Figure 5Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( A ) EVs isolated by UC of medium from HeLa cells cultured in glutamine - replete ( 2 . 00 mM ) and - depleted ( 0 . 02 mM ) conditions for 24 h ( see Appendix Fig S7A ' ' for analysis of SEC - isolated EVs ) . Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( B ) EVs isolated by size - exclusion chromatography ( SEC ; fractions two to seven ) from LNCaP cells cultured in glutamine - replete ( 2 . 00 mM ) and - depleted ( 0 . 02 mM ) conditions for 24Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( C ) EVs isolated by SEC from LNCaP cells cultured in the presence or absence of 120 nM Torin 1 for 24 h . Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( D ) EVs isolated by UC from HCT116 cells cultured for 24 h following three days of raptor or non - targeting ( NT ) shRNA knockdown . Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( E ) EVs isolated by SEC from HCT116 cells cultured in the presence or absence of 3 µM Akt inhibitor AZD5363 for 24 h . Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . ( F ) EVs isolated by SEC from HCT116 cells cultured under glutamine - replete conditions in the presence or absence of 10 µM KRAS inhibitor BI - 2852 ( or control compound Bl2853 ) for 24 h .

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

Figure 5Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (A) EVs isolated by UC of medium from HeLa cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24 h (see Appendix Fig S7A'' for analysis of SEC-isolated EVs).Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (B) EVs isolated by size-exclusion chromatography (SEC; fractions two to seven) from LNCaP cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (C) EVs isolated by SEC from LNCaP cells cultured in the presence or absence of 120 nM Torin 1 for 24 h.Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (D) EVs isolated by UC from HCT116 cells cultured for 24 h following three days of raptor or non-targeting (NT) shRNA knockdown.Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (E) EVs isolated by SEC from HCT116 cells cultured in the presence or absence of 3 µM Akt inhibitor AZD5363 for 24 h.Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. (F) EVs isolated by SEC from HCT116 cells cultured under glutamine-replete conditions in the presence or absence of 10 µM KRAS inhibitor BI-2852 (or control compound Bl2853) for 24 h.


Figure 6 ( A ) Growth curves for HCT116 recipient cells in reduced ( 1 % ) serum conditions following 30 min pre - incubation with EVs isolated by UC ( 104 per cell ; top ) or by SEC ( 4 x 103 per cell ; bottom ) from glutamine - replete and - depleted HCT116 cells or with vehicle ( PBS ) . Fold change in confluency is a measure of cell area occupying well relative to zero time , as measured by IncuCyte software . ( B ) Cumulative tube length for HUVEC recipient cells following treatment with 104 EVs isolated by UC from glutamine - replete and - depleted HCT116 cells or with vehicle ( PBS ) . Both EV preparations promote tubulation , but the network is more stable with EVs from glutamine - depleted HCT116 cells . ( C ) Western blot analysis of EVs isolated by UC from HCT116 cells cultured in glutamine - replete and - depleted medium for 24 h , following transduction with a Rab11a or control non - targeting ( NT ) shRNA knockdown construct over previous two days . Bar chart shows change in putative exosome proteins in EVs secreted from Rab11a knockdown cells relative to NT - treated cells in glutamine - depleted conditions , following initial normalisation to cell lysate protein . ( C ' ) Growth curves are for HCT116 recipient cells pre - treated with EVs isolated as in ( C ) or with vehicle ( PBS ) . * * * * P colour denotes significant increase relative to EVs from glutamine - replete cells ( black ) and Rab11a knockdown cells ( red ) . ( D ) Left hand group of four images show cells grown in glutamine - replete ( 2 . 00 mM ) and glutamine - depleted ( 0 . 15 mM ) conditions for 24 h , then incubated with an anti - CD63 antibody ( yellow ) for 30 min at 4oC , washed with PBS , chased at 37oC for 30 min , then fixed , immunostained and imaged . Right hand group of four images show cells grown in glutamine - replete and glutamine - depleted conditions for 24 h , incubated with Tfn - Alexa Fluor ® 488 ( yellow ) for 30 min at 4oC , shifted to 37oC for 30 min , then washed , immediately fixed and imaged . ( E ) Western blot showing levels of anti - CD63 heavy chain and biotin - conjugated Tfn in HCT116 cells cultured for 24 h in glutamine - replete or - depleted conditions , incubated for 30 min in medium containing these molecules at 4oC , then chased at 37oC for 15 min ( the chase step was not performed for Tfn in D , to reduce loss due to rapid recycling of Tfn ) . ( F ) Western blot analysis of EVs isolated by UC from HCT116 cells in glutamine - replete medium , transduced with a Rab7 or non - targeting ( NT ) control shRNA knockdown construct . Bar charts show changes in putative exosome proteins in EVs isolated by ultracentrifugation , following normalisation to cell lysate protein . ( F ' ) Growth curves are for HCT116 recipient cells pre - treated with EVs isolated as in ( F ) or with vehicle ( PBS ) .

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

Figure 6(A) Growth curves for HCT116 recipient cells in reduced (1%) serum conditions following 30 min pre-incubation with EVs isolated by UC (104 per cell; top) or by SEC (4 x 103 per cell; bottom) from glutamine-replete and -depleted HCT116 cells or with vehicle (PBS). Fold change in confluency is a measure of cell area occupying well relative to zero time, as measured by IncuCyte software.(B) Cumulative tube length for HUVEC recipient cells following treatment with 104 EVs isolated by UC from glutamine-replete and -depleted HCT116 cells or with vehicle (PBS). Both EV preparations promote tubulation, but the network is more stable with EVs from glutamine-depleted HCT116 cells.(C) Western blot analysis of EVs isolated by UC from HCT116 cells cultured in glutamine-replete and -depleted medium for 24 h, following transduction with a Rab11a or control non-targeting (NT) shRNA knockdown construct over previous two days. Bar chart shows change in putative exosome proteins in EVs secreted from Rab11a knockdown cells relative to NT-treated cells in glutamine-depleted conditions, following initial normalisation to cell lysate protein. (C') Growth curves are for HCT116 recipient cells pre-treated with EVs isolated as in (C) or with vehicle (PBS). ****P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and Rab11a knockdown cells (red).(D) Left hand group of four images show cells grown in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) conditions for 24 h, then incubated with an anti-CD63 antibody (yellow) for 30 min at 4oC, washed with PBS, chased at 37oC for 30 min, then fixed, immunostained and imaged. Right hand group of four images show cells grown in glutamine-replete and glutamine-depleted conditions for 24 h, incubated with Tfn-Alexa Fluor® 488 (yellow) for 30 min at 4oC, shifted to 37oC for 30 min, then washed, immediately fixed and imaged.(E) Western blot showing levels of anti-CD63 heavy chain and biotin-conjugated Tfn in HCT116 cells cultured for 24 h in glutamine-replete or -depleted conditions, incubated for 30 min in medium containing these molecules at 4oC, then chased at 37oC for 15 min (the chase step was not performed for Tfn in D, to reduce loss due to rapid recycling of Tfn).(F) Western blot analysis of EVs isolated by UC from HCT116 cells in glutamine-replete medium, transduced with a Rab7 or non-targeting (NT) control shRNA knockdown construct. Bar charts show changes in putative exosome proteins in EVs isolated by ultracentrifugation, following normalisation to cell lysate protein. (F') Growth curves are for HCT116 recipient cells pre-treated with EVs isolated as in (F) or with vehicle (PBS).


Figure 7 ( A ) Western blot analysis of ERK phosphorylation ( p - ERK ) in recipient serum - deprived HCT116 cells pre - treated with EVs isolated by SEC from glutamine - replete or - depleted HCT116 cells or vehicle ( PBS ) . Bar chart shows ratio of p - ERK to ERK levels from triplicate independent experiments . ( B ) Bar chart shows HCT116 recipient cell growth over 120 h , after pre - treatment with EV preparations isolated as in ( A ) or with PBS , and then incubation for the first 24 h of culture in the presence or absence of the ERK inhibitor SCH772984 ( 1 . 00 µM ) . ( C ) Western blot analysis showing levels of the EGFR ligand , Amphiregulin ( AREG ) in EVs isolated by SEC of medium from HCT116 cells cultured in glutamine - replete ( 2 . 00 mM ) or - depleted ( 0 . 15 mM ) conditions for 24 h . Gel loading was normalised to cell lysate protein levels . AREG ' s molecular weight ( approximately 26 - 28 kDa ) suggests it is in its membrane - associated form ( see Appendix Fig S8D ) . In the bar chart , the levels of putative exosome proteins were normalised to cell lysate protein levels . Significantly decreased levels are in blue and increased levels are in red . ( C ' ) Growth curves in low ( 1 % ) serum are for HCT116 recipient cells pre - treated with the EV preparations [ isolated as in ( C ) ] , which had themselves been pre - treated with and without anti - AREG neutralising antibody or with a control IgG . Solid red line shows growth - promoting effect of EVs isolated under glutamine depletion , which is blocked by anti - AREG antibody ( red dashed line ) . * * * P colour denotes significant increase relative to EVs from glutamine - replete cells ( black ) and anti - AREG - treated cells ( red ) . ( C ' ' ) Western blot analysis of ERK phosphorylation ( p - ERK ) in recipient serum - deprived HCT116 cells pre - treated with EVs isolated by SEC from glutamine - replete ( 2 . 00 mM ) or - depleted ( 0 . 15 mM ) HCT116 cells or vehicle ( PBS ) , which were pre - treated with anti - AREG ( AREGab ) or a control immunoglobulin ( Ig ) . Note increase in ERK phosphorylation using EVs from glutamine - depleted cells , which is blunted by the addition of the anti - AREG antibody .

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

Figure 7(A) Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete or -depleted HCT116 cells or vehicle (PBS). Bar chart shows ratio of p-ERK to ERK levels from triplicate independent experiments.(B) Bar chart shows HCT116 recipient cell growth over 120 h, after pre-treatment with EV preparations isolated as in (A) or with PBS, and then incubation for the first 24 h of culture in the presence or absence of the ERK inhibitor SCH772984 (1.00 µM).(C) Western blot analysis showing levels of the EGFR ligand, Amphiregulin (AREG) in EVs isolated by SEC of medium from HCT116 cells cultured in glutamine-replete (2.00 mM) or -depleted (0.15 mM) conditions for 24 h. Gel loading was normalised to cell lysate protein levels. AREG's molecular weight (approximately 26-28 kDa) suggests it is in its membrane-associated form (see Appendix Fig S8D). In the bar chart, the levels of putative exosome proteins were normalised to cell lysate protein levels. Significantly decreased levels are in blue and increased levels are in red.(C') Growth curves in low (1%) serum are for HCT116 recipient cells pre-treated with the EV preparations [isolated as in (C)], which had themselves been pre-treated with and without anti-AREG neutralising antibody or with a control IgG. Solid red line shows growth-promoting effect of EVs isolated under glutamine depletion, which is blocked by anti-AREG antibody (red dashed line). ***P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and anti-AREG-treated cells (red). (C'') Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete (2.00 mM) or -depleted (0.15 mM) HCT116 cells or vehicle (PBS), which were pre-treated with anti-AREG (AREGab) or a control immunoglobulin (Ig). Note increase in ERK phosphorylation using EVs from glutamine-depleted cells, which is blunted by the addition of the anti-AREG antibody.


Figure 8HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three - day intervals with vehicle ( PBS ) , or EVs isolated by UC from glutamine - replete or glutamine - depleted HCT116 cells . Tumours were excised 24 h after last of four injections for analysis . Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System . ( A ) Sections immunostained for CD31 , which labels endothelial cells and blood vessels , with blood vessel number ( upper ) and total area ( lower ) represented in bar charts . HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three - day intervals with vehicle ( PBS ) , or EVs isolated by UC from glutamine - replete or glutamine - depleted HCT116 cells . Tumours were excised 24 h after last of four injections for analysis . Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System . ( B ) Sections immunostained for Ki67 , which stains proliferative cells . Proportion of tumour cells with Ki69 staining is represented in bar chart . HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three - day intervals with vehicle ( PBS ) , or EVs isolated by UC from glutamine - replete or glutamine - depleted HCT116 cells . Tumours were excised 24 h after last of four injections for analysis . Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System . ( C ) Sections stained with haematoxylin and eosin , which highlights necrotic regions ( pale staining ) . Proportion of tumour area that is necrotic is represented in bar chart . HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three - day intervals with vehicle ( PBS ) , or EVs isolated by UC from glutamine - replete or glutamine - depleted HCT116 cells . Tumours were excised 24 h after last of four injections for analysis . Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System . ( D ) Sections immunostained for CA9 , which is expressed in hypoxic regions . Proportion of tumour cells with CA9 staining is represented in bar chart .

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

Figure 8HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (A) Sections immunostained for CD31, which labels endothelial cells and blood vessels, with blood vessel number (upper) and total area (lower) represented in bar charts.HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (B) Sections immunostained for Ki67, which stains proliferative cells. Proportion of tumour cells with Ki69 staining is represented in bar chart.HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (C) Sections stained with haematoxylin and eosin, which highlights necrotic regions (pale staining). Proportion of tumour area that is necrotic is represented in bar chart.HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (D) Sections immunostained for CA9, which is expressed in hypoxic regions. Proportion of tumour cells with CA9 staining is represented in bar chart.


Figure 1C - Left : Percentage of total live cells expressing ChAT - eGFP derived from IWAT ( including inguinal lymph node ; n = 7 ) , IWAT alone ( n = 8 ) and lymph node alone ( n = 8 ) . Right : Proportion of ChAT - eGFP + cells comprised by T cells , B cells and MΦ in each tissue type . F - Left : relative mRNA expression of Chat in ChATfl / fl and ChATfl / fl ; Vav - iCre IWAT ( n = 9 ) . Chat expression was analyzed by qPCR and normalized to levels of Tbp using the 2 - ΔΔCt method . Right : quantification by LC / MS - MS of Ach concentration in SVF isolated from ChATfl / fl and ChATfl / fl ; Vav - iCre IWAT ( n = 6 ) .

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

Figure 1C - Left: Percentage of total live cells expressing ChAT-eGFP derived from IWAT (including inguinal lymph node; n = 7), IWAT alone (n = 8) and lymph node alone (n = 8). Right: Proportion of ChAT-eGFP+ cells comprised by T cells, B cells and MΦ in each tissue type.F - Left: relative mRNA expression of Chat in ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 9). Chat expression was analyzed by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: quantification by LC/MS-MS of Ach concentration in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 6).


Figure 2Body weight ( C ) of ChATfl / fl ; LysM - Cre ( n = 7 for ChATfl / fl , n = 8 for Cre ChATfl / fl ; Cd4 - Cre ( n = 6 for ChATfl / fl and n = 7 for Cre ChATfl / fl ; Mb1 - Cre ( n = 12 in C , n = 9 for ChATfl / fl , n = 8 for Cre and littermate ChATfl / fl mice housed at RT . E - G - mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl / fl ; LysM - Cre ( n = 24 for ChATfl / fl _ RT , n = 25 for Cre _ RT , n = 20 for ChATfl / fl _ CE , n = 20 for Cre _ CE ) ( E ) , ChATfl / fl ; Cd4 - Cre ( n = 17 for ChATfl / fl _ RT , n = 22 - 23 for Cre _ RT , n = 19 for ChATfl / fl _ CE , n = 22 for Cre _ CE ) ( F ) , ChATfl / fl ; Mb1 - Cre ( n = 10 for ChATfl / fl _ RT , n = 11 for Cre _ RT , n = 9 - 12 for ChATfl / fl _ CE , n = 13 for Cre _ CE ) ( G ) and littermate ChATfl / fl mice housed at RT or 4oC ( CE ) for 6 h . An insert graph in ( E ) highlights mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl / fl and ChATfl / fl ; LysM - Cre mice after 6 h CE . K - Average whole - body oxygen consumption rate ( OCR ) of ChATfl / fl ( n = 10 ) and ChATfl / fl ; LysM - Cre ( n = 14 ) mice housed in metabolic chambers at RT or CE for 6 h ( from 9 a . m . to 3 p . m . ) .

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

Figure 2Body weight (C) of ChATfl/fl;LysM-Cre (n = 7 for ChATfl/fl, n = 8 for Cre ChATfl/fl;Cd4-Cre (n = 6 for ChATfl/fl and n = 7 for Cre ChATfl/fl;Mb1-Cre (n = 12 in C, n = 9 for ChATfl/fl, n = 8 for Cre and littermate ChATfl/fl mice housed at RT.E-G - mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl/fl;LysM-Cre (n = 24 for ChATfl/fl_RT, n = 25 for Cre_RT, n = 20 for ChATfl/fl_CE, n = 20 for Cre_CE) (E), ChATfl/fl;Cd4-Cre (n = 17 for ChATfl/fl_RT, n = 22-23 for Cre_RT, n = 19 for ChATfl/fl_CE, n = 22 for Cre_CE) (F), ChATfl/fl;Mb1-Cre (n = 10 for ChATfl/fl_RT, n = 11 for Cre_RT, n = 9-12 for ChATfl/fl_CE, n = 13 for Cre_CE) (G) and littermate ChATfl/fl mice housed at RT or 4oC (CE) for 6 h. An insert graph in (E) highlights mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl/fl and ChATfl/fl;LysM-Cre mice after 6 h CE.K - Average whole-body oxygen consumption rate (OCR) of ChATfl/fl (n = 10) and ChATfl/fl;LysM-Cre (n = 14) mice housed in metabolic chambers at RT or CE for 6 h (from 9 a.m. to 3 p.m.).


Figure 3A - ChAT - eGFP + cells as a percentage of total IWAT SVF cells from ChAT - eGFP mice housed at RT ( n = 7 ) or 10 ° C for 4 h ( n = 3 ) . B - Percentages of total T cells , B cells and MΦ that are ChAT - eGFP + in IWAT from ChAT - eGFP mice housed at RT ( n = 7 ) or 10 ° C for 4 h ( n = 3 ) . C - ChAT - eGFP + cells as a percentage of total IWAT SVF cells from β - less ChAT - eGFP mice housed at RT or 10 ° C for 4 h ( n = 4 ) . D - Percentages of total T cells , B cells and MΦ that are ChAT - eGFP + in IWAT from β - less ChAT - eGFP mice housed at RT or 10 ° C for 4 h ( n = 4 ) . E - ChAT - eGFP + cells as a percentage of total IWAT SVF cells from ChAT - eGFP mice treated with vehicle ( veh ) or 1 mg / kg NE for 2 h ( n = 6 ) . F - Percentages of total T cells , B cells and MΦ that are ChAT - eGFP + in IWAT from ChAT - eGFP mice treated with veh or 1 mg / kg NE for 2 h ( n = 6 ) . J - Relative mRNA expression of Adrb1 , Adrb2 and Adrb3 in IWAT MΦ sorted from WT mice ( n = 4 ) . Expression was measured by qPCR and normalized to levels of Tbp using the 2 - ΔΔCt method .

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

Figure 3A - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3). B - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3). C - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4). D - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4). E - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice treated with vehicle (veh) or 1 mg/kg NE for 2 h (n = 6). F - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice treated with veh or 1 mg/kg NE for 2 h (n = 6). J - Relative mRNA expression of Adrb1, Adrb2 and Adrb3 in IWAT MΦ sorted from WT mice (n = 4). Expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.


Figure 4D - E - Percentage of ( D ) total IWAT SVF cells and ( E ) IWAT MΦ expressing ChAT - eGFP in reporter mice with genetic deletion combinations of β - ARs 1 , 2 and 3 following treatment with veh ( white bar : n = 7 ) or 1 mg / kg NE ( black bars : from left to right , n = 7 , 5 , 4 , 5 , 4 , 7 ) for 2 h . F - LC / MS - MS quantification of acetylcholine levels secreted by SVF cells derived from IWAT of β2WT and β2KO mice treated with veh or 1 mg / kg Form for 2 h ( n = 3 ) . M - Left : BMDMs were treated for 2 h with veh or pan β - AR agonist ( 100 μM NE ) , β2 - AR antagonist ( 5 μM butoxamine , Buto ) or a combination of NE and β2 antagonist ( Buto ) ( n = 6 ) . Chat mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2 - ΔΔCt method . Right : Total number of ChAT - eGFP + BMDMs . ChAT - eGFP BMDMs were treated for 2 h with veh or pan β - AR agonist ( 100 μM NE ) , β2 - AR antagonist ( 5 μM butoxamine , Buto ) or a combination of NE and β2 - AR antagonist ( Buto ) ( n = 4 ) . An equal number of events ( 50 , 000 ) were analyzed by flow cytometry . N - Left : Bicompartmental co - culture system with media alone ( Ctrl ) or WT SVF cells isolated from IWAT in the upper compartment ( transwell insert ) and freshly isolated IWAT explants from β - less mice in the lower compartment . Cells were co - cultured for 4 h in the presence or absence of β2 - AR agonist ( 2 . 5 μM Form ) . 150 μM rivastigmine was added to the media to prevent degradation of Ach . Right : qPCR analyses of Chat and Ucp1 mRNA levels in β ‑ less explants following co - culture with media ( n = 4 ) , vehicle ( n = 10 ) or β2 - AR agonist ( n = 10 ) treated SVF cells . mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2 - ΔΔCt method .

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

Figure 4D-E - Percentage of (D) total IWAT SVF cells and (E) IWAT MΦ expressing ChAT-eGFP in reporter mice with genetic deletion combinations of β-ARs 1, 2 and 3 following treatment with veh (white bar: n = 7) or 1 mg/kg NE (black bars: from left to right, n = 7, 5, 4, 5, 4, 7) for 2 h.F - LC/MS-MS quantification of acetylcholine levels secreted by SVF cells derived from IWAT of β2WT and β2KO mice treated with veh or 1 mg/kg Form for 2 h (n = 3).M - Left: BMDMs were treated for 2 h with veh or pan β-AR agonist (100 μM NE), β2-AR antagonist (5 μM butoxamine, Buto) or a combination of NE and β2 antagonist (Buto) (n = 6). Chat mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: Total number of ChAT-eGFP+ BMDMs. ChAT-eGFP BMDMs were treated for 2 h with veh or pan β-AR agonist (100 μM NE), β2-AR antagonist (5 μM butoxamine, Buto) or a combination of NE and β2-AR antagonist (Buto) (n = 4). An equal number of events (50,000) were analyzed by flow cytometry.N - Left: Bicompartmental co-culture system with media alone (Ctrl) or WT SVF cells isolated from IWAT in the upper compartment (transwell insert) and freshly isolated IWAT explants from β-less mice in the lower compartment. Cells were co-cultured for 4 h in the presence or absence of β2-AR agonist (2.5 μM Form). 150 μM rivastigmine was added to the media to prevent degradation of Ach. Right: qPCR analyses of Chat and Ucp1 mRNA levels in β‑less explants following co-culture with media (n = 4), vehicle (n = 10) or β2-AR agonist (n = 10) treated SVF cells. mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.


Figure 1C Western blots of the lysates of HEK293T cells , resolved on SDS - PAGE and stained with a pan - arrestin antibody . mt = mock transfected . Bpa : Benzoyl - Phenylalanine . Arrestins were equipped with a 3xHA tag at the C - terminus , which increases their size of about 3 kDa compared to the endogenous proteins ( βarr1 47 kDa , βarr2 46 kDa ) . βarr1 and βarr2 were transfected at 1 / 3 DNA compared to the corresponding TAG - mutants .

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Figure 1C Western blots of the lysates of HEK293T cells, resolved on SDS-PAGE and stained with a pan-arrestin antibody. mt = mock transfected. Bpa: Benzoyl-Phenylalanine. Arrestins were equipped with a 3xHA tag at the C-terminus, which increases their size of about 3 kDa compared to the endogenous proteins (βarr1 47 kDa, βarr2 46 kDa). βarr1 and βarr2 were transfected at 1/3 DNA compared to the corresponding TAG-mutants.


Figure 2Each panel represents immunoblots of whole cell lysates stained with an anti - HA antibody detecting arrestin . In the left panels GPCRs were combined with βarr1 variants , in the right panels with βarr2 variants . Residues replaced by Bpa are indicated above each lane . Covalent arrestin - receptor complexes are expected between 100 - 200 kDa , considering the size of the receptors ( V2R 41 . 5 kDa , PTH1R 63 . 8 kDa , CRF1R , 46 . 3 kDa ) and their glycosylation . A PTH1R , agonist PTH ( 1 - 34 ) B CRF1R , agonist Urocortin 1 ( Ucn1 ) C V2R , agonist arginine - vasopressin ( AVP ) D No receptor .

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Figure 2Each panel represents immunoblots of whole cell lysates stained with an anti-HA antibody detecting arrestin. In the left panels GPCRs were combined with βarr1 variants, in the right panels with βarr2 variants. Residues replaced by Bpa are indicated above each lane. Covalent arrestin-receptor complexes are expected between 100-200 kDa, considering the size of the receptors (V2R 41.5 kDa, PTH1R 63.8 kDa, CRF1R, 46.3 kDa) and their glycosylation. A PTH1R, agonist PTH(1-34) B CRF1R, agonist Urocortin 1 (Ucn1) C V2R, agonist arginine-vasopressin (AVP) D No receptor.


Figure 3Aliquots from agonist - treated samples shown in Figure 2 were deglycosylated with PNGaseF , run on SDS - PAGE and immunoblotted with antibodies detecting arrestin ( upper panels , α - HA ) and the GPCRs ( as indicated , CRF1R is equipped with a N - terminal FLAG , V2R carries a C - terminal 1D4 epitope ) .

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Figure 3Aliquots from agonist-treated samples shown in Figure 2 were deglycosylated with PNGaseF, run on SDS-PAGE and immunoblotted with antibodies detecting arrestin (upper panels, α-HA) and the GPCRs (as indicated, CRF1R is equipped with a N-terminal FLAG, V2R carries a C-terminal 1D4 epitope).


Figure 4A SDS - PAGE and immunoblots of immunoprecipitated samples . Sample 1 was immunoprecipitated without previous UV treatment . Sample 2 is the product of irradiation of sample 1 . Sample 3 was treated with UV light before immunoprecipitation . All samples were run in parallel on the same gel . B Size exclusion chromatography ( SEC ) of Sample 3 . C Western blot of the fractions eluted by SEC . MALDI - TOF analysis of the SEC fractions VR2 and VR3 . The spectrum of sample VR2 shows a [ M + H ] + peak featuring twice the mass ( ~ 108 000 Da ) of the [ M + H ] + peak of the βarr1 monomer ( sample VR3 , ~ 50 400 Da ) .

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Figure 4A SDS-PAGE and immunoblots of immunoprecipitated samples. Sample 1 was immunoprecipitated without previous UV treatment. Sample 2 is the product of irradiation of sample 1. Sample 3 was treated with UV light before immunoprecipitation. All samples were run in parallel on the same gel.B Size exclusion chromatography (SEC) of Sample 3.C Western blot of the fractions eluted by SEC.MALDI-TOF analysis of the SEC fractions VR2 and VR3. The spectrum of sample VR2 shows a [M+H]+ peak featuring twice the mass (~108 000 Da) of the [M+H]+ peak of the βarr1 monomer (sample VR3, ~50 400 Da).


Figure 5C Western blot of whole cell lysates from HEK293T cells expressing βarr1 mutated as indicated in the top lines . All samples were run in parallel on the same gel . ( E ) Western blot of whole cells lysates stained with an α - HA antibody . Residues exchanged with BrEtY are indicated in the upper row , mutations at PTH1R are indicated on the left side . The red arrows indicate the two most prominent signals . Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R .

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Figure 5C Western blot of whole cell lysates from HEK293T cells expressing βarr1 mutated as indicated in the top lines. All samples were run in parallel on the same gel.(E) Western blot of whole cells lysates stained with an α-HA antibody. Residues exchanged with BrEtY are indicated in the upper row, mutations at PTH1R are indicated on the left side. The red arrows indicate the two most prominent signals. Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R.


Figure 1 ( A ) Tandem affinity purification was used to isolate KDF1 associated proteins from SILAC - labelled keratinocytes . Precipitated proteins were resolved by SDS - PAGE and subjected to identification with LC - MS / MS . Arrows denote IgG heavy and light chains . Star denotes KDF1 . Ctrl : control . Kd : kilodalton for molecular weight markers . ( B - C ) HEK293 cells were transfected with empty vector or plasmids encoding HA tagged KDF1 and / or GST - tagged IKKα . Immunoprecipitation ( α - HA or α - GST ) was carried out to determine their interaction . Immunoprecipitates ( IP ) and whole cell lysate ( WCL ) were immunoblotted ( IB ) with different antibodies as indicated . ( D ) Immunoprecipitation was carried out with α - KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium ( Ca ) . IPs were blotted with different antibodies as indicated . Star denotes KDF1 . ( E ) HEK293 cells were transfected to co - express HA - tagged KDF1 with GST - tagged IKKα or its kinase dead ( KD ) mutant . IP and WCL were blotted with different antibodies as indicated . ( F ) HEK293 cells were transfected to co - express HA - tagged KDF1 with GST - tagged IKKα or its different truncation mutants ( EV Fig . 1B ) . IP and WCL were blotted with different antibodies as indicated . Arrow denotes antibody heavy chain . ( G ) HEK293 cells were transfected to co - express GST - tagged IKKα with HA - tagged KDF1 or its different truncation mutants ( EV Fig . 1C ) . IP and WCL were blotted with different antibodies as indicated .

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

Figure 1(A) Tandem affinity purification was used to isolate KDF1 associated proteins from SILAC-labelled keratinocytes. Precipitated proteins were resolved by SDS-PAGE and subjected to identification with LC-MS/MS. Arrows denote IgG heavy and light chains. Star denotes KDF1. Ctrl: control. Kd: kilodalton for molecular weight markers.(B-C) HEK293 cells were transfected with empty vector or plasmids encoding HA tagged KDF1 and/or GST-tagged IKKα. Immunoprecipitation (α-HA or α-GST) was carried out to determine their interaction. Immunoprecipitates (IP) and whole cell lysate (WCL) were immunoblotted (IB) with different antibodies as indicated.(D) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca). IPs were blotted with different antibodies as indicated. Star denotes KDF1.(E) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its kinase dead (KD) mutant. IP and WCL were blotted with different antibodies as indicated.(F) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its different truncation mutants (EV Fig. 1B). IP and WCL were blotted with different antibodies as indicated. Arrow denotes antibody heavy chain.(G) HEK293 cells were transfected to co-express GST-tagged IKKα with HA-tagged KDF1 or its different truncation mutants (EV Fig. 1C). IP and WCL were blotted with different antibodies as indicated.


Figure 2 ( A ) H / E staining of E18 . 5 skin sections from WT , IKKα KO , and KDF1 KO mice . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 50 μm . ( B ) E18 . 5 skin sections from WT , IKKα KO , and KDF1 KO mice were immunostained with different antibodies as indicated ( Krt14 : Keratin 14 , Krt10 : Keratin 10 , Lor : Loricrin , β4 : β4 - integrin , CD104 ) . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 100 μm . ( C ) Immunoblots of WCL collected at 0 , 1 and 2 days after calcium shift with different antibodies as indicated .

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Figure 2(A) H/E staining of E18.5 skin sections from WT, IKKα KO, and KDF1 KO mice. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm. (B) E18.5 skin sections from WT, IKKα KO, and KDF1 KO mice were immunostained with different antibodies as indicated (Krt14: Keratin 14, Krt10: Keratin 10, Lor: Loricrin, β4: β4-integrin, CD104). Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100 μm. (C) Immunoblots of WCL collected at 0, 1 and 2 days after calcium shift with different antibodies as indicated.


Figure 3A ) Immunoblots of WCL from IKKα KO cells with rescued expression of HA - tagged IKKα or its mutant using different antibodies as indicated . B ) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated . Expression f exogenous KDF1 was induced by doxycycline ( DOX ) at different concentration . C - D ) Expression of loricrin is determined by immunoblots and quantified by densitometry . The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ( C - D ) Expression of loricrin is determined by immunoblots and quantified by densitometry . The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05E ) Sections of regenerated skin developed from engrafted IKKα KO , KDF1 KO , and their rescued cells were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 50 μm .

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

Figure 3A) Immunoblots of WCL from IKKα KO cells with rescued expression of HA-tagged IKKα or its mutant using different antibodies as indicated.B) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated. Expression f exogenous KDF1 was induced by doxycycline (DOX) at different concentration.C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05(C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05E) Sections of regenerated skin developed from engrafted IKKα KO, KDF1 KO, and their rescued cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm.


Figure 4 ( A ) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated . Lo : low calcium ; Hi : high calcium . ( B - C ) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide ( CHX ) . WCL was collected at 0 , 30 , 60 , 90 , 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody ( B ) . Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified ( C ) . Statistical analysis is conducted using 2 - way ANOVA . N = 3 ( biological replicates ) . * , p < 0 . 05 . Error bar represents S . D . ( standard deviation ) . ( D - E ) WT and KDF1 KO keratinocyte ( right panels ) or cells transfected with plasmid encoding exogenous IKKα ( left panels ) were treated with MG132 at 10µM for 6 hours , then were subjected to immunoprecipitation using anti - ubiquitin ( Ub ) antibody . IP and WCL were analyzed by immunoblots with α - IKKα antibody ( D ) . Overall intensity of all ubiquitinated IKKα bands was determined by densitometry . Ratio of ubiquitinated IKKα was quantified and presented as bar graphs ( E ) . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * , p < 0 . 05 . Kd : kilodalton for molecular weight markers . ( F ) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant , under both low and high calcium conditions , was examined and quantified by western blotting ( G ) PiggyBac transposon was used to ectopically express HA - tagged IKKα in KDF1 deficient cells . WCL were collected and analyzed by immunoblots with different antibodies as indicated . ( H ) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification . Skin sections from grafted tissue were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bars = 50 µm .

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

Figure 4(A) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated. Lo: low calcium; Hi: high calcium.(B-C) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide (CHX). WCL was collected at 0, 30, 60, 90, 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody (B). Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified (C). Statistical analysis is conducted using 2-way ANOVA. N=3 (biological replicates). *, p<0.05. Error bar represents S.D. (standard deviation).(D-E) WT and KDF1 KO keratinocyte (right panels) or cells transfected with plasmid encoding exogenous IKKα (left panels) were treated with MG132 at 10µM for 6 hours, then were subjected to immunoprecipitation using anti-ubiquitin (Ub) antibody. IP and WCL were analyzed by immunoblots with α-IKKα antibody (D). Overall intensity of all ubiquitinated IKKα bands was determined by densitometry. Ratio of ubiquitinated IKKα was quantified and presented as bar graphs (E). Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). *, p<0.05. Kd: kilodalton for molecular weight markers.(F) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant, under both low and high calcium conditions, was examined and quantified by western blotting(G) PiggyBac transposon was used to ectopically express HA-tagged IKKα in KDF1 deficient cells. WCL were collected and analyzed by immunoblots with different antibodies as indicated.(H) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification. Skin sections from grafted tissue were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bars = 50 µm.


Figure 5A ) HEK293 cells were transfected to co - express KDF1 with USP7 . IP and WCL were blotted with different antibodies as indicated . B ) Immunoprecipitation was carried out with α - KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium ( Ca ) , same as in Fig . 1D . IPs were blotted with different antibodies as indicated . The star denotes KDF1 . C ) WCL from WT and USP7 CRISPR KO cells were subjected to immunoblotting with different antibodies as indicated . D ) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift . E ) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours , then subjected to immunoprecipitation using anti - ubiquitin antibody . Precipitated product was analyzed by immunoblotting with IKKα antibody . Band intensity was determined by densitometry and shown as bar graphs . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * , p < 0 . 01 . Kd : kilodalton for molecular weight markers . F - G ) Immunoblot of Krt10 and Loricrin ( Lor ) with WT and USP7 KO cells before and after calcium shift ( F ) . Star denotes an unspecific band in α - Loricrin blots . Band intensity was determined by densitometry and shown as bar graphsStatistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * * , p < 0 . 0001 ; * * , p < 0 . 01 . H ) Sections of engrafted skin developed from USP7 KO or control WT cells were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 100μm .

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

Figure 5A) HEK293 cells were transfected to co-express KDF1 with USP7. IP and WCL were blotted with different antibodies as indicated.B) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca), same as in Fig. 1D. IPs were blotted with different antibodies as indicated. The star denotes KDF1.C) WCL from WT and USP7 CRISPR KO cells were subjected to immunoblotting with different antibodies as indicated.D) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift.E) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours, then subjected to immunoprecipitation using anti-ubiquitin antibody. Precipitated product was analyzed by immunoblotting with IKKα antibody. Band intensity was determined by densitometry and shown as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). **, p<0.01. Kd: kilodalton for molecular weight markers.F-G) Immunoblot of Krt10 and Loricrin (Lor) with WT and USP7 KO cells before and after calcium shift (F). Star denotes an unspecific band in α-Loricrin blots. Band intensity was determined by densitometry and shown as bar graphsStatistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ****, p<0.0001; **, p<0.01.H) Sections of engrafted skin developed from USP7 KO or control WT cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100μm.


Figure 2 ( H , I ) Induction of Ctcflos and Ucp1 in vivo during cold - induced iWAT browning . Relative expression levels of ( H ) Ctcflos transcript 1 and ( I ) Ucp1 in iWAT of C57BL / 6J mice held at 5 ° C for one week compared to age and weight matched mice held at 30 ° C or 23 ° C , assessed by quantitative PCR ( qPCR ) . Mean and individual values , n = 7 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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Figure 2(H, I) Induction of Ctcflos and Ucp1 in vivo during cold-induced iWAT browning. Relative expression levels of (H) Ctcflos transcript 1 and (I) Ucp1 in iWAT of C57BL/6J mice held at 5°C for one week compared to age and weight matched mice held at 30°C or 23°C, assessed by quantitative PCR (qPCR). Mean and individual values, n=7 (biological replicates), one-way ANOVA (Šídák-test), **p< 0.01, ****p<0.0001.


Figure 3 ( D ) Relative transcript levels of Ctcflos and Ucp1 in various tissues of 129S6 mice , assessed by qPCR . Mean values ± SD , n = 1 ( biological replicate ) for Ucp1n = 3 ( biological replicates ) for Ctcflos . ( E ) Ctcflos , 45SrRNA and 12SrRNA transcript abundance in nuclear , cytosolic and mitochondrial subfractions of primary brite adipocytes of 129S6 mice , assessed by qPCR . Mean values ± SD , n = 6 ( biological replicates ) , two - way ANOVA ( Tukey - test ) , * * * * p < 0 . 0001 .

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Figure 3(D) Relative transcript levels of Ctcflos and Ucp1 in various tissues of 129S6 mice, assessed by qPCR. Mean values ± SD, n=1 (biological replicate) for Ucp1n=3 (biological replicates) for Ctcflos .(E) Ctcflos, 45SrRNA and 12SrRNA transcript abundance in nuclear, cytosolic and mitochondrial subfractions of primary brite adipocytes of 129S6 mice, assessed by qPCR. Mean values ± SD, n=6 (biological replicates), two-way ANOVA (Tukey-test), ****p< 0.0001.


Figure 4 ( E ) Relative Ucp1 transcript levels in response to Ctcflos tr1 , 3 and 4 KD by ASO 1 , 3 , 4 and 2 , 5 6 , respectively , compared to nontargeting controls , assessed by qPCR . Mean values ± SD , n = 3 - 4 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( F ) Relative expression levels of cell death - inducing DNA fragmentation factor alpha - like effector A ( Cidea ) and cytochrome c oxidase subunit 7a1 ( Cox7a1 ) in Ctcflos tr1 , 3 and 4 KD compared to control samples , assessed by qPCR . Mean values ± SD , n = 3 ( biological replicates ) - 4 , unpaired t tests or in grey parenthesis two - way ANOVA ( Šídák - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( H ) Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 ( UCP1 ( red ) ; DNA staining by DAPI ( green ) ; phase contrast ( PC ) ( gray ) ) . Impact of Ctcflos KD on mitochondrial biogenesis . ( R ) Microscopic images of Mito Tracker stained brite adipocytes ( Mito tracker ( blue ) ; DNA staining by DAPI ( green ) ; phase contrast ( PC ) ( gray ) )

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

Figure 4(E) Relative Ucp1 transcript levels in response to Ctcflos tr1, 3 and 4 KD by ASO 1, 3, 4 and 2, 5 6, respectively, compared to nontargeting controls, assessed by qPCR. Mean values ± SD, n=3-4 (biological replicates), one-way ANOVA (Šídák-test), ***p<0.001, ****p<0.0001.(F) Relative expression levels of cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea) and cytochrome c oxidase subunit 7a1 (Cox7a1) in Ctcflos tr1, 3 and 4 KD compared to control samples, assessed by qPCR. Mean values ± SD, n=3 (biological replicates)-4, unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.(H) Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 (UCP1 (red); DNA staining by DAPI (green); phase contrast (PC) (gray)).Impact of Ctcflos KD on mitochondrial biogenesis. (R) Microscopic images of Mito Tracker stained brite adipocytes (Mito tracker (blue); DNA staining by DAPI (green); phase contrast (PC) (gray))


Figure 5 ( D ) Relative expression levels of brite adipocyte marker genes uncoupling protein 1 ( Ucp1 ) , cell death - inducing DNA fragmentation factor alpha - like effector A ( Cidea ) , cytochrome c oxidase subunit 7a1 ( Cox7a1 ) , peroxisome proliferator activated receptor γ coactivator 1α ( Pgc1a ) and elongation of very long chain fatty acids - like 3 ( Elovl3 ) , comparing Ctcflos tr1 ( ASO 1 ) and tr 3 and 4 ( ASO 2 ) KD with their respective controls . Mean values ± SD , n = 6 ( biological replicates ) , unpaired t tests or in grey parenthesis two - way ANOVA ( Šídák - test ) n . s . p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( K - M ) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression . Primary iWAT cells infected with Prdm16 - or turbo green fluorescent protein ( GFP ) - expressing viral particles at the second day of proliferation , followed by reverse transfection at the first day of differentiation using nontargeting control ( NC ) or Ctcflos - targeting ASO1 . Gene expression analyzed 72 hours later by qPCR ( K ) Transcript levels of Ctcflos tr1 relative to Fabp4 ( L ) Transcript levels of Prdm16 relative to Fabp4 ( M ) Transcript levels of Ucp1 relative to Fabp4 . Mean and individual values . Each graph presents pooled data from two experiments slightly varying virus titers . RM One - way ANOVA ( Tukey - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 5(D) Relative expression levels of brite adipocyte marker genes uncoupling protein 1 (Ucp1), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), cytochrome c oxidase subunit 7a1 (Cox7a1), peroxisome proliferator activated receptor γ coactivator 1α (Pgc1a) and elongation of very long chain fatty acids-like 3 (Elovl3), comparing Ctcflos tr1 (ASO 1) and tr 3 and 4 (ASO 2) KD with their respective controls. Mean values ± SD, n=6 (biological replicates), unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test) n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001.(K-M) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression. Primary iWAT cells infected with Prdm16- or turbo green fluorescent protein (GFP)-expressing viral particles at the second day of proliferation, followed by reverse transfection at the first day of differentiation using nontargeting control (NC) or Ctcflos-targeting ASO1. Gene expression analyzed 72 hours later by qPCR (K) Transcript levels of Ctcflos tr1 relative to Fabp4 (L) Transcript levels of Prdm16 relative to Fabp4 (M) Transcript levels of Ucp1 relative to Fabp4. Mean and individual values. Each graph presents pooled data from two experiments slightly varying virus titers. RM One-way ANOVA (Tukey-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.


Figure 6 ( B - D ) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis , ( B ) expressed as Ucp1 relative to Fabp4 mRNA levels , ( C ) Ucp1 relative to Gtf2b and ( D ) Fabp4 relative to Gtf2b . Mean values ± SD , n = 3 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Effect of Ctcflos KD on Serine / Arginine rich Splicing factor 2 ( SC35 / SRSF2 ) and nuclear speckle abundance in Ctcflos tr1 ( ASO 1 ) KD and control cells ( KD day 1 of differentiation , analysis after 48h ) . ( E ) Immunocytochemistry of SC35 . SC35 signal ( blue ) alone ( middle images ) and SC35 ( blue ) , DNA staining by DAPI ( green ) and phase contrast ( PC ) ( gray ) combined ( outer images ) . Influence of Ctcflos KD on Prdm16 alternative splicing . ( O ) Relative Prdm16 total , ( P ) Relative Prdm16 long transcript levels 24 hours after Ctcflos KD , comparing Ctcflos tr1 ( ASO1 ) and tr3 , 4 ( ASO2 ) KD and control samples , assessed by qPCR , Mean values ± SD , n = 3 - 4 ( biological replicates ) , unpaired t tests , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Influence of Ctcflos KD on Prdm16 alternative splicing . ( R , S ) Fold change of Prdm16 short and Prdm16 long ( R ) ASO1 to NC and ( S ) ASO2 and NC 24 hours after Ctcflos KD , n = 3 - 4 ( biological replicates ) , unpaired t tests , n . s . p > 0 . 05 , * * p < 0 . 01 .

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

Figure 6(B-D) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis, (B) expressed as Ucp1 relative to Fabp4 mRNA levels, (C) Ucp1 relative to Gtf2b and (D) Fabp4 relative to Gtf2b. Mean values ± SD, n=3 (biological replicates), one-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Effect of Ctcflos KD on Serine/Arginine rich Splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (E) Immunocytochemistry of SC35. SC35 signal (blue) alone (middle images) and SC35 (blue), DNA staining by DAPI (green) and phase contrast (PC) (gray) combined (outer images).Influence of Ctcflos KD on Prdm16 alternative splicing. (O) Relative Prdm16 total, (P) Relative Prdm16 long transcript levels 24 hours after Ctcflos KD, comparing Ctcflos tr1 (ASO1) and tr3, 4 (ASO2) KD and control samples, assessed by qPCR, Mean values ± SD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, * p<0.05, **p<0.01, ***p<0.001.Influence of Ctcflos KD on Prdm16 alternative splicing. (R, S) Fold change of Prdm16 short and Prdm16 long (R) ASO1 to NC and (S) ASO2 and NC 24 hours after Ctcflos KD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, ** p<0.01.


Figure 1 ( B ) Representative emission spectra of mixtures containing ECFP - D4H , mVenus - Lact - C2 , and liposomes containing indicated compositions of lipids . Note that FRET signal was increased in the presence of both cholesterol ( 50 % ) and phosphatidylserine ( DOPS ) ( 20 % ) and that FRET signal was decreased when sphingomyelin ( SM ) ( 25 % ) was additionally incorporated into the liposomes . Asterisks indicate positions of emission maximum of ECFP ( * 477 nm ) and FRET ( * * 525 nm ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol ; SM , sphingomyelin ( N - oleoyl - D - erythro - sphingosylphosphorylcholine ) . ( C ) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm ( FRET / ECFP ) ( see Materials and Methods ) from mixtures as shown in ( D ) ( mean ± SEM , n = 4 independent experiments ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ) . ( D ) Left : Time course of normalized mCherry signal , as assessed by TIRF microscopy , from GRAMD1 triple knockout ( TKO ) HeLa cells expressing mCherry - GRAM1b . Cells were pre - incubated with either control imaging buffer ( for control ) or imaging buffer containing purified ECFP - D4H proteins ( 3 µM ) ( for masking accessible PM cholesterol ) for 30 min at 37 ° C and imaged with the same buffer conditions . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 32 cells ( Control ) , n = 35 cells ( Pre - incubation w / ECFP - D4H ) ; data are pooled from two independent experiments for each condition ; two - tailed unpaired Student ' s t - test , * * P < 0 . 0001 ] . ( E ) Left : Time course of normalized EGFP signal , as assessed by TIRF microscopy , from GRAMD1 triple knockout ( TKO ) HeLa cells expressing EGFP - GRAM1b together with either mCherry for control or mCherry - Lact - C2 for PS masking . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 53 cells ( mCherry ) , n = 32 cells ( mCherry - Lact - C2 OE ) ; data are pooled from four independent experiments for each condition ; two - tailed unpaired Student ' s t - test , * * P < 0 . 0001 ] .

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

Figure 1(B) Representative emission spectra of mixtures containing ECFP-D4H, mVenus-Lact-C2, and liposomes containing indicated compositions of lipids. Note that FRET signal was increased in the presence of both cholesterol (50%) and phosphatidylserine (DOPS) (20%) and that FRET signal was decreased when sphingomyelin (SM) (25%) was additionally incorporated into the liposomes. Asterisks indicate positions of emission maximum of ECFP (*477 nm) and FRET (**525 nm). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; SM, sphingomyelin (N-oleoyl-D-erythro-sphingosylphosphorylcholine). (C) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm (FRET/ECFP) (see Materials and Methods) from mixtures as shown in (D) (mean ± SEM, n = 4 independent experiments; Dunnett's multiple comparisons test, **P < 0.0001).(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing mCherry-GRAM1b. Cells were pre-incubated with either control imaging buffer (for control) or imaging buffer containing purified ECFP-D4H proteins (3 µM) (for masking accessible PM cholesterol) for 30 min at 37°C and imaged with the same buffer conditions. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 32 cells (Control), n = 35 cells (Pre-incubation w/ ECFP-D4H); data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].(E) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing EGFP-GRAM1b together with either mCherry for control or mCherry-Lact-C2 for PS masking. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 53 cells (mCherry), n = 32 cells (mCherry-Lact-C2 OE); data are pooled from four independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].


Figure 2 ( C - E ) Liposome sedimentation assays of wild - type GRAM1b proteins ( WT ) and mutant versions of GRAM1b proteins carrying indicated mutations ( K161A , R191A , and K161A / R191A ) . Liposomes containing the indicated mole % lipids were incubated with purified GRAM1b proteins as shown . Bound proteins [ pellet , ( P ) ] were separated from the unbound proteins [ supernatant , ( S ) ] , run on SDS - PAGE and visualized by colloidal blue staining ( mean ± SEM , n = 3 independent experiments for all conditions in phosphatidylserine ( PS ) sensitivity , co - incidence detection , and anionic lipid selectivity assays ; n = 12 independent experiments for WT , n = 3 independent experiments for K161A and R191A , and n = 6 independent experiments for K161A / R191A in the cholesterol sensitivity assay ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol ; PA , phosphatidic acid ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphate ) ; PI ( 4 ) P , phosphatidylinositol 4 - phosphate ; PI ( 4 , 5 ) P2 , phosphatidylinositol 4 , 5 - bisphosphate ; PI , L - α - phosphatidylinositol .

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

Figure 2(C-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (K161A, R191A, and K161A/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 3 independent experiments for all conditions in phosphatidylserine (PS) sensitivity, co-incidence detection, and anionic lipid selectivity assays; n = 12 independent experiments for WT, n = 3 independent experiments for K161A and R191A, and n = 6 independent experiments for K161A/R191A in the cholesterol sensitivity assay). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; PA, phosphatidic acid (1,2-dioleoyl-sn-glycero-3-phosphate); PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI, L-α-phosphatidylinositol.


Figure 3 ( B ) Sterol specificity of the GRAM domain of GRAMD1b ( GRAM1b ) as assessed via liposome sedimentation assays . Liposomes containing the indicated mole % lipids were incubated with purified GRAM1b proteins as indicated . Bound proteins [ pellet , ( P ) ] were separated from the unbound proteins [ supernatant , ( S ) ] , run on SDS - PAGE and visualized by colloidal blue staining ( mean ± SEM , n = 6 independent experiments for liposomes containing only DOPC , n = 3 independent experiments for the rest ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) . ( D - E ) Liposome sedimentation assays of wild - type GRAM1b proteins ( WT ) and mutant versions of GRAM1b proteins carrying indicated mutations ( R189W , R189W / R191A ) . Liposomes containing the indicated mole % lipids were incubated with purified GRAM1b proteins as shown . Bound proteins [ pellet , ( P ) ] were separated from the unbound proteins [ supernatant , ( S ) ] , run on SDS - PAGE and visualized by colloidal blue staining [ mean ± SEM , n = 3 independent experiments for all conditions in the co - incidence detection assay ; n = 3 independent experiments for R189W and R189W / R191A , n = 6 independent experiments for WT in phosphatidylserine ( PS ) sensitivity assay ; n = 3 independent experiments for R189W and R189W / R191A , n = 5 independent experiments for WT in cholesterol sensitivity assay ] . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol . ( F ) Confocal images of live GRAMD1 TKO HeLa cells expressing either wild - type ( WT ) or mutant versions of EGFP - tagged GRAM domain of GRAMD1b ( EGFP - GRAM1b ) constructs as indicated . Cells were treated with SMase ( 100 mU / ml for 1 hour at 37 ° C ) before imaging . Insets show at higher magnification the regions indicated by white dashed boxes . Anionic lipid sensing defective mutants ( K161A , R191A , and K161A / R191A ) , a cholesterol sensing defective mutant ( R189W ) , and a cholesterol / anionic lipid sensing defective mutant ( R189W / R191A ) are shown . Note the absence of ( R189W , R189W / R191A , and K161A / R191A ) or significantly reduced ( K161A , R191A ) PM recruitment of the mutant versions of EGFP - GRAM1b compared to the strong PM recruitment of wild - type EGFP - GRAM1b ( WT ) . Scale bars , 10 µm . ( G ) Quantification of the ratio of PM EGFP - GRAM1b signals to the cytosolic EGFP - GRAM1b signals , as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing either wild - type ( WT ) or mutant versions of EGFP - GRAM1b with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) as shown in ( F ) and Appendix Fig S1B ( mean ± SEM , n = 40 cells for WT , and n = 30 cells for other conditions ; data are pooled from four independent experiments for WT and three independent experiments for the rest ; two - tailed unpaired Student ' s t - test , * * P < 0 . 0001 . n . s . denotes not significant ) . ( H ) Left : Time course of normalized EGFP signal , as assessed by TIRF microscopy , from GRAMD1 TKO HeLa cells expressing either wild - type ( WT ) or mutant versions of EGFP - GRAM1b constructs as indicated . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 126 cells ( WT ) , n = 38 cells ( K161A ) , n = 33 cells ( R191A ) , n = 43 cells ( K161A / R191A ) , n = 46 cells ( R189W ) , n = 44 cells ( R189W / R191A ) ; data are pooled from six independent experiments for WT and two independent experiments for the rest ; Dunnett ' s multiple comparisons test , * * P = 0 . 0002 ( WT vs . K161A ) and * * P < 0 . 0001 for the rest ] .

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

Figure 3(B) Sterol specificity of the GRAM domain of GRAMD1b (GRAM1b) as assessed via liposome sedimentation assays. Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as indicated. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 6 independent experiments for liposomes containing only DOPC, n = 3 independent experiments for the rest). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine).(D-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (R189W, R189W/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining [mean ± SEM, n = 3 independent experiments for all conditions in the co-incidence detection assay; n = 3 independent experiments for R189W and R189W/R191A, n = 6 independent experiments for WT in phosphatidylserine (PS) sensitivity assay; n = 3 independent experiments for R189W and R189W/R191A, n = 5 independent experiments for WT in cholesterol sensitivity assay]. DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol.(F) Confocal images of live GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-tagged GRAM domain of GRAMD1b (EGFP-GRAM1b) constructs as indicated. Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Anionic lipid sensing defective mutants (K161A, R191A, and K161A/R191A), a cholesterol sensing defective mutant (R189W), and a cholesterol/anionic lipid sensing defective mutant (R189W/R191A) are shown. Note the absence of (R189W, R189W/R191A, and K161A/R191A) or significantly reduced (K161A, R191A) PM recruitment of the mutant versions of EGFP-GRAM1b compared to the strong PM recruitment of wild-type EGFP-GRAM1b (WT). Scale bars, 10 µm.(G) Quantification of the ratio of PM EGFP-GRAM1b signals to the cytosolic EGFP-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b with or without SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (F) and Appendix Fig S1B (mean ± SEM, n = 40 cells for WT, and n = 30 cells for other conditions; data are pooled from four independent experiments for WT and three independent experiments for the rest; two-tailed unpaired Student's t-test, **P < 0.0001. n.s. denotes not significant).(H) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b constructs as indicated. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 126 cells (WT), n = 38 cells (K161A), n = 33 cells (R191A), n = 43 cells (K161A/R191A), n = 46 cells (R189W), n = 44 cells (R189W/R191A); data are pooled from six independent experiments for WT and two independent experiments for the rest; Dunnett's multiple comparisons test, **P = 0.0002 (WT vs. K161A) and **P < 0.0001 for the rest].


Figure 4 ( B ) Liposome tethering by purified GRAMD1b proteins . Left : Representative time course of liposome tethering , as assessed by turbidity of mixtures containing both LPM and LER together with either wild - type GRAMD1b proteins ( WT ) , mutant GRAMD1b R189W proteins ( R189W ) , mutant GRAMD1b R189W / R191A proteins ( RW / RA ) , or buffer alone as indicated . Turbidity , as assessed by measuring absorbance values at 405 nm , reflects clustering of liposomes due to tethering of LPM and LER . Right : Values of turbidity corresponding to the end of the experiment as indicated by the arrow ( mean ± SEM , n = 6 independent experiments for WT , n = 3 independent experiments for buffer alone , R189W , and RW / RA ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ) ( C ) DHE transfer between liposomes by purified GRAMD1b proteins . Left : Representative time course of DHE transfer from LPM to LER mediated by either wild - type GRAMD1b proteins ( WT ) , mutant GRAMD1b R189W proteins ( R189W ) , mutant GRAMD1b R189W / R191A proteins ( RW / RA ) , or buffer alone as indicated . Methyl - β - cyclodextrin ( MCD ) at 1 mM was used to determine DHE equilibration . Transfer of DHE from LPM to LER resulted in a decrease in fluorescence resonance energy transfer ( FRET ) between DHE and DNS - PE in LPM . A series of liposomes with different DHE mol % were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred ( see Appendix Fig S2D and Materials and Methods ) . Right : Values of DHE transfer rate of GRAMD1b ( WT ) , GRAMD1b ( R189W ) , and GRAMD1b ( RW / RA ) , as estimated by the FRET - based lipid transfer assay [ mean ± SEM , n = 6 independent experiments for WT , n = 3 independent experiments for R189W and RW /

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

Figure 4(B) Liposome tethering by purified GRAMD1b proteins. Left: Representative time course of liposome tethering, as assessed by turbidity of mixtures containing both LPM and LER together with either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Turbidity, as assessed by measuring absorbance values at 405 nm, reflects clustering of liposomes due to tethering of LPM and LER. Right: Values of turbidity corresponding to the end of the experiment as indicated by the arrow (mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for buffer alone, R189W, and RW/RA; Dunnett's multiple comparisons test, **P < 0.0001)(C) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Transfer of DHE from LPM to LER resulted in a decrease in fluorescence resonance energy transfer (FRET) between DHE and DNS-PE in LPM. A series of liposomes with different DHE mol% were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred (see Appendix Fig S2D and Materials and Methods). Right: Values of DHE transfer rate of GRAMD1b (WT), GRAMD1b (R189W), and GRAMD1b (RW/RA), as estimated by the FRET-based lipid transfer assay [mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for R189W and RW/RA