR as measured by Frequency Distribution Analysis (E) and linear density (LD, in F) (data are means± SEM, number of Golgi stacks is n=15 at 0 min, n=14 at 10 min, n=17 at 20 min. (E) The red arrows indicate the fraction of LCS-GR gold particles (%) in vesicles at each indicated time-point.


G . HeLa cells expressing LCS - GR mock or GOLPH3 - KD were treated and stained as in D . Black arrowheads indicate GM130 ; peri - Golgi vesicles containing LCS - GR are marked by red arrowheads . LCS - GR in the TGN is marked by blue arrowheads . Scale bar , 150 nm .

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G. HeLa cells expressing LCS-GR mock or GOLPH3-KD were treated and stained as in D. Black arrowheads indicate GM130; peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. LCS-GR in the TGN is marked by blue arrowheads. Scale bar, 150 nm.


H . Quantification of LCS - GR distribution ( linear density , LD ) across the stack and in peri - Golgi vesicles ( data are means ± SEM , number of Golgi stacks is n = 17 at 20 min for the CTRL , n = 13 at 20 min for the GOLPH3 KD ; * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR 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 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

H. Quantification of LCS-GR distribution (linear density, LD) across the stack and in peri-Golgi vesicles (data are means ± SEM, number of Golgi stacks is n=17 at 20 min for the CTRL, n=13 at 20 min for the GOLPH3 KD; *p <0.05, **p < 0.01; ***p <0.001 [Student's t-test]).


A . PHFs GOLPH3 OE ( left ) , or KD ( right ) , were fixed and processed for IF : Endogenous LCS ( green ) , GOLPH3 ( red ) , TGN46 or DAPI ( blue ) . Dashed lines indicate cells overexpressing or interfered for GOLPH3 . Scale bar , 20 µm . Quantification of the amount of endogenous LCS ( right graph ) at the Golgi ; data are means ± SEM derived from two biological replicates , n = 42 in CTRL , n = 49 in GOLPH3 KD and n = 27 in GOLPH3 OE ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

O O B-CELL_TYPE B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE 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 B-GENEPROD 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 B-GENEPROD 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

A. PHFs GOLPH3 OE (left), or KD (right), were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), TGN46 or DAPI (blue). Dashed lines indicate cells overexpressing or interfered for GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi; data are means ± SEM derived from two biological replicates, n=42 in CTRL, n=49 in GOLPH3 KD and n=27 in GOLPH3 OE; ***p <0.001 [Student's t-test]).


B . GOLPH3 - OE HeLa cells were fixed and processed for IF : Endogenous LCS ( green ) , GOLPH3 ( red ) , and TGN46 ( blue ) . Dashed lines indicate cells overexpressing GOLPH3 . Scale bar , 20 µm . Quantification of the amount of endogenous LCS ( right graph ) at the Golgi ( data are means ± SEM derived from two biological replicates , n = 21 in CTRL , n = 11 in GOLPH3 OE ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

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

B. GOLPH3-OE HeLa cells were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), and TGN46 (blue). Dashed lines indicate cells overexpressing GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi (data are means ± SEM derived from two biological replicates, n=21 in CTRL, n=11 in GOLPH3 OE; ***p <0.001 [Student's t-test]).


C . GOLPH3 OE or KD HeLa cells expressing LCS - HA were fixed and processed for IF : HA ( green ) . Asterisks indicate GOLPH3 OE cells . Scale bar , 20 µm .

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C. GOLPH3 OE or KD HeLa cells expressing LCS-HA were fixed and processed for IF: HA (green). Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.


D . Assessment of LCS - GR synchronized transport in GOLPH3 OE or KD HeLa cells . Micrographs show cells at the indicated trafficking time points . Scale bar , 20 µm .

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D. Assessment of LCS-GR synchronized transport in GOLPH3 OE or KD HeLa cells. Micrographs show cells at the indicated trafficking time points. Scale bar, 20 µm.


E . Representative WB of cells treated as in ( D ) ( n = 3 ) .

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E. Representative WB of cells treated as in (D) (n = 3).


F . Plots of the densitometric quantification of the amount of LCS - GR ( Golgi mature band ) over time , starting from 2 hr chase ( data are means of at least 3 experiments ± SD ) .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD 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

F. Plots of the densitometric quantification of the amount of LCS-GR (Golgi mature band) over time, starting from 2 hr chase (data are means of at least 3 experiments ± SD).


G . GOLPH3 OE or KD HeLa cells expressing LCS - HA were treated with bafilomycin A1 ( BafA1 , 10 nM ) for 16 hr , fixed , and processed for IF labelling : LCS ( green ) , LAMP1 ( red ) , and DAPI ( blue ) . Arrowheads indicate LCS and LAMP1 co - localisation . Scale bar , 20 µm .

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

G. GOLPH3 OE or KD HeLa cells expressing LCS-HA were treated with bafilomycin A1 (BafA1, 10 nM) for 16 hr, fixed, and processed for IF labelling: LCS (green), LAMP1 (red), and DAPI (blue). Arrowheads indicate LCS and LAMP1 co-localisation. Scale bar, 20 µm.


H . WB of cells treated as in ( G ) . Densitometric quantification of the blot is plotted in the top panel ( data are means of at least 3 experiments ± SD ) .

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H. WB of cells treated as in (G). Densitometric quantification of the blot is plotted in the top panel (data are means of at least 3 experiments ± SD).


I . GOLPH3 OE or KD HeLa cells expressing the indicated HA - tagged glycoenzymes were fixed and processed for IF . Asterisks indicate GOLPH3 OE cells . Scale bar , 20 µm .

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

I. GOLPH3 OE or KD HeLa cells expressing the indicated HA-tagged glycoenzymes were fixed and processed for IF. Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.


J . Number of glycoenzymes - HA positive spots per cell in GOLPH3 - KD cells ( left panel ) and percentage of cells with glycoenzymes - HA in the ER in GOLPH3 - OE cells ( right panel ) are plotted ( n is indicated in the graph ; data are means ± SEM ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR 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

J. Number of glycoenzymes-HA positive spots per cell in GOLPH3-KD cells (left panel) and percentage of cells with glycoenzymes-HA in the ER in GOLPH3-OE cells (right panel) are plotted (n is indicated in the graph; data are means ± SEM; ***p <0.001 [Student's t-test]).


A . Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [ 3H ] - sphingosine pulse ( 2 hr ) and chase ( indicated times ) , lipid extraction and HPTLC separation and radioactive counting ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) .

O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O

A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]).


B . Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC / MS of HeLa cells ( top ) and PHFs ( middle ) . Effects of LCS KD or OE on sphingolipid levels as assessed by LC / MS of HeLa cells ( bottom ) ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs : ST001673 for HeLa cells , and ST001672 for PHFs .

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

B. Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (bottom) (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


C . Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI - MS after lipid extraction . Profiles are representative of 3 independent experiments . Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID : ST001674 .

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

C. Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI-MS after lipid extraction. Profiles are representative of 3 independent experiments. Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID: ST001674.


D . Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig . 1F ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 [ Student ' s t - test ] ) .

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

D. Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig. 1F (data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001 [Student's t-test]).


E . Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC / MS of HeLa cells ( top ) and PHFs ( middle ) . Effects of LCS KD or OE on ceramide levels as assessed by LC / MS of HeLa cells ( bottom ) . The dotted black box highlights the effects on C16 : 0 Cer ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs : ST001673 for HeLa cells , and ST001672 for PHFs .

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

E. Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (bottom). The dotted black box highlights the effects on C16:0 Cer (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


A . Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS - PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway . Data are representative of at least 3 independent experiments .

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

A. Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS-PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway. Data are representative of at least 3 independent experiments.


B . Clonogenic assay of HeLa cells in adherence . 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days . Afterward , colonies were fixed and stained as detailed in Materials and Methods . The graph on right reports the number of colonies per well in each indicated condition ( data are means ± SEM of 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 [ Student ' s t - test ] ) .

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

B. Clonogenic assay of HeLa cells in adherence. 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days. Afterward, colonies were fixed and stained as detailed in Materials and Methods. The graph on right reports the number of colonies per well in each indicated condition (data are means ± SEM of 3 independent experiments; *p <0.05, **p <0.01 [Student's t-test]).


C . Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 ( left upper graph ) or LCS ( left bottom graph ) encoding plasmids . Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS ( right upper graph ) . Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD ( right bottom graph ) . Colonies were stained and quantified as described in Materials and Methods ; ( individual data points are shown in the graphs ; data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 , n . s . = not significant [ Student ' s t - test ] ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD 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 O O O O O 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. Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 (left upper graph) or LCS (left bottom graph) encoding plasmids. Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS (right upper graph). Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD (right bottom graph). Colonies were stained and quantified as described in Materials and Methods; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001, n.s. = not significant [Student's t-test]).


D . Protein lysates from parental ( left ) , GCS - KO ( middle ) , and LCS - KO ( right ) HeLa cells , CTRL or GOLPH3 - OE were processed for SDS - PAGE and WB with the indicated antibodies . Data are representative of at least 3 independent experiments .

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

D. Protein lysates from parental (left), GCS-KO (middle), and LCS-KO (right) HeLa cells, CTRL or GOLPH3-OE were processed for SDS-PAGE and WB with the indicated antibodies. Data are representative of at least 3 independent experiments.


E . Soft agar colony formation assay of parental and LCS - KO HeLa cells expressing LCS - HA or GOLPH3 . Colony number was quantified ; ( individual data points are shown in the graphs ; data are means ± SD of at least 3 independent experiments ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

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

E. Soft agar colony formation assay of parental and LCS-KO HeLa cells expressing LCS-HA or GOLPH3. Colony number was quantified; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; ***p <0.001 [Student's t-test]).


F . Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS ( data are means ± SEM of at least 3 independent experiments ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-CELL_LINE B-DISEASE I-DISEASE 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 O O O O O O O O O O O O O O O O O

F. Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS (data are means ± SEM of at least 3 independent experiments; ***p <0.001 [Student's t-test]).


D . Representative images of NSCLC sections stained for GOLPH3 and LCS . Scale bar , 100 µm .

O O O B-EXP_ASSAY O B-DISEASE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O

D. Representative images of NSCLC sections stained for GOLPH3 and LCS. Scale bar, 100 µm.


( B ) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence ( magenta ) and the effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , fluorescence ) . Dotted lines indicate the electroporated area of the VZ . Single optical sections ; scale bars , 200 μm .

O O O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O O O O O O O

(B) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.


( C ) Higher magnification of the VZ and SVZ of the electroporated area shown in ( B ) , with DAPI staining ( blue ) depicted in addition to PaprikaRFP ( Cas9 ) and GFP fluorescence . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O B-GENEPROD B-EXP_ASSAY 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 O O O

(C) Higher magnification of the VZ and SVZ of the electroporated area shown in (B), with DAPI staining (blue) depicted in addition to PaprikaRFP (Cas9) and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


( D ) Quantification of the proportion of Cas9 - positive cells in the VZ plus SVZ that are GFP - positive 48 h after control ( Con , white ) or gGFP ( black ) Cas9 plasmid electroporation . Data are the mean of 4 independent experiments ( 7 embryos per condition in total , from 4 litters ) .

O O O O O O O O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O

(D) Quantification of the proportion of Cas9-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 plasmid electroporation. Data are the mean of 4 independent experiments (7 embryos per condition in total, from 4 litters).


( F ) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence ( magenta ) showing the effects of Cas9 protein together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green , fluorescence ) . Dotted lines indicate the electroporated area of the VZ . Single optical sections ; scale bars , 200 μm .

O O O O O O O O B-TISSUE O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O O O O O O O

(F) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence (magenta) showing the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.


( G ) Higher magnification of the VZ and SVZ of the electroporated area shown in ( F ) , with DAPI staining ( blue ) depicted in addition to mCherry and GFP fluorescence . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O B-GENEPROD B-EXP_ASSAY 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 O O O

(G) Higher magnification of the VZ and SVZ of the electroporated area shown in (F), with DAPI staining (blue) depicted in addition to mCherry and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


H ) Quantification of the proportion of mCherry - positive cells in the VZ plus SVZ that are GFP - positive 48 h after control ( Con , white ) or gGFP ( black ) Cas9 protein electroporation . Data are the mean of 4 independent experiments ( 5 embryos per condition in total , from 4 litters ) .

O O O O O O O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O

H) Quantification of the proportion of mCherry-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 protein electroporation. Data are the mean of 4 independent experiments (5 embryos per condition in total, from 4 litters).


( I ) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence ( magenta ) and the effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , fluorescence ) ; blue , DAPI staining . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

O O O B-TISSUE O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY 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 B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O B-GENEPROD B-EXP_ASSAY 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 O O O

(I) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


( J ) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence ( magenta ) and the effects of Cas9 protein together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green ) ; blue , DAPI staining . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

O O O B-TISSUE O B-TISSUE O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD 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 B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O B-GENEPROD B-EXP_ASSAY 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 O O O

(J) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence (magenta) and the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


K ) Quantification of the proportion of Cas9 - positive cells ( left ) and mCherry - positive cells ( right ) in the VZ plus SVZ that are GFP - positive 24 h after control ( Con , white ) or gGFP ( black ) Cas9 plasmid ( left ) or protein ( right ) electroporation . Data are the mean of 3 independent experiments each ( 3 embryos per condition in total , from 3 litters ) . ( D , H , K ) Controls were set to 100 % and the gGFP conditions expressed relative to control ( D , 11 % ; H , 24 % ; K left , 40 % K right , 28 % ) . Error bars indicate SD ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD 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 B-TISSUE O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K) Quantification of the proportion of Cas9-positive cells (left) and mCherry-positive cells (right) in the VZ plus SVZ that are GFP-positive 24 h after control (Con, white) or gGFP (black) Cas9 plasmid (left) or protein (right) electroporation. Data are the mean of 3 independent experiments each (3 embryos per condition in total, from 3 litters).(D, H, K) Controls were set to 100% and the gGFP conditions expressed relative to control (D, 11%; H, 24%; K left, 40% K right, 28%). Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.


( B ) Daughter cells of single aRGCs microinjected with either Cas9 / control gRNA ( top ) or Cas9 / gGFP ( bottom ) revealed by Dx - A555immunofluorescence ( magenta ) ; Cells 1 , aRGC daughter ; Cells 2 , BP daughter . Dashed lines indicate ventricular surface . Images are maximum intensity projections of 20 ( Control ) and 24 ( gGFP ) optical sections . Scale bars , 20 µm .

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

(B) Daughter cells of single aRGCs microinjected with either Cas9/control gRNA (top) or Cas9/gGFP (bottom) revealed by Dx-A555immunofluorescence (magenta); Cells 1, aRGC daughter; Cells 2, BP daughter. Dashed lines indicate ventricular surface. Images are maximum intensity projections of 20 (Control) and 24 (gGFP) optical sections. Scale bars, 20 µm.


( C ) Single optical sections of Cells 1 and Cells 2 depicted in ( B ) , showing the effects of Cas9 and control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( as revealed by immunofluorescence , green ) . Cells 1 and Cells 2 are indicated by dotted lines ; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9 / gGFP microinjection . Scale bars , 5 µm .

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(C) Single optical sections of Cells 1 and Cells 2 depicted in (B), showing the effects of Cas9 and control gRNA (top) or gGFP (bottom) on GFP expression (as revealed by immunofluorescence, green). Cells 1 and Cells 2 are indicated by dotted lines; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9/gGFP microinjection. Scale bars, 5 µm.


( D ) Quantification of the proportion of daughter cells ( Dx - A555 + ) of microinjected cells that show GFP expression 24 h after control ( Con , white ) or gGFP ( black ) microinjection . Control is set to 100 % and the gGFP condition expressed relative to control ( 39 % ) . Data are the mean of two independent experiments ( 3 embryos and 1 litter per experiment , total number of daughter cells scored : control 142 , gGFP 88 ) ; bars indicate the variation of the two individual values from the mean ( P < 0 . 05 ) .

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(D) Quantification of the proportion of daughter cells (Dx-A555+) of microinjected cells that show GFP expression 24 h after control (Con, white) or gGFP (black) microinjection. Control is set to 100% and the gGFP condition expressed relative to control (39%). Data are the mean of two independent experiments (3 embryos and 1 litter per experiment, total number of daughter cells scored: control 142, gGFP 88); bars indicate the variation of the two individual values from the mean (P<0.05).