Effect of HK2 targeting on in vitro tumorigenicity . HK2 - silencing with two different shRNAs ( shHK2 - 1 and shHK2 - 2 ) in CT26 colon cancer cells ( A ) Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells (A) Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . HK2 - silencing with two different shRNAs ( shHK2 - 1 and shHK2 - 2 ) in CT26 colon cancer cells inhibits growth in soft agar ( B ; colony area ± SEM ; n = 6 replicates obtained from 3 independent experiments ; Student ' s t test * * * p < 0 . 001 ; A . U . , arbitrary units ) . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells inhibits growth in soft agar (B; colony area±SEM; n=6 replicates obtained from 3 independent experiments; Student's t test ***p<0.001; A.U., arbitrary units). Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . Treatment with cl - HK2pep ( 50 µM , 6 h ; C , scale bar : 100 µm ) reverts formation of foci ; dead cells are highlighted by Trypan Blue staining . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of HK2 targeting on in vitro tumorigenicity. Treatment with cl-HK2pep (50 µM, 6 h; C, scale bar: 100 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . Treatment with uncleaved HK2pep ( 2 µM , 48 h ; D , scale bar : 200 µm ) reverts formation of foci ; dead cells are highlighted by Trypan Blue staining . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of HK2 targeting on in vitro tumorigenicity. Treatment with uncleaved HK2pep (2 µM, 48 h; D, scale bar: 200 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 - targeting on in vivo neoplastic growth . HK2 - and MMP - 2 - expressing CT26 cells ( E , scale bar : 100 µm ) are allografted in Balb / c male mice . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of HK2-targeting on in vivo neoplastic growth. HK2- and MMP-2-expressing CT26 cells (E, scale bar: 100 µm) are allografted in Balb/c male mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 - targeting on in vivo neoplastic growth . Intratumor administration ( 5 injections of 2 nmol peptide every 12 h ) of either cl - HK2pep or HK2pep ( F ; CNT n = 4 ; cl - HK2pep n = 5 ; HK2pep n = 5 mice ) in Balb / c female mice . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control . In F , mean tumor volume ± SD is reported ; Student ' s t test * * * p < 0 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of HK2-targeting on in vivo neoplastic growth. Intratumor administration (5 injections of 2 nmol peptide every 12 h) of either cl-HK2pep or HK2pep (F; CNT n=4; cl-HK2pep n=5; HK2pep n=5 mice) in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In F, mean tumor volume±SD is reported; Student's t test ***p<0.001


Effect of HK2 - targeting on in vivo neoplastic growth . intraperitoneal administration of HK2pep ( G ; 5 injections of 60 nmol peptide every 12 h ; SCRpep n = 6 ; HK2pep n = 7 mice ) reduce neoplastic growth in Balb / c female mice . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control . In G , representative ultrasound inspections and 3D reconstructions of tumor masses are shown ( light grey : SCRpep treated animal ; light blue : HK2pep treated animal ) . normalized volume ± SEM ; a two - way ANOVA is performed ( p < 0 . 001 - HK2pep vs SCRpep ) ; Bonferroni post - test in the graphs * p < 0 . 05 ; * * * p < 0 . 001 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of HK2-targeting on in vivo neoplastic growth. intraperitoneal administration of HK2pep (G; 5 injections of 60 nmol peptide every 12 h; SCRpep n=6; HK2pep n=7 mice) reduce neoplastic growth in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In G, representative ultrasound inspections and 3D reconstructions of tumor masses are shown (light grey: SCRpep treated animal; light blue: HK2pep treated animal). normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.


Effect of HK2 - targeting on in vivo neoplastic growth . the same effect is observed on allografts of breast carcinoma 4T1 cells ( H ; 5 injections of 60 nmol peptide every 12 h ; SCRpep n = 5 ; HK2pep n = 7 mice ) in Balb / c female mice . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control . normalized volume ± SEM ; a two - way ANOVA is performed ( p < 0 . 001 - HK2pep vs SCRpep ) ; Bonferroni post - test in the graphs * p < 0 . 05 ; * * * p < 0 . 001 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of HK2-targeting on in vivo neoplastic growth. the same effect is observed on allografts of breast carcinoma 4T1 cells (H; 5 injections of 60 nmol peptide every 12 h; SCRpep n=5; HK2pep n=7 mice) in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.


( D ) RNF145 and ADIPOR2 respond to a range of FAs . HEK - 293T cells were treated for 6h with saturated FAs ( SFAs ; 400 µM ( C12 : 0 - C18 : 0 ) ; 200 µM ( C20 : 0 ) ) or unsaturated FAs ( UFAs ; 400 µM ( C18 : 1 - n9 ) ; 200 µM ( C20 : 4 - n6 , C20 : 5 - n3 ) ) . * non - specific bands .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNF145 and ADIPOR2 respond to a range of FAs. HEK-293T cells were treated for 6h with saturated FAs (SFAs; 400 µM (C12:0 - C18:0); 200 µM (C20:0)) or unsaturated FAs (UFAs; 400 µM (C18:1-n9); 200 µM (C20:4-n6, C20:5-n3)). * non-specific bands.


( E ) Inhibition of the primary lipid desaturase SCD1 affects RNF145 and ADIPOR2 levels . HEK - 293T cells were treated with the SCD1 inhibitor MF - 438 ( SCDi , 100 nM , 18h ) and RNF145 / ADIPOR2 expression analysed by immunoblotting . * non - specific bands .

O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O

(E) Inhibition of the primary lipid desaturase SCD1 affects RNF145 and ADIPOR2 levels. HEK-293T cells were treated with the SCD1 inhibitor MF-438 (SCDi, 100 nM, 18h) and RNF145/ADIPOR2 expression analysed by immunoblotting. * non-specific bands.


( F ) SCD1 overexpression affects RNF145 / ADIPOR2 . HEK - 293T cells containing an empty control vector or expressing SCD1 - HA were cultured in FCS - free DMEM for 24h and whole - cell lysates analysed . * non - specific bands .

O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR 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) SCD1 overexpression affects RNF145/ADIPOR2. HEK-293T cells containing an empty control vector or expressing SCD1-HA were cultured in FCS-free DMEM for 24h and whole-cell lysates analysed. * non-specific bands.


( A ) Endogenous ADIPOR2 is stabilised in RNF145 knockout cells . Representative 35S pulse - chase analysis in HeLa WT , RNF145 knockout ( ∆ RNF145 ) or ADIPOR2 knockout ( ∆ AR2 ) cells . Quantification of 35S - labelled ADIPOR2 normalised to t = 0 h is shown below .

O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

(A) Endogenous ADIPOR2 is stabilised in RNF145 knockout cells. Representative 35S pulse-chase analysis in HeLa WT, RNF145 knockout (∆RNF145) or ADIPOR2 knockout (∆AR2) cells. Quantification of 35S-labelled ADIPOR2 normalised to t = 0 h is shown below.


( B ) ADIPOR2 regulation requires the E3 ligase activity of RNF145 . HEK - 293T RNF145 knockout cells ( ∆ RNF145 ) were stably complemented with WT RNF145 ( RNF145 - V5 ) or an inactive RNF145C552A , H554A RING domain mutant ( mRNF145 - V5 ) . * non - specific bands .

O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(B) ADIPOR2 regulation requires the E3 ligase activity of RNF145. HEK-293T RNF145 knockout cells (∆RNF145) were stably complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). * non-specific bands.


( C ) RNF145 binds ADIPOR2 . RNF145 was immunoprecipitated from HeLa RNF145 knockout cells complemented with WT RNF145 ( RNF145 - V5 ) or an inactive RNF145C552A , H554A RING domain mutant ( mRNF145 - V5 ) . Cells were treated with VCP inhibitor ( NMS - 873 , 10 µM , 90 min ) prior to immunoprecipitation with an anti - V5 antibody .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(C) RNF145 binds ADIPOR2. RNF145 was immunoprecipitated from HeLa RNF145 knockout cells complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). Cells were treated with VCP inhibitor (NMS-873, 10 µM, 90 min) prior to immunoprecipitation with an anti-V5 antibody.


( D ) The E2 ubiquitin - conjugating enzyme UBE2G2 is involved in RNF145 - mediated ADIPOR2 degradation . Immunoblot analysis of HEK - 293T cells depleted of RNF145 ( gRNF145 ) , UBE2G2 ( gUBE2G2 ) or B2M ( control ; gCTR ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(D) The E2 ubiquitin-conjugating enzyme UBE2G2 is involved in RNF145-mediated ADIPOR2 degradation. Immunoblot analysis of HEK-293T cells depleted of RNF145 (gRNF145), UBE2G2 (gUBE2G2) or B2M (control; gCTR).


( E ) ADIPOR2 degradation depends on UBXD8 . HEK - 293T cells were depleted of B2M ( gCTR ) or UBXD8 ( gUBXD8 ) and analysed by immunoblotting .

O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(E) ADIPOR2 degradation depends on UBXD8. HEK-293T cells were depleted of B2M (gCTR) or UBXD8 (gUBXD8) and analysed by immunoblotting.


( A ) FA - mediated regulation of RNF145 is proteasome - dependent . HEK - 293T cells were treated with BSA , PA ( 400 µM , 6h ) ± MG132 ( 10 µg / ml , 6h ) and analysed by immunoblotting . * non - specific bands .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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) FA-mediated regulation of RNF145 is proteasome-dependent. HEK-293T cells were treated with BSA, PA (400 µM, 6h) ± MG132 (10 µg/ml, 6h) and analysed by immunoblotting. *non-specific bands.


( B ) UBXD8 participates in PA - modulated RNF145 degradation . CRISPR / Cas9 - mediated depletion of UBXD8 ( gUBXD8 ) or B2M ( gB2M , control ) in HEK - 293T cells , followed by exposure to PA ( 400 µM , 6h ) or BSA ( 6h , vehicle control ) .

O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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) UBXD8 participates in PA-modulated RNF145 degradation. CRISPR/Cas9-mediated depletion of UBXD8 (gUBXD8) or B2M (gB2M, control) in HEK-293T cells, followed by exposure to PA (400 µM, 6h) or BSA (6h, vehicle control).


( C ) PA and OA treatment alter RNF145 ubiquitination . Immunoprecipitation of RNF145 from cells treated with BSA ( control ) , PA or OA ( each 200 µM , 3h ) . During FA treatments , VCP inhibitor ( NMS - 873 , 10 µM ) was present to stabilise ubiquitinated proteins . RNF145 ubiquitination ( ubi ) was detected using an anti - ubiquitin antibody ( VU - 1 ) .

O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(C) PA and OA treatment alter RNF145 ubiquitination. Immunoprecipitation of RNF145 from cells treated with BSA (control), PA or OA (each 200 µM, 3h). During FA treatments, VCP inhibitor (NMS-873, 10 µM) was present to stabilise ubiquitinated proteins. RNF145 ubiquitination (ubi) was detected using an anti-ubiquitin antibody (VU-1).


( D ) HEK - 293T cells were treated with cycloheximide ( CHX , 1 µg / ml ) and OA ( 400 µM , 6h ) ± MG132 ( 10 µg / ml ) for the indicated times . LE , long exposure ; * non - specific bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK-293T cells were treated with cycloheximide (CHX, 1 µg/ml) and OA (400 µM, 6h) ± MG132 (10 µg/ml) for the indicated times. LE, long exposure; * non-specific bands.


( E ) Lysine - less RNF145 is stabilised under PA treatment . RNF145 knockout HEK - 293T cells ( ∆ RNF145 ) were complemented with RNF145 - V5 or a lysine - less variant of RNF145 ( RNF145 - V5 K > R ) and treated BSA or PA ( 400 µM , 6h ) . Dotted lines demarcate the different cell lines .

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(E) Lysine-less RNF145 is stabilised under PA treatment. RNF145 knockout HEK-293T cells (∆RNF145) were complemented with RNF145-V5 or a lysine-less variant of RNF145 (RNF145-V5 K > R) and treated BSA or PA (400 µM, 6h). Dotted lines demarcate the different cell lines.


( F ) Adjustment of RNF145 levels to PA requires its ubiquitin ligase activity . Immunoblot of whole - cell lysates from RNF145 knockout HEK - 293T ( ∆ RNF145 ) cells stably complemented with RNF145 - V5 or RNF145C552A , H554A - V5 ( mRNF145 - V5 ) and treated with BSA or PA ( 400 µM , 6h ) . LE , long exposure .

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(F) Adjustment of RNF145 levels to PA requires its ubiquitin ligase activity. Immunoblot of whole-cell lysates from RNF145 knockout HEK-293T (∆RNF145) cells stably complemented with RNF145-V5 or RNF145C552A, H554A-V5 (mRNF145-V5) and treated with BSA or PA (400 µM, 6h). LE, long exposure.


( A ) ADIPOR2 regulation by FAs is completely dependent on RNF145 . WT or RNF145 knockout HEK - 293T cells ( ∆ RNF145 ) were treated with BSA , PA or OA ( 400 µM , 6h ) and analysed by immunoblotting . Non - specific bands are indicated ( * ) .

O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O 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) ADIPOR2 regulation by FAs is completely dependent on RNF145. WT or RNF145 knockout HEK-293T cells (∆RNF145) were treated with BSA, PA or OA (400 µM, 6h) and analysed by immunoblotting. Non-specific bands are indicated (*).


( B ) RNF145 - ADIPOR2 interaction is FA - modulated . Immunoprecipitation of endogenous RNF145 HEK - 293T cells depleted of UBE2G2 ( gUBE2G2 ) and / or RNF145 ( gRNF145 ) and treated with 400 µM of the indicated PA , OA or BSA ( control ) for 3h . LE , long exposure . * non - specific bands .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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) RNF145 - ADIPOR2 interaction is FA-modulated. Immunoprecipitation of endogenous RNF145 HEK-293T cells depleted of UBE2G2 (gUBE2G2) and/or RNF145 (gRNF145) and treated with 400 µM of the indicated PA, OA or BSA (control) for 3h. LE, long exposure. * non-specific bands.


( C ) Immunoprecipitation ( IP ) of endogenous ADIPOR2 from WT or ∆ RNF145 cells ( see ( C ) ) treated with MG132 ( 5 µg / ml , 6h ) ± OA ( 200 µM , 6h ) . Ubiquitinated ADIPOR2 was detected using a specific anti - ubiquitin antibody ( clone VU - 1 ) . Note that ADIPOR2 levels in RNF145 knockout HEK - 293T cells are increased .

O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O

(C) Immunoprecipitation (IP) of endogenous ADIPOR2 from WT or ∆RNF145 cells (see (C)) treated with MG132 (5 µg/ml, 6h) ± OA (200 µM, 6h). Ubiquitinated ADIPOR2 was detected using a specific anti-ubiquitin antibody (clone VU-1). Note that ADIPOR2 levels in RNF145 knockout HEK-293T cells are increased.


( D ) ADIPOR2 knockout HEK - 293T cells were stably complemented with ADIPOR2 - HA , an ADIPOR2 mutant in which all lysines were mutated to arginines ( K - less ADIPOR2 - HA ) , or an empty vector control and exposed to OA ( 200 µM , 6h ) or BSA ( control ) . ADIPOR2 variants were visualised by immunoblotting using a specific anti - HA antibody . LE , long exposure .

O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(D) ADIPOR2 knockout HEK-293T cells were stably complemented with ADIPOR2-HA, an ADIPOR2 mutant in which all lysines were mutated to arginines (K-less ADIPOR2-HA), or an empty vector control and exposed to OA (200 µM, 6h) or BSA (control). ADIPOR2 variants were visualised by immunoblotting using a specific anti-HA antibody. LE, long exposure.


( A ) Loss of ADIPOR2 sensitises cells to PA - induced cytotoxicity . HEK - 293T cells were depleted of RNF145 ( purple ) , ADIPOR2 ( blue ) , B2M ( CTR ; grey ) , or both ADIPOR2 and RNF145 ( blue + purple ) , exposed to PA ( 200 µM , 96h ) and stained with crystal violet . Quantification of crystal - violet stained cells is shown alongside a representative micrograph ( below ) . The response of each cell line to PA was normalised to its BSA - treated condition ( dotted line ) . Statistically significant differences to cells containing gCTR cells are indicated ( robust one - way ANOVA with Benjamini - Hochberg multiple testing correction ) . Mean ± SD are shown . Boxplots represent the median , first and third quantiles . Upper and lower whiskers extend to values up to 1 . 5 - fold interquartile range . N = 3 biological replicates , * * q ≤ 0 . 01 . Double knockdowns were generated by sequential constitutive CRISPR / Cas9 - mediated depletion of RNF145 or ADIPOR2 . Knockdown efficiencies are shown in Appendix Fig S1A .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

(A) Loss of ADIPOR2 sensitises cells to PA-induced cytotoxicity. HEK-293T cells were depleted of RNF145 (purple), ADIPOR2 (blue), B2M (CTR; grey), or both ADIPOR2 and RNF145 (blue + purple), exposed to PA (200 µM, 96h) and stained with crystal violet. Quantification of crystal-violet stained cells is shown alongside a representative micrograph (below). The response of each cell line to PA was normalised to its BSA-treated condition (dotted line). Statistically significant differences to cells containing gCTR cells are indicated (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction). Mean ± SD are shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. N = 3 biological replicates, ** q ≤ 0.01. Double knockdowns were generated by sequential constitutive CRISPR/Cas9-mediated depletion of RNF145 or ADIPOR2. Knockdown efficiencies are shown in Appendix Fig S1A.


( B ) Cells were treated as in ( A ) and lysates analysed by immunoblotting . LE , long exposure ; * non - specific bands .

O O O O 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) Cells were treated as in (A) and lysates analysed by immunoblotting. LE, long exposure; *non-specific bands.


( C ) HEK - 293T cells depleted of B2M ( gCTR ) or ADIPOR2 ( gADIPOR2 ) were complemented with empty vector ( blue ) , full - length ADIPOR2 - HA ( green ) , or a catalytically inactive variant of ADIPOR2 - HA ( dead ADIPOR2 ; red ) and treated with PA ( 200 µM , 96h ) or BSA ( vehicle control , 96h ) . Measurements from PA - treated cells were normalised to their BSA - treated condition ( dotted line ) . Cell growth was quantified , and representative crystal violet staining is shown . Boxplots represent the median , first and third quantiles . Upper and lower whiskers extend to values up to 1 . 5 - fold interquartile range . * * q ≤ 0 . 01 ( robust one - way ANOVA with Benjamini - Hochberg multiple testing correction ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK-293T cells depleted of B2M (gCTR) or ADIPOR2 (gADIPOR2) were complemented with empty vector (blue), full-length ADIPOR2-HA (green), or a catalytically inactive variant of ADIPOR2-HA (dead ADIPOR2; red) and treated with PA (200 µM, 96h) or BSA (vehicle control, 96h). Measurements from PA-treated cells were normalised to their BSA-treated condition (dotted line). Cell growth was quantified, and representative crystal violet staining is shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. ** q ≤ 0.01 (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction).


Expression level of Shox2 , Tbx15 , HoxC8 , and HoxC5 , mRNA was compared using quantitative real - time PCR ( qPCR ) of RNA isolated from different fat depots . Two subcutaneous depots ( inguinal and scapular white ) , three intra - abdominal fat depots ( perigonadal , perirenal , and mesenteric ) , and interscapular brown adipose tissue of 8 week - old male C57BL / 6 mice . Data are shown as mean ± SEM of six samples . Expression level of Shox2 , Tbx15 , HoxC8 , and HoxC5 , mRNA was compared using qPCR of RNA isolated from cultured stromovascular cells derived from adipose tissue of the Immortomouse ™ . The depot - specific cell lines were made from inguinal , scapular white , perigonadal , perirenal , mesenteric , and interscapular brown adipose tissue of 8 week - old male mice . Data are shown as mean ± SEM of cell lines from three individual mice .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using quantitative real-time PCR (qPCR) of RNA isolated from different fat depots. Two subcutaneous depots (inguinal and scapular white), three intra-abdominal fat depots (perigonadal, perirenal, and mesenteric), and interscapular brown adipose tissue of 8 week-old male C57BL/6 mice. Data are shown as mean ± SEM of six samples. Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using qPCR of RNA isolated from cultured stromovascular cells derived from adipose tissue of the Immortomouse™. The depot-specific cell lines were made from inguinal, scapular white, perigonadal, perirenal, mesenteric, and interscapular brown adipose tissue of 8 week-old male mice. Data are shown as mean ± SEM of cell lines from three individual mice.


Bright - field image of depot - derived cell lines after in vitro adipogenic differentiation and Oil Red O staining ( Lee et al . , 2017a ) . Original photographs were taken at 10x magnification ( left panel ) . Scale bar = 100 μm .

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Bright-field image of depot-derived cell lines after in vitro adipogenic differentiation and Oil Red O staining (Lee et al., 2017a). Original photographs were taken at 10x magnification (left panel). Scale bar = 100 μm.


qPCR analysis for adiponectin ( Adipoq ) , peroxisome proliferator - activated receptor gamma ( Pparγ2 ) , and fatty acid synthase ( FAS ) in RNA isolated from depot - derived cell lines after in vitro differentiation . Data are shown as mean ± SEM of 3 cell lines / group .

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qPCR analysis for adiponectin (Adipoq), peroxisome proliferator-activated receptor gamma (Pparγ2), and fatty acid synthase (FAS) in RNA isolated from depot-derived cell lines after in vitro differentiation. Data are shown as mean ± SEM of 3 cell lines/group.


Representative picture of preadipocyte clonal cell lines showing media color 48 hours after last media change . Cells in all wells were 100 % confluent when media was changed .

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Representative picture of preadipocyte clonal cell lines showing media color 48 hours after last media change. Cells in all wells were 100% confluent when media was changed.


Heatmap depicting the 50 most variable genes among all preadipocyte clonal cell lines . Red represents high expression of genes , while blue represents low expression of genes . Heatmap depicting the 50 most variable genes among Type 2 and 3 preadipocyte clonal cell lines . Red represents high expression of genes , while blue represents low expression of genes .

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Heatmap depicting the 50 most variable genes among all preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes. Heatmap depicting the 50 most variable genes among Type 2 and 3 preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes.


Expression of Wt1 , leucine rich repeat neuronal 4 ( Lrrn4 ) , Uroplakin 3b ( Upk3b ) , in Immortomouse clonal cell line clusters as preadipocytes ( d0 ) and during six days of adipogenic differentiation .

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Expression of Wt1, leucine rich repeat neuronal 4 (Lrrn4), Uroplakin 3b (Upk3b), in Immortomouse clonal cell line clusters as preadipocytes (d0) and during six days of adipogenic differentiation.


Bright - field images of preadipocyte cell lines under standard culture conditions . The photographs were taken at 10x magnification .

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Bright-field images of preadipocyte cell lines under standard culture conditions. The photographs were taken at 10x magnification.


Expression of Tagln , connective tissue growth factor ( Ctgf ) , Keratin 19 ( Krt19 ) , Mx1 , caspase 1 ( Casp1 ) , and C - X - C Motif Chemokine Ligand 12 ( Cxcl12 ) in Immortomouse clonal cell line clusters as preadipocytes ( d0 ) and during six days of adipogenic differentiation .

Expression of Tagln, connective tissue growth factor (Ctgf), Keratin 19 (Krt19), Mx1, caspase 1 (Casp1), and C-X-C Motif Chemokine Ligand 12 (Cxcl12) in Immortomouse clonal cell line clusters as preadipocytes (d0) and during six days of adipogenic differentiation.


Triglyceride content of cell cultures after six days of differentiation using the protocol described in Methods . Data are shown as mean ± SEM of 3 - 7 samples , and the entire experiment was repeated twice .

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Triglyceride content of cell cultures after six days of differentiation using the protocol described in Methods. Data are shown as mean ± SEM of 3-7 samples, and the entire experiment was repeated twice.


[ 3H ] - labeled 2 - deoxyglucose uptake in preadipocyte cell lines in the basal state and after pretreatment for 20 minutes with 100 nM insulin . Uptake was measured for one hour , and data are shown as mean ± SEM of 3 - 7 samples and repeated twice . [ 3H ] - labeled 2 - deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin . Uptake was measured for one hour , and data are shown as mean ± SEM of 3 - 7 samples . The experiment was repeated twice .

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[3H]-labeled 2-deoxyglucose uptake in preadipocyte cell lines in the basal state and after pretreatment for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples and repeated twice. [3H]-labeled 2-deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.


[ 14C ] D - glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin . [ 14C ] D - glucose incorporation was measured after three hours , and data are shown as mean ± SEM of 3 - 7 samples . The experiment was repeated twice .

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[14C] D-glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. [14C] D-glucose incorporation was measured after three hours, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.


Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation . Data are shown as mean ± SEM of 3 - 7 replicates and normalized by lipid content of the cells . The entire experiment was repeated twice .

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Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation. Data are shown as mean ± SEM of 3-7 replicates and normalized by lipid content of the cells. The entire experiment was repeated twice.


Extracellular acidification rate ( ECAR ) of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve ( AUC ) during measurements of basal respiration . The whole experiment was repeated three times . Data are shown as mean ± SEM of 3 - 7 cell lines per group .

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Extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of basal respiration. The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.


Basal respiration of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve ( AUC ) during measurements of oxygen consumption rate ( OCR ) . The whole experiment was repeated three times . Data are shown as mean ± SEM of 3 - 7 cell lines per group .

Basal respiration of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.


Ratio of oxygen consumption rate ( OCR ) to extracellular acidification rate ( ECAR ) of preadipocytes and adipocytes after 4 days of in vitro differentiation . Data are shown as mean ± SEM of 3 - 7 cell lines per group .

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Ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation. Data are shown as mean ± SEM of 3-7 cell lines per group.


Representative Western blot analysis of three independent adipocyte cell lines after 6 days of in vitro differentiation for : pJnk T183 / Y185 and total Jnk after treatment with 10 nM TNFα for 15 minutes ( Inflammation ) ; pAkt S473 and total Akt after treatment with 100 nM insulin for 15 minutes ( Insulin Signaling ) ; pStat5 Y694 and total Stat5 after treatment with 500 nM GH for 20 minutes ( Growth Hormone ) .

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Representative Western blot analysis of three independent adipocyte cell lines after 6 days of in vitro differentiation for: pJnk T183/Y185 and total Jnk after treatment with 10 nM TNFα for 15 minutes (Inflammation); pAkt S473 and total Akt after treatment with 100 nM insulin for 15 minutes (Insulin Signaling); pStat5 Y694 and total Stat5 after treatment with 500 nM GH for 20 minutes (Growth Hormone).


Expression of Wt1 , Lrrn4 , Upk3b , Tagln , Ctgf , Krt19 , Mx1 , Casp1 , and Cxcl12 in primary preadipocytes isolated from white adipose tissue of 5 - 6 month old Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , and Mx1 - cre ; Rosa26mT / mG male mice . mGFP and mTomato positive preadipocytes were isolated from the pooled visceral fat depots ( perigonadal , perirenal , mesenteric , and pericardial ) of Wt1 - creERT2 ; Rosa26mT / mG mice , the pooled subcutaneous fat depots ( subcutaneous and scapular white ) of Mx1 - cre ; Rosa26mT / mG , and both the pooled visceral ( Tagln - V ) and subcutaneous depots ( Tagln - S ) from Tagln - cre ; Rosa26mT / mG mice . Data are shown as mean ± SEM of 4 - 6 mice / group .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Expression of Wt1, Lrrn4, Upk3b, Tagln, Ctgf, Krt19, Mx1, Casp1, and Cxcl12 in primary preadipocytes isolated from white adipose tissue of 5-6 month old Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG male mice. mGFP and mTomato positive preadipocytes were isolated from the pooled visceral fat depots (perigonadal, perirenal, mesenteric, and pericardial) of Wt1-creERT2;Rosa26mT/mG mice, the pooled subcutaneous fat depots (subcutaneous and scapular white) of Mx1-cre;Rosa26mT/mG, and both the pooled visceral (Tagln-V) and subcutaneous depots (Tagln-S) from Tagln-cre;Rosa26mT/mG mice. Data are shown as mean ± SEM of 4-6 mice/group.


Bright - field image of immortalized mGFP and mTomato positive preadipocytes after in vitro adipogenic differentiation . Neutral lipoids were stained with Oil Red O and original photographs were at 10x magnification ( left panel ) . Scale bar = 100 μm .

Bright-field image of immortalized mGFP and mTomato positive preadipocytes after in vitro adipogenic differentiation. Neutral lipoids were stained with Oil Red O and original photographs were at 10x magnification (left panel). Scale bar = 100 μm.


Triglyceride content of immortalized mGFP and mTomato positive preadipocytes after six days of differentiation . Data are shown as mean ± SEM of 5 - 8 replicates , and the entire experiment was repeated with three independent cell lines .

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Triglyceride content of immortalized mGFP and mTomato positive preadipocytes after six days of differentiation. Data are shown as mean ± SEM of 5-8 replicates, and the entire experiment was repeated with three independent cell lines.


Basal extracellular acidification rate ( ECAR ) of immortalized mGFP and mTomato positive preadipocytes was determined by calculating the area under the curve ( AUC ) in basal conditions . The whole experiment was repeated twice . Data are shown as mean ± SEM of 3 cell lines per group .

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

Basal extracellular acidification rate (ECAR) of immortalized mGFP and mTomato positive preadipocytes was determined by calculating the area under the curve (AUC) in basal conditions. The whole experiment was repeated twice. Data are shown as mean ± SEM of 3 cell lines per group.


Basal respiration of preadipocytes was of immortalized mGFP and mTomato positive preadipocytes determined by calculating the area under the curve ( AUC ) during measurements of oxygen consumption rate ( OCR ) . The whole experiment was repeated twice . Data are shown as mean ± SEM of 3 cell lines per group .

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

Basal respiration of preadipocytes was of immortalized mGFP and mTomato positive preadipocytes determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated twice. Data are shown as mean ± SEM of 3 cell lines per group.


Ratio of basal oxygen consumption rate ( OCR ) to basal extracellular acidification rate ( ECAR ) of immortalized mGFP and mTomato positive preadipocytes . Data are shown as mean ± SEM of 3 cell lines per group .

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Ratio of basal oxygen consumption rate (OCR) to basal extracellular acidification rate (ECAR) of immortalized mGFP and mTomato positive preadipocytes. Data are shown as mean ± SEM of 3 cell lines per group.


Number of mGFP and mTomato positive preadipocytes isolated by FACS from the indicated white adipose depots from 5 - 6 month old male Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , and Mx1 - cre ; Rosa26mT / mG mice . Data are shown as mean ± SEM of 4 - 9 mice .

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Number of mGFP and mTomato positive preadipocytes isolated by FACS from the indicated white adipose depots from 5-6 month old male Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG mice. Data are shown as mean ± SEM of 4-9 mice.


Representative images of whole - mount preparations of the indicated white adipose tissue depots of 5 - 6 month old Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , and Mx1 - cre ; Rosa26mT / mG male mice . The photographs were taken at 10x magnification . Scale bar = 100 μm .

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Representative images of whole-mount preparations of the indicated white adipose tissue depots of 5-6 month old Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG male mice. The photographs were taken at 10x magnification. Scale bar = 100 μm.


Quantitation of mGFP and mTomato positive adipocytes from each of the indicated white adipose depots from 5 - 6 month old male Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , and Mx1 - cre ; Rosa26mT / mG mice . Adipocytes were counted from four non - overlapping images per depot per mouse .

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Quantitation of mGFP and mTomato positive adipocytes from each of the indicated white adipose depots from 5-6 month old male Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG mice. Adipocytes were counted from four non-overlapping images per depot per mouse.


Number of mGFP positive preadipocytes isolated by FACS from each of the indicated white adipose depots from 5 - 6 month - old Wt1 - creERT2 , Tagln - cre , and Mx1 - cre crossed to Rosa26mT / mG mice from the experiment shown in Figure 6B . Data are shown as mean ± SEM of 4 - 9 mice .

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Number of mGFP positive preadipocytes isolated by FACS from each of the indicated white adipose depots from 5-6 month-old Wt1-creERT2, Tagln-cre, and Mx1-cre crossed to Rosa26mT/mG mice from the experiment shown in Figure 6B. Data are shown as mean ± SEM of 4-9 mice.


Number of mGFP positive adipocytes from each of the indicated white adipose depots from 5 - 6 month - old Wt1 - creERT2 , Tagln - cre , and Mx1 - cre crossed to Rosa26mT / mG mice . Four separate non - overlapping images / depot / mouse from the experiment shown in Figure 6C . Data are shown as mean ± SEM of 4 - 9 mice .

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Number of mGFP positive adipocytes from each of the indicated white adipose depots from 5-6 month-old Wt1-creERT2, Tagln-cre, and Mx1-cre crossed to Rosa26mT/mG mice. Four separate non-overlapping images/depot/mouse from the experiment shown in Figure 6C. Data are shown as mean ± SEM of 4-9 mice.


( F ) TIRF images of Ramos cells transduced with endophilin A2 - GFP and mCherry - ClathrinLC on antigen - loaded PMS . Scale bar = 5 μm . White arrowheads highlight endophilin A2 colocalization with antigen clusters .

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(F) TIRF images of Ramos cells transduced with endophilin A2-GFP and mCherry-ClathrinLC on antigen-loaded PMS. Scale bar = 5 μm. White arrowheads highlight endophilin A2 colocalization with antigen clusters.


( J ) IgM - BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs . Anti - endophilin A2 blots detect overexpressed endophilin A2 - GFP in streptavidin immunoprecipitates and total supernatants from anti - IgM - biotin - stimulated Ramos and DG75 cells . Representative experiment and quantification from 3 independent pulldowns in WT , BLNK - or GRB2 - knockout ( KO ) DG75 cells . Pulldown intensity is calculated as a percentage of total endophilin A2 protein and normalized to WT in each experiment . Data show mean and SEM ; p values calculated using one - way ANOVA .

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(J) IgM-BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs. Anti-endophilin A2 blots detect overexpressed endophilin A2-GFP in streptavidin immunoprecipitates and total supernatants from anti-IgM-biotin-stimulated Ramos and DG75 cells. Representative experiment and quantification from 3 independent pulldowns in WT, BLNK- or GRB2-knockout (KO) DG75 cells. Pulldown intensity is calculated as a percentage of total endophilin A2 protein and normalized to WT in each experiment. Data show mean and SEM; p values calculated using one-way ANOVA.


( E ) Quantification of surface CD86 and CD69 mean fluorescence intensity ( MFI ) . Data show mean and SEM from 6 mice across 2 experiments .

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(E) Quantification of surface CD86 and CD69 mean fluorescence intensity (MFI). Data show mean and SEM from 6 mice across 2 experiments.


( F ) Fractions of mCherry + cells in GC , PC , IgG1 + class - switched and follicular B cells at 4 - , 8 - and 14 - days post SRBC - HEL immunization . N = 8 - 10 mice ; data shows mean and SEM . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 using 2 - way ANOVA with multiple comparisons .

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(F) Fractions of mCherry+ cells in GC, PC, IgG1+ class-switched and follicular B cells at 4-, 8- and 14-days post SRBC-HEL immunization. N = 8-10 mice; data shows mean and SEM. *p<0.05; ***p<0.001; ****p<0.0001 using 2-way ANOVA with multiple comparisons.


( C ) Surface levels of MHCII , BAFFR , CD40 and LPAM1 in Sh3gl1 - / - and WT littermates . N = 4 - 5 mice . P , statistical significance from unpaired t tests . Data show mean ± SEM .

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(C) Surface levels of MHCII, BAFFR, CD40 and LPAM1 in Sh3gl1-/- and WT littermates. N = 4-5 mice. P, statistical significance from unpaired t tests. Data show mean ± SEM.


( D ) Quantification of B220 + Fas + CD38 - GC population as percentage of total splenic B cells and NP - specific cells as a percentage of the GC population 14 days after NP - CGG immunization in alum . Data show mean ± SEM from N = 6 mice , p , significance in unpaired t tests .

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(D) Quantification of B220+Fas+CD38- GC population as percentage of total splenic B cells and NP-specific cells as a percentage of the GC population 14 days after NP-CGG immunization in alum. Data show mean ± SEM from N = 6 mice, p, significance in unpaired t tests.


( E - F ) Serum antibodies collected pre - immunization and 7 - or 14 - days post NP - CGG immunization , detected by NP7 - BSA ELISA using isotype - specific secondary antibodies . Data show mean ± SEM from N = 3 experiments with statistical significance from 2 - way ANOVA with multiple comparisons .

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(E-F) Serum antibodies collected pre-immunization and 7- or 14-days post NP-CGG immunization, detected by NP7-BSA ELISA using isotype-specific secondary antibodies. Data show mean ± SEM from N = 3 experiments with statistical significance from 2-way ANOVA with multiple comparisons.


( H ) Numbers of Tfh cells 14 days post - immunization , identified as a CD4 + CD44 + PD - 1 + CXCR5 + population . Mean and SEM of N = 4 mice .

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(H) Numbers of Tfh cells 14 days post-immunization, identified as a CD4+CD44+PD-1+CXCR5+ population. Mean and SEM of N = 4 mice.


( A ) Soluble anti - IgM internalization in CRISPR - targeted follicular B cells . N = 11 - 12 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA . ( B ) Soluble anti - IgD internalization . N = 3 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA . ( C ) Soluble anti - IgG internalization in GC B cells from SRBC - immunized mice . N = 4 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA .

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(A) Soluble anti-IgM internalization in CRISPR-targeted follicular B cells. N = 11- 12 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA. (B) Soluble anti-IgD internalization. N = 3 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA. (C) Soluble anti-IgG internalization in GC B cells from SRBC-immunized mice. N = 4 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA.


( F ) IgM degradation following total cell surface biotinylation , measured as loss of biotin - labelled membrane IgM ( mIgM ) in cell lysates over time . ( G ) Western densitometry quantifying IgM degradation assay . Proportion of biotin detected is normalized to the initial WT condition ( left ) or to the initial amount for each genotype ( right ) . Data show mean ± SEM from three independent tests . P , statistical significance from two - way ANOVA .

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(F) IgM degradation following total cell surface biotinylation, measured as loss of biotin-labelled membrane IgM (mIgM) in cell lysates over time. (G) Western densitometry quantifying IgM degradation assay. Proportion of biotin detected is normalized to the initial WT condition (left) or to the initial amount for each genotype (right). Data show mean ± SEM from three independent tests. P, statistical significance from two-way ANOVA.


( K ) Proliferation ( CFSE dilution ) of OTII CD4 cells in co - cultures with anti - Igκ - OVA pulsed B cells ; and quantification from 6 - 8 mice . Data show mean ± SEM .

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(K) Proliferation (CFSE dilution) of OTII CD4 cells in co-cultures with anti-Igκ-OVA pulsed B cells; and quantification from 6-8 mice. Data show mean ± SEM.


( C - D ) Isolated naïve B cells from CRISPR - targeted chimeras after 3 - day culture in stated cytokines . ( C ) mCherry percentage in targeted primary cell cultures . N = 15 mice . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 using two - way ANOVA . ( D ) AnnexinV surface stain in targeted cells . N = 3 - 4 mice . Data information : All data show means ± SEM .

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(C-D) Isolated naïve B cells from CRISPR-targeted chimeras after 3-day culture in stated cytokines. (C) mCherry percentage in targeted primary cell cultures. N = 15 mice. *p<0.05, **p<0.01, ****p<0.0001 using two-way ANOVA. (D) AnnexinV surface stain in targeted cells. N = 3-4 mice. Data information: All data show means ± SEM.


( G ) Oxygen consumption rate ( OCR ) in CRISPR - targeted Ramos cells . N = 2 independent infections . P , two - way ANOVA of basal and maximum OCR . ( H ) OCR in B cells isolated from Sh3gl1 - / - and WT littermates . N = 9 mice . P , two - way ANOVA of basal and maximum OCR .

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(G) Oxygen consumption rate (OCR) in CRISPR-targeted Ramos cells. N = 2 independent infections. P, two-way ANOVA of basal and maximum OCR. (H) OCR in B cells isolated from Sh3gl1-/- and WT littermates. N = 9 mice. P, two-way ANOVA of basal and maximum OCR.


Cell cultures with full RPMI media supplemented with 50 μg / ml FeSO4 or 5 μM hemin chloride . ( J ) SH3GL1 - targeted Ramos cells . N = 4 independent infections . P , statistical significance from two - way ANOVA from both treatments versus RPMI . ( K ) Control sgRNA - targeted Ramos cells .

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Cell cultures with full RPMI media supplemented with 50 μg/ml FeSO4 or 5 μM hemin chloride. (J) SH3GL1-targeted Ramos cells. N = 4 independent infections. P, statistical significance from two-way ANOVA from both treatments versus RPMI. (K) Control sgRNA-targeted Ramos cells.


( O ) TfR internalization assay in Ramos cells using biotinylated anti - CD71 . 4 independent CRISPR infections in 2 experiments . P , significance calculated using two - way ANOVA and indicated in corresponding color .

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(O) TfR internalization assay in Ramos cells using biotinylated anti-CD71. 4 independent CRISPR infections in 2 experiments. P, significance calculated using two-way ANOVA and indicated in corresponding color.


( P , Q ) Mouse transferrin internalization in B cells from SRBC - immunized WT and Sh3gl1 - / - littermates . N = 7 mice per genotype in 2 experiments . P , statistical significance analyzed using two - way ANOVA . ( P ) Naïve B cells . ( Q ) GC B cells .

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(P,Q) Mouse transferrin internalization in B cells from SRBC-immunized WT and Sh3gl1-/- littermates. N = 7 mice per genotype in 2 experiments. P, statistical significance analyzed using two-way ANOVA. (P) Naïve B cells. (Q) GC B cells.


A Robust immunofluorescence detection of ubiquitination on LAMP1 - positive compartments with pan - ubiquitin FK2 or K48 chain - specific antibodies . HeLa cells were treated with 250 µM LLOMe or EtOH ( untreated ) for 3 h , fixed and stained with indicated antibodies . Confocal microscopy was followed by automated image analysis . Graphs represent data from three independent experiments with ≥ 50 cells per condition ( mean ± SD ) . Scale bar : 20 µm .

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A Robust immunofluorescence detection of ubiquitination on LAMP1-positive compartments with pan-ubiquitin FK2 or K48 chain-specific antibodies. HeLa cells were treated with 250 µM LLOMe or EtOH (untreated) for 3 h, fixed and stained with indicated antibodies. Confocal microscopy was followed by automated image analysis. Graphs represent data from three independent experiments with ≥ 50 cells per condition (mean ± SD). Scale bar: 20 µm.


C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes . HeLa cells were seeded in 384 - well plates , transfected with a siRNA library in quadruplicates for 72 h . Cells were LLOMe - treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope . Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined . Robust z - scores were calculated for LLOMe - treated samples for each depletion against the values of the whole plate . Samples exceeding the threshold of - 2 of robust z - score ( highlighted in red ) were considered for the secondary screen .

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C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes. HeLa cells were seeded in 384-well plates, transfected with a siRNA library in quadruplicates for 72 h. Cells were LLOMe-treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope. Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined. Robust z-scores were calculated for LLOMe-treated samples for each depletion against the values of the whole plate. Samples exceeding the threshold of -2 of robust z-score (highlighted in red) were considered for the secondary screen.


D Selected candidates were re - screened with the same work pipeline but with the four single siRNAs of pools of each candidate from the primary screen . Candidates exceeding the threshold in both stainings are highlighted in red .

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D Selected candidates were re-screened with the same work pipeline but with the four single siRNAs of pools of each candidate from the primary screen. Candidates exceeding the threshold in both stainings are highlighted in red.


E Damage - induced translocation of UBE2QL1 to lysosomes . The three best candidates ( from D ) were transiently expressed with N - terminal HA - tag in HeLa cells . Cells were treated with LLOMe or EtOH ( untreated ) for 3 h , fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy . Scale bar : 10 µm . F Automated quantification of ( E ) . Shown are Pearson correlation coefficients ( P . C . C ) of HA and LAMP1 signals . Graph represents data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) . * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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E Damage-induced translocation of UBE2QL1 to lysosomes. The three best candidates (from D) were transiently expressed with N-terminal HA-tag in HeLa cells. Cells were treated with LLOMe or EtOH (untreated) for 3 h, fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy. Scale bar: 10 µm. F Automated quantification of (E). Shown are Pearson correlation coefficients (P.C.C) of HA and LAMP1 signals. Graph represents data from three independent experiments with ≥ 30 cells per condition (mean ± SD). ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


G Western blot detection of endogenous UBE2QL1 and depletion efficiency . Cells were transfected with control siRNA ( Ctrl ) , two UBE2QL1 siRNAs from the screen ( # 2 or # 4 ) or an additional siRNA ( # 5 ) , and lysates probed with an antibody to UBE2QL1 as indicated . GAPDH was probed as loading control .

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G Western blot detection of endogenous UBE2QL1 and depletion efficiency. Cells were transfected with control siRNA (Ctrl), two UBE2QL1 siRNAs from the screen (#2 or #4) or an additional siRNA (#5), and lysates probed with an antibody to UBE2QL1 as indicated. GAPDH was probed as loading control.


A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h , and LLOMe or control - treated ( untreated ) for 3 h . Cells were fixed and immuno - stained for LAMP1 along with antibodies specific for K63 or K48 - linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope . Note the strong reduction of damage - induced K48 ubiquitination and decreased intensity of the K63 signal . Scale bar : 20 µm . B , C Automated image quantification of ( A ) . Shown are percentages of K48 or K63 - positive LAMP1 vesicles per cell . Graphs represent data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h, and LLOMe or control-treated (untreated) for 3 h. Cells were fixed and immuno-stained for LAMP1 along with antibodies specific for K63 or K48-linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope. Note the strong reduction of damage-induced K48 ubiquitination and decreased intensity of the K63 signal. Scale bar: 20 µm. B, C Automated image quantification of (A). Shown are percentages of K48 or K63-positive LAMP1 vesicles per cell. Graphs represent data from three independent experiments with ≥ 70 cells per condition (mean ± SD). **P<0.01; ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


A HeLa cells expressing UBE2QL1 - HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy . Cells were stained with antibodies specific for LAMP1 ( not shown ) , and combinations of HA with K48 chains , K63 chains or p62 as indicated . Note the delayed co - emergence of K48 chains and UBE2QL1 vesicles , compared to the earlier appearance of K63 chains and p62 . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 10 µm , 2 µm for inlays . B Automated quantification of ( A ) . Shown are the Pearson correlation coefficients ( P . C . C . ) representing colocalization of UBE2QL1 - HA , K48 chains , K63 chains or p62 with LAMP1 - positive vesicles . Graphs represent data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) . C Automated quantification of ( A ) . P . C . C . ' s representing colocalization of K48 chains , K63 chains or p62 with UBE2QL1 - HA in cells treated with LLOMe or vehicle alone ( untreated ) for 180 min . Graphs represent data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) .

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A HeLa cells expressing UBE2QL1-HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy. Cells were stained with antibodies specific for LAMP1 (not shown), and combinations of HA with K48 chains, K63 chains or p62 as indicated. Note the delayed co-emergence of K48 chains and UBE2QL1 vesicles, compared to the earlier appearance of K63 chains and p62. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 10 µm, 2 µm for inlays. B Automated quantification of (A). Shown are the Pearson correlation coefficients (P.C.C.) representing colocalization of UBE2QL1-HA, K48 chains, K63 chains or p62 with LAMP1-positive vesicles. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD). C Automated quantification of (A). P.C.C.'s representing colocalization of K48 chains, K63 chains or p62 with UBE2QL1-HA in cells treated with LLOMe or vehicle alone (untreated) for 180 min. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD).


Immuno - electron microscopy of LLOMe - treated HeLa cells . Cells in ( A ) overexpress mCherry - Gal3 and a dominant - negative C160S mutant of GFP - YOD1 , and were immunostained for p62 ( A ) p62 ( 10 nm gold particles ) localizes as a cap ( arrowheads ) to the outer limiting membrane of lysosomes ( L ) . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (A) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 (A) p62 (10 nm gold particles) localizes as a cap (arrowheads) to the outer limiting membrane of lysosomes (L). Scale bars: 200 nm.


Immuno - electron microscopy of LLOMe - treated HeLa cells . Cells in ( B ) overexpress mCherry - Gal3 and a dominant - negative C160S mutant of GFP - YOD1 , and were immunostained for p62 and K48 . ( B ) K48 ubiquitin chains ( 15 nm gold particles , arrows ) are found in the lumen of lysosomes , that also contain p62 ( 10 nm gold particles , arrowheads ) at their outer membrane . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (B) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 and K48. (B) K48 ubiquitin chains (15 nm gold particles, arrows) are found in the lumen of lysosomes, that also contain p62 (10 nm gold particles, arrowheads) at their outer membrane. Scale bars: 200 nm.


Immuno - electron microscopy of LLOMe - treated HeLa cells . ( C ) Overexpressed UBE2QL1 - GFP ( 10 nm gold particles ) is found in late endosomes ( LE , Inset ) and lysosomes . M = mitochondrion . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. (C) Overexpressed UBE2QL1-GFP (10 nm gold particles) is found in late endosomes (LE, Inset) and lysosomes. M = mitochondrion. Scale bars: 200 nm.


D , E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry . HeLa cells expressing a UBE2QL1 - APEX2 fusion were EtOH ( light - labelled , L ) - or LLOMe ( heavy - labelled , H ) - treated and pulsed with biotin phenol ( 30 min ) and H2O2 ( 1 min ) . Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram ( D ) and displayed high correlation coefficients ( E ) .

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D, E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram (D) and displayed high correlation coefficients (E).


, F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry . HeLa cells expressing a UBE2QL1 - APEX2 fusion were EtOH ( light - labelled , L ) - or LLOMe ( heavy - labelled , H ) - treated and pulsed with biotin phenol ( 30 min ) and H2O2 ( 1 min ) . ( F ) Results from the four experiments are summarized in a volcano plot . Horizontal dotted line represents the significance threshold ( p > 0 . 05 ) . The vertical lines indicate the fold change cutoff ( log2 ( H / L ) ≥ 1 . 5 ) . Red dots indicate proteins above the significance and fold - change thresholds . Protein names are color - coded in blue ( endosome / lysosome - associated ) , green ( endolysosomal damage / autophagy - associated ) and orange ( VCP / p97 and its cofactors ) .

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, F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). (F) Results from the four experiments are summarized in a volcano plot. Horizontal dotted line represents the significance threshold (p > 0.05). The vertical lines indicate the fold change cutoff (log2 (H/L) ≥ 1.5). Red dots indicate proteins above the significance and fold-change thresholds. Protein names are color-coded in blue (endosome/lysosome-associated), green (endolysosomal damage/autophagy-associated) and orange (VCP/p97 and its cofactors).


A HeLa cells stably expressing p97 - GFP were control or UBE2QL1 - depleted for 48 h and expression of p97 - GFP was induced for the last 24 h . Cells were LLOMe or control treated for 1 h , fixed after a chase of 2 h , and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated . Note the reduction of the K48 and p97 - GFP signals on damaged lysosomes in UBE2QL1 - depleted cells . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 10 µm for inlays .

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A HeLa cells stably expressing p97-GFP were control or UBE2QL1-depleted for 48 h and expression of p97-GFP was induced for the last 24 h. Cells were LLOMe or control treated for 1 h, fixed after a chase of 2 h, and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated. Note the reduction of the K48 and p97-GFP signals on damaged lysosomes in UBE2QL1-depleted cells. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 10 µm for inlays.


B Automated quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of p97 - GFP and LAMP1 signals , normalized to untreated siCtrl . Data are from four independent experiments with ≥ 30 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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B Automated quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p97-GFP and LAMP1 signals, normalized to untreated siCtrl. Data are from four independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


C HeLa cells were control or UBE2QL1 - depleted for 60 h , fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 5 µm for inlays .

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C HeLa cells were control or UBE2QL1-depleted for 60 h, fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.


D Quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of p62 on LAMP1 signals , normalized to untreated siCtrl . Graph represents data from three independent experiments with ≥ 30 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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D Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p62 on LAMP1 signals, normalized to untreated siCtrl. Graph represents data from three independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


E HeLa cells were treated but fixed after additional 2 h of chase . Samples were stained for Galectin - 3 ( Gal3 ) to detect damaged lysosomes , and LC3B as marker for autophagosomes . Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 5 µm for inlays .

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E HeLa cells were treated but fixed after additional 2 h of chase. Samples were stained for Galectin-3 (Gal3) to detect damaged lysosomes, and LC3B as marker for autophagosomes. Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.


F Quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of LC3B and Gal3 signals , normalized to untreated siCtrl , representing LC3B colocalization with Gal3 . Graph represents data from three independent experiments with ≥ 50 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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F Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of LC3B and Gal3 signals, normalized to untreated siCtrl, representing LC3B colocalization with Gal3. Graph represents data from three independent experiments with ≥ 50 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h , treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A / B . GAPDH was probed as loading control .

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G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h, treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A/B. GAPDH was probed as loading control.


A Gal3 puncta assay for damaged lysosomes . HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone ( untreated ) for 1 h . Cells were fixed at indicated times after washout , stained with DAPI and Gal3 and LAMP1 antibodies , and processed for confocal microscopy . Note increased number of Gal3 puncta in untreated UBE2QL1 - depleted cells and their persistence 10 h after LLOMe - induced damage . Scale bars : 10 µm , 2 µm for inlays . Arrows indicate Gal3 and LAMP1 colocalizing vesicles .

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A Gal3 puncta assay for damaged lysosomes. HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone (untreated) for 1 h. Cells were fixed at indicated times after washout, stained with DAPI and Gal3 and LAMP1 antibodies, and processed for confocal microscopy. Note increased number of Gal3 puncta in untreated UBE2QL1-depleted cells and their persistence 10 h after LLOMe-induced damage. Scale bars: 10 µm, 2 µm for inlays. Arrows indicate Gal3 and LAMP1 colocalizing vesicles.


B Automated quantification The percentage of cells with more than three Gal3 puncta is shown . Graph represents data from three independent experiments with ≥ 50 cells per condition ( mean ± SD ) . * P < 0 . 05 ; * * P < 0 . 01 ( One - way - ANOVA with Dunnett ' s multiple comparison test ) .

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B Automated quantification The percentage of cells with more than three Gal3 puncta is shown. Graph represents data from three independent experiments with ≥ 50 cells per condition (mean ± SD). *P<0.05; **P<0.01 (One-way-ANOVA with Dunnett's multiple comparison test).


C Survival assay . Control or UBE2QL1 - depleted HeLa cells ( 48 h ) were treated with increasing concentrations of LLOMe as indicated . Cell viability was measured with the MTS assay . Graph represents data from one experiment including three replicates ( mean ± SD ) . * P < 0 . 05 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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C Survival assay. Control or UBE2QL1-depleted HeLa cells (48 h) were treated with increasing concentrations of LLOMe as indicated. Cell viability was measured with the MTS assay. Graph represents data from one experiment including three replicates (mean ± SD). *P<0.05; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


A Control or UBE2QL1 - depleted ( for 60 h ) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy . Note nuclear translocation of TFEB upon loss of UBE2QL1 . Scale bar : 20µm .

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A Control or UBE2QL1-depleted (for 60 h) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy. Note nuclear translocation of TFEB upon loss of UBE2QL1. Scale bar: 20µm.


B Automated quantification The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions . Graph represents data from four independent experiments with ≥ 100 cells per condition ( mean ± SD ) . * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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B Automated quantification The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions. Graph represents data from four independent experiments with ≥ 100 cells per condition (mean ± SD). ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


C Cells were stained for LAMP1 and mTOR . Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells . Scale bar : 20 µm .

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C Cells were stained for LAMP1 and mTOR. Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells. Scale bar: 20 µm.


D Automated quantification The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients ( P . C . C . ) of the signals . Graph represents data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) . * P < 0 . 05 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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D Automated quantification The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients (P.C.C.) of the signals. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD). *P<0.05 (One-way-ANOVA with Bonferroni's multiple comparison test).


E Automated quantification Number of LAMP1 - positive vesicles per cell is shown . Graph represents data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) .

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E Automated quantification Number of LAMP1-positive vesicles per cell is shown. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD).


F Western blot analysis of lysates of cells treated As a positive control , cells were treated with the mTOR - inhibitor Torin1 ( 1 µM ) for 2 h . GAPDH was probed as loading control .

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F Western blot analysis of lysates of cells treated As a positive control, cells were treated with the mTOR-inhibitor Torin1 (1 µM) for 2 h. GAPDH was probed as loading control.


G C . elegans wild - type ( N2 ) and mutant strains for ubc - 25 ( ok1732 ) , for scav - 3 ( qx193 ) or the ubc - 25 and scav - 3 double mutant ( ok1732 ; qx193 ) carrying a EGFP - Gal3 transgene were imaged in adult worms ( L4 + 24 h ) . 3D reconstructions of the hypodermis . Small and big units represent 1 and 5 µm , respectively .

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G C.elegans wild-type (N2) and mutant strains for ubc-25(ok1732), for scav-3(qx193) or the ubc-25 and scav-3 double mutant (ok1732;qx193) carrying a EGFP-Gal3 transgene were imaged in adult worms (L4 + 24 h). 3D reconstructions of the hypodermis. Small and big units represent 1 and 5 µm, respectively.


H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified . At least 20 animals were scored in each strain . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Dunnett ' s multiple comparison test or Student ' s two - tailed unpaired t test ) .

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H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified. At least 20 animals were scored in each strain. *P<0.05; **P<0.01; ****P<0.0001 (One-way-ANOVA with Dunnett's multiple comparison test or Student's two-tailed unpaired t test).


I Populations of worms with different extent of lysosome permeabilization were analyzed . Graph represents percentage of worms with less than 5 , 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length .

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I Populations of worms with different extent of lysosome permeabilization were analyzed. Graph represents percentage of worms with less than 5, 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length.


RT - PCR analysis ( top ) and representative direct sequencing ( bottom ) of Ttn exon 326 transcripts from HL - 1 cardiomyocytes transiently transfected with different 2OMePS AONs . Only 2OMePS - AON1 and 2OMePS - AON1 + 3 ( * ) lead to a correct excision of exon 326 as confirmed by direct sequencing .

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RT-PCR analysis (top) and representative direct sequencing (bottom) of Ttn exon 326 transcripts from HL-1 cardiomyocytes transiently transfected with different 2OMePS AONs. Only 2OMePS-AON1 and 2OMePS-AON1 + 3 (*) lead to a correct excision of exon 326 as confirmed by direct sequencing.


Immunofluorescence images of sarcomeric α - actinin in untransfected and 2OMePS - AON1 + 3 - transfected HL - 1 cardiomyocytes . Scale bars , 10 μm .

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Immunofluorescence images of sarcomeric α-actinin in untransfected and 2OMePS-AON1 + 3-transfected HL-1 cardiomyocytes. Scale bars, 10 μm.


RT - PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS - AON1 , 2OMePS - AON3 , and 2OMePS - AON1 + 3 .

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RT-PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.


RT - PCR analysis ( left ) and representative direct sequencing ( right ) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA - TTNAONs - IRES - GFP lentiviral vector carrying the AON1 and 3 sequences ( TTN - AON ) or with a control vector ( U7snRNA - ScrAONs - IRES - GFP ) .

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RT-PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).


Mass spectrometry - based analysis of titin peptides in cells infected with the U7snRNA - ScrAONs - IRES - GFP ( Scr - AON ) and U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) vectors . Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down - regulated in DCM Scr - AON cardiomyocytes compared to CTR Scr - AON cardiomyocytes ( n = 3 , P = 9 . 03E − 8 , Fisher ' s exact test , FDR = 0 . 04 , top ) . Down - regulation of exon 326 was also detected in DCM TTN - AON cells when compared to DCM Scr - AON cells ( n = 3 , P = 0 . 02 , Fisher ' s exact test , bottom ) .

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Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).


Percentage of iPSC - derived cardiomyocytes with cross - striated myofibrils occupying a fraction ( partially organized , dark gray ) or the whole cytoplasm ( fully organized , light gray ) from two control and two patient clones . Statistical difference was tested using the two - sided chi - squared test ( CTR1 basal : n = 283 , CTR2 basal : n = 250 , DCM1 basal : n = 255 , DCM2 basal : n = 236 ; * * * P = 1 . 93E − 16 ) .

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Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).


Immunofluorescence images of titin ( Z - disk portion ) and cardiac troponin T ( cTNT , A - band ) in well - organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions . Scale bars , 5 μm .

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Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.


Immunofluorescence images ( left ) of α - actinin and cTNT in CTR and DCM single cardiomyocytes , illustrating 3 different levels of sarcomeric organization ( perinuclear , fully and peripherally organized ) . Percentage of cells with different levels of sarcomeric organization ( right ) under basal and stress conditions . Statistical difference was tested using the chi - squared test ( CTR1 basal : n = 283 , CTR2 basal : n = 250 , DCM1 basal : n = 255 , DCM2 basal : n = 236 , CTR1 Iso : n = 245 , CTR2 Iso : n = 230 ; DCM1 Iso : n = 242 and DCM2 Iso : n = 269 ; * * * P = 1 . 93E − 16 CTR basal versus DCM basal ; * * * P = 9 . 61E − 34 CTR Iso versus DCM Iso , * * P = 0 . 001 DCM basal versus DCM Iso ) . No significant differences were observed comparing CTR basal and CTR Iso groups . Scale bars , 25 μm .

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Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.


Percentage of perinuclear , fully , and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA - ScrAONs - IRES - GFP and U7snRNA - TTNAONs - IRES - GFP lentiviruses . Statistical difference was tested using the two - sided chi - squared test ( CTR1 Scr - AON : n = 190 , CTR2 Scr - AON : n = 200 , DCM1 Scr - AON : n = 221 , DCM2 Scr - AON : n = 115 , CTR1 TTN - AON : n = 223 , CTR2 TTN - AON : n = 187 , DCM1 TTN - AON : n = 171 , DCM2 TTN - AON : n = 243 ; * * * P = 4 . 22E − 15 CTR Scr - AON versus DCM Scr - AON ; * * * P = 4 . 61E − 02 DCM Scr - AON versus DCM TTN - AON ) . No significant differences were observed comparing CTR Scr - AON and CTR TTN - AON groups .

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Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.