Figure 2Relative mRNA levels of immune related genes in Vgll4 - knockdown LLC or MB49 cells by qRT - PCR analysisKnockdown of Vgll4 in MB49 cells decreased the surface expression of PD - L1 protein on control or shVgll4 MB49 cells measured by flow cytometry analysis with anti - mPD - L1 antibodyRelative mRNA levels of immune related genes in Vgll4 - knockdown A549 cells by qRT - PCR analysisImmunoblot analysis revealed the reduced expression of PD - L1 protein in VGLL4 knockdown A549 , 16HBE and H358 cells compared with control cellsqRT - PCR analysis revealed a reduction of PD - L1 mRNA levels in VGLL4 - knockdown 16HBE and H358 cells by siRNAFlow cytometry analysis of surface PD - L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated timesImmunoblot analysis revealed the reduced IFNγ - inducible PD - L1 expression in VGLL4 knockdown A549 and 16HBE cellsImmunoblot analysis of total cell lysates with the indicated antibodies revealed the attenuated IFNγ - inducible PD - L1 expression in VGLL4 knockout ( KO ) A549 cells generated by CRISPR / Cas9Loss of VGLL4 dampened the activity of PD - L1 promoter reporter in a luciferase assay . A549 cells were transfected with human PD - L1 - promoter luciferase plasmid and indicated siRNA , treated with IFNγ for 12 h , and subjected to a luciferase assayOverexpression of mouse PD - L1 in Vgll4 knockdown LLC cells restored the tumor growth in C57BL / 6 mice . Tumor weights of shVgll4 or shVgll4 - lenti - mPD - L1 LLC cells were determined 21 days after tumor cell transplantation into C57BL / 6 mice ( left panel ) . Expression of mPD - L1 was shown in the right panel by immunoblot analysis with anti - mPD - L1 antibody . n = 10 tumors for each groupKnockdown of VGLL4 in A549 cells enhances T cell - meditated tumor cell killing . Activated T cells and A549 cells were co - cultured in 24 - well plates for 4 days and then surviving tumor cells were visualized by crystal violet staining . Relative fold ratios of surviving cell intensities are shown in right

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Figure 2Relative mRNA levels of immune related genes in Vgll4-knockdown LLC or MB49 cells by qRT-PCR analysisKnockdown of Vgll4 in MB49 cells decreased the surface expression of PD-L1 protein on control or shVgll4 MB49 cells measured by flow cytometry analysis with anti-mPD-L1 antibodyRelative mRNA levels of immune related genes in Vgll4-knockdown A549 cells by qRT-PCR analysisImmunoblot analysis revealed the reduced expression of PD-L1 protein in VGLL4 knockdown A549, 16HBE and H358 cells compared with control cellsqRT-PCR analysis revealed a reduction of PD-L1 mRNA levels in VGLL4-knockdown 16HBE and H358 cells by siRNAFlow cytometry analysis of surface PD-L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated timesImmunoblot analysis revealed the reduced IFNγ-inducible PD-L1 expression in VGLL4 knockdown A549 and 16HBE cellsImmunoblot analysis of total cell lysates with the indicated antibodies revealed the attenuated IFNγ-inducible PD-L1 expression in VGLL4 knockout (KO) A549 cells generated by CRISPR/Cas9Loss of VGLL4 dampened the activity of PD-L1 promoter reporter in a luciferase assay. A549 cells were transfected with human PD-L1-promoter luciferase plasmid and indicated siRNA, treated with IFNγ for 12 h, and subjected to a luciferase assayOverexpression of mouse PD-L1 in Vgll4 knockdown LLC cells restored the tumor growth in C57BL/6 mice. Tumor weights of shVgll4 or shVgll4-lenti-mPD-L1 LLC cells were determined 21 days after tumor cell transplantation into C57BL/6 mice (left panel). Expression of mPD-L1 was shown in the right panel by immunoblot analysis with anti-mPD-L1 antibody. n=10 tumors for each groupKnockdown of VGLL4 in A549 cells enhances T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and then surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensities are shown in right panel


Figure 3The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti - VGLL4 antibody and visualized by anti - IRF2BP2 antibody in A549 cellsThe endogenous IRF2BP2 was immunoprecipitated by VGLL4 - FLAG proteins in HEK293T cellsInteraction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co - immunoprecipitation of differentially tagged proteins in HEK293T cellsC - terminus of IRF2BP2 interacted with VGLL4 by co - immunoprecipitation assay in HEK293T cellsInteraction of IRF2BP2 with VGLL1 - 4 was revealed by co - immunoprecipitation of differentially tagged proteins in HEK293T cellsTDU domains in VGLL4 are required for the interaction with TEADs by co - immunoprecipitation assay in HEK293T cellsTDU - deleted or HF4A VGLL4 mutations interacted with IRF2BP by co - immunoprecipitation analysis in HEK293T cellsTDU - deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co - immunoprecipitation analysis in HEK293T cellsExpression of VGLL4 - WT or VGLL4 - HF4A enhances PD - L1 expression in A549 cells by immunoblot analysis . A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated antibodiesVGLL4 - HF4A rescues PD - L1 expression in VGLL4 - knockdown A549 cells . A549 cells were transfected with siRNA targeting to the 3 " UTR of VGLL4 mRNA and followed by transduction with lenti - VGLL4 - HF4A virus . Cell lysates were analyzed by western blot with indicated antibExpression of VGLL4 - HF4A rescues tumor growth of VGLL4 - knockdown LLC cells in C57BL / 6 mice . Control , Vgll4 knockdown or Vgll4 knockdown together with VGLL4 - HF4A overexpressing LLC cells were transplanted into nude mice or C57BL6 mice . Tumor volumes were measured 15 days for nude mice and 21 days for C57BL / 6 mice after tumor cell inoculationExpression of VGLL4 - HF4A attenuates the T cell - meditated tumor cell killing . Activated T cells and A549 cells were co - cultured in 24 - well plates for 4 days and Relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal

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Figure 3The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti-VGLL4 antibody and visualized by anti-IRF2BP2 antibody in A549 cellsThe endogenous IRF2BP2 was immunoprecipitated by VGLL4-FLAG proteins in HEK293T cellsInteraction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cellsC-terminus of IRF2BP2 interacted with VGLL4 by co-immunoprecipitation assay in HEK293T cellsInteraction of IRF2BP2 with VGLL1-4 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cellsTDU domains in VGLL4 are required for the interaction with TEADs by co-immunoprecipitation assay in HEK293T cellsTDU-deleted or HF4A VGLL4 mutations interacted with IRF2BP by co-immunoprecipitation analysis in HEK293T cellsTDU-deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co-immunoprecipitation analysis in HEK293T cellsExpression of VGLL4-WT or VGLL4-HF4A enhances PD-L1 expression in A549 cells by immunoblot analysis. A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated antibodiesVGLL4-HF4A rescues PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with siRNA targeting to the 3"UTR of VGLL4 mRNA and followed by transduction with lenti-VGLL4-HF4A virus. Cell lysates were analyzed by western blot with indicated antibExpression of VGLL4-HF4A rescues tumor growth of VGLL4-knockdown LLC cells in C57BL/6 mice. Control, Vgll4 knockdown or Vgll4 knockdown together with VGLL4-HF4A overexpressing LLC cells were transplanted into nude mice or C57BL6 mice. Tumor volumes were measured 15 days for nude mice and 21 days for C57BL/6 mice after tumor cell inoculationExpression of VGLL4-HF4A attenuates the T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and Relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal violet


Figure 4Correlation between protein expression of VGLL4 and IRF2BP2 in 6 murine tumor cell line with the indicated antibodiesCorrelation between protein expression of VGLL4 and IRF2BP2 i human cell line with the indicated antibodiesImmunoblot analysis of A549 cells transfected with VGLL4 - FLAG or VGLL4 - HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control cellsDeficiency of VGLL4 reduces IRF2BP2 protein levels . Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and VGLL4The indicated cell lines were transfected with control or VGLL4 siRNA and subjected to immunoblot analysis with the indicated antibodiesqRT - PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549 cellsA549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies . Relative IRF2BP2 expression levels were quantified in the right panelLys48 - linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells . HEK293T cells were transfected with indicated plasmids , treated with MG132 for 3 h before harvest , and subjected to immunoprecipitation and immunoblot analysis as indicatedLys48 - linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells . HEK293T cells were transfected with indicated siRNA and plasmids , and subjected to immunoprecipitation and immunoblot analysis as

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Figure 4Correlation between protein expression of VGLL4 and IRF2BP2 in 6 murine tumor cell line with the indicated antibodiesCorrelation between protein expression of VGLL4 and IRF2BP2 i human cell line with the indicated antibodiesImmunoblot analysis of A549 cells transfected with VGLL4-FLAG or VGLL4-HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control cellsDeficiency of VGLL4 reduces IRF2BP2 protein levels. Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and VGLL4The indicated cell lines were transfected with control or VGLL4 siRNA and subjected to immunoblot analysis with the indicated antibodiesqRT-PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549 cellsA549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies. Relative IRF2BP2 expression levels were quantified in the right panelLys48-linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells. HEK293T cells were transfected with indicated plasmids, treated with MG132 for 3 h before harvest, and subjected to immunoprecipitation and immunoblot analysis as indicatedLys48-linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells. HEK293T cells were transfected with indicated siRNA and plasmids, and subjected to immunoprecipitation and immunoblot analysis as indicated


Figure 5IRF2 inhibits IRF1 transcriptional activity . A549 cells transfected with human PD - L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase assayKnockdown IRF2 restores PD - L1 expression in VGLL4 - knockdown A549 cells . Gene expression levels were analyzed by qRT - PCR in A549 cells transfected or transduced as indicatedOverexpression of IRF1 increases PD - L1 expression in VGLL4 - knockdown A549 cells . A549 cells were transfected with VGLL4 siRNA or together with HA - IRF1 plasmid and total cell lysates were analyzed by western blotIRF2 is released from PD - L1 promoter upon IFNγ stimulation . IRF2 - 3 × HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP - qPCR analysis in the PD - L1 promoter using control and HA antibodies . Normal mouse IgG was used as controlIncreased association of IRF2 with PD - L1 promoter in IRF2BP2 knockout ( KO ) A549 cells revealed by ChIP - qPCR analysis . Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower panelDynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti - HA antibody in IRF2 - 3 × HA knockin A549 cellsLoss of IRF2BP2 attenuated IFNγ - inducible PD - L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD - L1 protein levelLoss of IRF2BP2 attenuated IFNγ - inducible PD - L1 mRNA expression revealed by qRT - PCR analysis in A549 control and IRF2BP2 knockout

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Figure 5IRF2 inhibits IRF1 transcriptional activity. A549 cells transfected with human PD-L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase assayKnockdown IRF2 restores PD-L1 expression in VGLL4-knockdown A549 cells. Gene expression levels were analyzed by qRT-PCR in A549 cells transfected or transduced as indicatedOverexpression of IRF1 increases PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with VGLL4 siRNA or together with HA-IRF1 plasmid and total cell lysates were analyzed by western blotIRF2 is released from PD-L1 promoter upon IFNγ stimulation. IRF2-3×HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP-qPCR analysis in the PD-L1 promoter using control and HA antibodies. Normal mouse IgG was used as controlIncreased association of IRF2 with PD-L1 promoter in IRF2BP2 knockout (KO) A549 cells revealed by ChIP-qPCR analysis. Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower panelDynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti-HA antibody in IRF2-3×HA knockin A549 cellsLoss of IRF2BP2 attenuated IFNγ-inducible PD-L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD-L1 protein levelLoss of IRF2BP2 attenuated IFNγ-inducible PD-L1 mRNA expression revealed by qRT-PCR analysis in A549 control and IRF2BP2 knockout cells


Figure 6miR - 130a represses VGLL4 and PD - L1 protein levels . A549 cells were transfected with control and miR - 130a mimic and subjected to immunoblot analysisInhibition of miR - 130a by its sponge enhances IFNγ - inducible PD - L1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected to immunoblotInhibition of miR - 130a by its sponge enhances IFNγ - inducible PD - L1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected t qRT - PCR analysisDecreased YAP target gene expression by inhibition of miR - 130a using its sponge in A549 cellsYAP5SA expression attenuates IFNγ - inducible PD - L1 expression . A549 cells were transduced with control , YAP - 5SA or YAP - S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot analysisqRT - PCR analysis of cell lysates from control , YAP - 5SA or YAP - S94A expressing A549 cells treated with IFNγ for the indicated timesControl or YAP - overexpressing LLC cells were transplanted into nude mice ( upper panel ) or C57BL / 6 ( lower panel ) mice , and tumor - growth kinetics was measured at the indicated times . Tumor volumes at the end time points were analyzed and shown at the right panelmiR - 130a regulates IRF1 3 ′ UTR sensor activity . A549 cells were transfected with WT or mutant IRF1 3 ′ UTR sensor plasmids together with miR - 130a mimic . Sensor activities were determined by dual luciferase assayYAP5SA induces miR - 130a expression and represses IRF1 expression . A549 cells were transduced with control or YAP - 5SA lentivirus and subjected to qRT - PCR analysisInhibition of miR - 130a by its sponge enhances IFNγ - inducible IRF1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected to qRT - PCR analysisSuppression of IFNγ - inducible PD - L1 expression by YAP5SA is compromised in miR - 130a KO A549 cells . PD - L1 expression levels were analyzed by qRT - PCR in IFNγ - treated control or miR - 130a KO A549 cells pre - transduced with lenti - YAP5SA

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Figure 6miR-130a represses VGLL4 and PD-L1 protein levels. A549 cells were transfected with control and miR-130a mimic and subjected to immunoblot analysisInhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to immunoblotInhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected t qRT-PCR analysisDecreased YAP target gene expression by inhibition of miR-130a using its sponge in A549 cellsYAP5SA expression attenuates IFNγ-inducible PD-L1 expression. A549 cells were transduced with control, YAP-5SA or YAP-S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot analysisqRT-PCR analysis of cell lysates from control, YAP-5SA or YAP-S94A expressing A549 cells treated with IFNγ for the indicated timesControl or YAP-overexpressing LLC cells were transplanted into nude mice (upper panel) or C57BL/6 (lower panel) mice, and tumor-growth kinetics was measured at the indicated times. Tumor volumes at the end time points were analyzed and shown at the right panelmiR-130a regulates IRF1 3′UTR sensor activity. A549 cells were transfected with WT or mutant IRF1 3′UTR sensor plasmids together with miR-130a mimic. Sensor activities were determined by dual luciferase assayYAP5SA induces miR-130a expression and represses IRF1 expression. A549 cells were transduced with control or YAP-5SA lentivirus and subjected to qRT-PCR analysisInhibition of miR-130a by its sponge enhances IFNγ-inducible IRF1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to qRT-PCR analysisSuppression of IFNγ-inducible PD-L1 expression by YAP5SA is compromised in miR-130a KO A549 cells. PD-L1 expression levels were analyzed by qRT-PCR in IFNγ-treated control or miR-130a KO A549 cells pre-transduced with lenti-YAP5SA virus


Figure 7Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non - small cell lung cancer tissues . IHC score is indicated . Scale bar : 50 μmStaining of VGLL4 and IRF2BP2 on human non - small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section . Correlation of VGLL4 and IRF2BP2 expressions was significant ( Pearson correlation test ; R = 0 . 6341 , P < 0 . 001 ) Correlation analysis of VGLL4 and PD - L1 expression on human non - small cell lung cancer tissue arrays containing 71 samplesKaplan - Meier survival analysis of patients with high ( n = 74 ) or low ( n = 130 ) VGLL4 mRNA levels from GSE31210 , a lung cancer data set . Survival curves are calculated according to the Kaplan - Meier

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Figure 7Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non-small cell lung cancer tissues. IHC score is indicated. Scale bar: 50 μmStaining of VGLL4 and IRF2BP2 on human non-small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section. Correlation of VGLL4 and IRF2BP2 expressions was significant (Pearson correlation test; R = 0.6341, P < 0.001) Correlation analysis of VGLL4 and PD-L1 expression on human non-small cell lung cancer tissue arrays containing 71 samplesKaplan-Meier survival analysis of patients with high (n=74) or low (n=130) VGLL4 mRNA levels from GSE31210, a lung cancer data set. Survival curves are calculated according to the Kaplan-Meier method


Figure 1 ( A , B ) Effect of MPC inhibition on cellular lipid droplet content . ( A ) Live - cell super - resolution confocal imaging of primary brown adipocytes pre - stained overnight with the fatty acid tracer BODIPY C12 558 / 568 ( Bodipy C12 , red ) . Cells were stained with mitotracker green ( MTG , green ) prior to imaging . Cells were imaged before and 120 minutes after treatment with either vehicle ( DMSO ) or 100 nM UK5099 . As a positive control , cells were imaged 30 min after treatment with 1 µM norepinephrine . LD , lipid droplet ; N , nucleus . Scale bar = 10 µm . ( B ) Quantification of changes in lipid droplet cross - sectional area with indicated treatments from images shown in ( A ) . Data are represented as percentage change in LD area at time = 120 min compared to time = 0 min . ( C , D ) Effect of MPC inhibition on fatty acid uptake and incorporation into triacylglycerides ( TAGs ) ( C ) Representative thin - layer chromatography ( TLC ) plate of lipids extracted from primary brown adipocytes and cell culture media . Cells were incubated with Bodipy C12 in presence of vehicle ( V ) , 100 nM UK5099 ( UK ) or 1 µM norepinephrine ( NE ) for 24 h and triacylglyceride ( TAG ) synthesis ( Lipids in cells ) and fatty acid uptake ( Lipids in media ) were detected . The relative polarity of the lipid species determines the motility , with nonpolar TAG migrating the furthest . ( D ) Quantification of C12 uptake from media , TAG in cells and TAG per amount of free fatty acid uptake shown in ( C ) , n = 5 individual experiments . UK5099 treatment decreases the amount of synthesized TAG per free fatty acid uptake , similarly to NE . ( E - G ) Effect of UK5099 treatment on cellular energy expenditure . Fully differentiated primary brown adipocytes were pre - treated with vehicle ( DMSO ) or UK5099 at indicated concentrations for 2 h . Oxygen consumption rates ( OCR ) were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK - 5099 . ( E ) Quantification of non - stimulated OCR from n = 3 - 7 individual experiments . Data were normalized to vehicle for each individual experiment . UK5099 increases OCR in non - stimulated brown adipocytes . vehicle . ( F ) Brown adipocytes were treated with 100 nM UK5099 or vehicle . Etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . Representative OCR traces averaging 6 technical replicates . ( G ) Quantification of non - stimulated OCR , etomoxir sensitive , and etomoxir insensitive OCR from n = 4 individual experiments . Data were normalized to vehicle for each individual experiment . * ( H , I ) Effect of MPC inhibition on adipocyte fuel preference towards fatty acid . ( H ) Schematic representation of metabolite tracing using [ U - 13C16 ] palmitate . ( I ) [ U - 13C16 ] palmitate tracing in fully differentiated primary brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h . Data shows mole percent enrichment ( MPE ) of isotope labeled substrate into respective metabolite .

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 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 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 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

Figure 1(A,B) Effect of MPC inhibition on cellular lipid droplet content. (A) Live-cell super-resolution confocal imaging of primary brown adipocytes pre-stained overnight with the fatty acid tracer BODIPY C12 558/568 (Bodipy C12, red). Cells were stained with mitotracker green (MTG, green) prior to imaging. Cells were imaged before and 120 minutes after treatment with either vehicle (DMSO) or 100 nM UK5099. As a positive control, cells were imaged 30 min after treatment with 1 µM norepinephrine. LD, lipid droplet; N, nucleus. Scale bar=10 µm. (B) Quantification of changes in lipid droplet cross-sectional area with indicated treatments from images shown in (A). Data are represented as percentage change in LD area at time=120 min compared to time=0 min.(C,D) Effect of MPC inhibition on fatty acid uptake and incorporation into triacylglycerides (TAGs) (C) Representative thin-layer chromatography (TLC) plate of lipids extracted from primary brown adipocytes and cell culture media. Cells were incubated with Bodipy C12 in presence of vehicle (V), 100 nM UK5099 (UK) or 1 µM norepinephrine (NE) for 24 h and triacylglyceride (TAG) synthesis (Lipids in cells) and fatty acid uptake (Lipids in media) were detected. The relative polarity of the lipid species determines the motility, with nonpolar TAG migrating the furthest. (D) Quantification of C12 uptake from media, TAG in cells and TAG per amount of free fatty acid uptake shown in (C), n=5 individual experiments. UK5099 treatment decreases the amount of synthesized TAG per free fatty acid uptake, similarly to NE.(E-G) Effect of UK5099 treatment on cellular energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or UK5099 at indicated concentrations for 2 h. Oxygen consumption rates (OCR) were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK-5099. (E) Quantification of non-stimulated OCR from n=3-7 individual experiments. Data were normalized to vehicle for each individual experiment. UK5099 increases OCR in non-stimulated brown adipocytes. vehicle. (F) Brown adipocytes were treated with 100 nM UK5099 or vehicle. Etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. Representative OCR traces averaging 6 technical replicates. (G) Quantification of non-stimulated OCR, etomoxir sensitive, and etomoxir insensitive OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment. *(H,I) Effect of MPC inhibition on adipocyte fuel preference towards fatty acid. (H) Schematic representation of metabolite tracing using [U-13C16] palmitate. (I) [U-13C16] palmitate tracing in fully differentiated primary brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h. Data shows mole percent enrichment (MPE) of isotope labeled substrate into respective metabolite.


Figure 2 ( A , B ) Effect of UK5099 treatment on norepinephrine - stimulated energy expenditure . Fully differentiated primary brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 4 technical replicates . ( B ) Quantification of non - stimulated OCR , NE - stimulated OCR , ATP - linked OCR , mitochondrial proton leak and Eto - sensitive OCR as shown in ( A ) from n = 9 individual experiments . The effects of UK5099 treatment were normalized to vehicle for each experiment . Primary brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble RNA ( Scramble ) . ( C ) Representative Western Blot analysis of MPC1 , MPC2 and actin . ( D ) Quantification of MPC1 and MPC2 expression normalized to actin from Western Blots in ( C ) from n = 3 individual experiments . Primary brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble RNA ( Scramble ) . ( E ) mRNA levels of MPC1 and MPC2 from n = 3 individual experiments . Primary brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble RNA ( Scramble ) . ( F ) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( G ) Quantification of basal , NE - stimulated and Eto - sensitive OCR as shown in ( F ) from n = 7 individual experiments . Data were normalized to Scramble RNA for each individual experiment . ( H ) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n = 5 individual experiments . Data were normalized to vehicle for each experiment .

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 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 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 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

Figure 2(A,B) Effect of UK5099 treatment on norepinephrine-stimulated energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 4 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and Eto-sensitive OCR as shown in (A) from n=9 individual experiments. The effects of UK5099 treatment were normalized to vehicle for each experiment.Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (C) Representative Western Blot analysis of MPC1, MPC2 and actin. (D) Quantification of MPC1 and MPC2 expression normalized to actin from Western Blots in (C) from n=3 individual experiments.Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (E) mRNA levels of MPC1 and MPC2 from n=3 individual experiments.Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (F) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (G) Quantification of basal, NE-stimulated and Eto-sensitive OCR as shown in (F) from n=7 individual experiments. Data were normalized to Scramble RNA for each individual experiment. (H) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n=5 individual experiments. Data were normalized to vehicle for each experiment.


Figure 3 ( A , B ) Requirement for glutamine in UK5099 - induced energy expenditure . Primary brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 h . OCR were measured in presence of either 5 mM glucose and 3 mM glutamine or 5 mM glucose alone . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 6 technical replicates . ( B ) Quantification of non - stimulated OCR , NE - stimulated OCR , ATP - linked OCR , mitochondrial proton leak and etomoxir - sensitive OCR after vehicle or UK5099 treatment as shown in ( A ) from n = 4 individual experiments . ( C ) Effect of glutaminase inhibitor on UK5099 - induced energy expenditure . Primary brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , or 100 nM UK5099 + 2 . 5 µM CB839 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle , UK5099 or UK5099 + CB839 from n = 5 individual experiments . Data were normalized to vehicle for each experiment . ( D ) Effect of MPC inhibition on glutamate / aspartate ratio measurement . Glutamate / aspartate ratio was quantified using GC - MS as a measure of glutamine catabolism . Primary brown adipocytes were treated for 24 h with vehicle ( DMSO ) or 10 µM UK5099 from n = 6 individual experiments . * *

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 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 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

Figure 3(A,B) Requirement for glutamine in UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in presence of either 5 mM glucose and 3 mM glutamine or 5 mM glucose alone. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and etomoxir-sensitive OCR after vehicle or UK5099 treatment as shown in (A) from n=4 individual experiments.(C) Effect of glutaminase inhibitor on UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, or 100 nM UK5099 + 2.5 µM CB839 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099 or UK5099 + CB839 from n=5 individual experiments. Data were normalized to vehicle for each experiment.(D) Effect of MPC inhibition on glutamate/aspartate ratio measurement. Glutamate/aspartate ratio was quantified using GC-MS as a measure of glutamine catabolism. Primary brown adipocytes were treated for 24 h with vehicle (DMSO) or 10 µM UK5099 from n=6 individual experiments. **


Figure 4 ( B ) Effect of transaminase inhibition on UK5099 - induced energy expenditure . Brown adipocytes were pre - treated with vehicle ( DMSO ) , 50 - 100 nM UK5099 , 1 mM aminooxyacetic acid ( AOA ) , or a combination of UK5099 and AOA for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of the tested compounds . Data show quantification of non - stimulated OCR from n = 5 individual experiments . ( C - E ) Effect OGC1 downregulation on UK5099 - induced energy expenditure . Primary brown adipocytes were transduced with Scramble RNA ( Scramble ) or shOGC1 ( OGC1 KD ) adenovirus . Cells were pre - treated for 2 h with vehicle ( DMSO ) or 100 nM UK5099 before OCR measurements . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM , ) oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( C ) Representative OCR traces averaging 4 technical replicates . ( D ) Quantification of non - stimulated OCR as measured in ( C ) from n = 6 individual experiments . ( E ) Quantification of Eto - sensitive OCR as measured in ( C ) from n = 6 individual experiments . Data were normalized to vehicle for each experiment . ( F - H ) Effect of Aralar1 downregulation on UK5099 - induced energy expenditure . Primary brown adipocytes were transfected with Scramble RNA ( Scramble ) or siRNA for Aralar1 ( Aralar1 KD ) . Cells were pre - treated for 2 h with vehicle ( DMSO ) or 100 nM UK5099 before OCR measurements . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM ) , oligomycin a ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin a ( AA ; 4 µM ) were injected where indicated . ( F ) Representative OCR traces averaging 4 technical replicates . ( G ) Quantification of non - stimulated OCR as measured ( G ) from n = 4 individual experiments . Data were normalized to vehicle for each experiment . ( H ) Quantification of Eto - sensitive OCR as measured in ( F ) from n = 4 individual experiments . ( I ) Brown adipocytes transfected with Scramble RNA or Aralar1 siRNA were treated for 24 hours with vehicle ( DMSO ) or 10 µM UK5099 . Data shows quantification of the ratio of aspartate to glutamate abundance as measured by GC - MS from n = 3 individual experiments .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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(B) Effect of transaminase inhibition on UK5099-induced energy expenditure. Brown adipocytes were pre-treated with vehicle (DMSO), 50-100 nM UK5099, 1 mM aminooxyacetic acid (AOA), or a combination of UK5099 and AOA for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of the tested compounds. Data show quantification of non-stimulated OCR from n=5 individual experiments.(C-E) Effect OGC1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transduced with Scramble RNA (Scramble) or shOGC1 (OGC1 KD) adenovirus. Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM,) oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (C) Representative OCR traces averaging 4 technical replicates. (D) Quantification of non-stimulated OCR as measured in (C) from n=6 individual experiments. (E) Quantification of Eto-sensitive OCR as measured in (C) from n=6 individual experiments. Data were normalized to vehicle for each experiment.(F-H) Effect of Aralar1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transfected with Scramble RNA (Scramble) or siRNA for Aralar1 (Aralar1 KD). Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin a (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin a (AA; 4 µM) were injected where indicated. (F) Representative OCR traces averaging 4 technical replicates. (G) Quantification of non-stimulated OCR as measured (G) from n=4 individual experiments. Data were normalized to vehicle for each experiment. (H) Quantification of Eto-sensitive OCR as measured in (F) from n=4 individual experiments.(I) Brown adipocytes transfected with Scramble RNA or Aralar1 siRNA were treated for 24 hours with vehicle (DMSO) or 10 µM UK5099. Data shows quantification of the ratio of aspartate to glutamate abundance as measured by GC-MS from n=3 individual experiments.


Figure 5 ( A , B ) Effect of UK5099 on energy expenditure contributed by ATP demand and by mitochondrial proton leak in non - stimulated brown adipocytes . Primary brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 hours . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099 . Oligomycin A ( Oligo ; 4 µM ) , mitochondrial uncoupler FCCP ( 2 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 6 technical replicates . ( B ) Quantification of mitochondrial proton leak and ATP - linked OCR ( oligomycin sensitive ) as measured in ( A ) from n = 9 individual experiments . Data were normalized to vehicle for each individual experiment . ( C ) To determine the contribution of ATP demand to increased energy expenditure under UK5099 cells were permeabilized and cytosolic ADP concentrations were clamped . OCR were measured in the presence of 5 mM pyruvate , 3 mM malate , 0 . 1 mM palmitoyl - CoA , 0 . 5 mM carnitine and 5 mM ADP . Cells were treated with either 100 nM , 1 µM or 10 µM UK5099 . Data shows maximal state 3 OCR normalized to maximal complex 4 activity from n = 3 individual experiments . ( E ) Effect of MPC inhibition on glycerol 3 - phosphate ( G3P ) and dihydroxyacetone phosphate ( DHAP ) generation . G3P / DHAP is used as a measure of acylglycerol synthesis . Brown adipocytes were treated with either vehicle , 100 nM UK5099 or 1 µM norepinephrine ( NE ) for 24 h from n = 4 individual experiments . ( F , G ) Contribution of lipolysis to UK5099 induced energy demand . Primary brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 40 µM Atglistatin or a combination of Atglistatin with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle , UK5099 , or Atglistatin . ( F ) Quantification of non - stimulated OCR from n = 4 individual experiments . ( G ) Quantification of ATP - linked respiration from n = 4 individual experiments . Data were normalized to vehicle for each individual experiment . ( H , I ) Contribution of TAG synthesis to UK5099 induced energy demand . Primary brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 1 µM DGAT1 and 1 µM DGAT2 or a combination of DGAT1 / 2 inhibitors with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle , UK5099 , DGAT1 / 2 inhibitors , or UK5099 + DGAT1 / 2 inhibitors . ( H ) Quantification of non - stimulated OCR from n = 6 individual experiments . ( I ) Quantification of ATP - linked OCR from n = 6 individual experiments . Data were normalized to vehicle for each individual experiment . ( J , K ) Contribution of ACS to UK5099 induced energy demand . Primary brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 5 µM Triacsin C or a combination of 5 µM Triacsin C with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle , UK5099 , Triacsin C , or UK5099 + Triacsin C . ( J ) Quantification of non - stimulated OCR from n = 4 individual experiments . ( K ) Quantification of ATP - linked OCR from n = 4 individual experiments . Data were normalized to vehicle for each individual experiment .

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

Figure 5(A,B) Effect of UK5099 on energy expenditure contributed by ATP demand and by mitochondrial proton leak in non-stimulated brown adipocytes. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 hours. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Oligomycin A (Oligo; 4 µM), mitochondrial uncoupler FCCP (2 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of mitochondrial proton leak and ATP-linked OCR (oligomycin sensitive) as measured in (A) from n=9 individual experiments. Data were normalized to vehicle for each individual experiment.(C) To determine the contribution of ATP demand to increased energy expenditure under UK5099 cells were permeabilized and cytosolic ADP concentrations were clamped. OCR were measured in the presence of 5 mM pyruvate, 3 mM malate, 0.1 mM palmitoyl-CoA, 0.5 mM carnitine and 5 mM ADP. Cells were treated with either 100 nM, 1 µM or 10 µM UK5099. Data shows maximal state 3 OCR normalized to maximal complex 4 activity from n=3 individual experiments.(E) Effect of MPC inhibition on glycerol 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) generation. G3P/DHAP is used as a measure of acylglycerol synthesis. Brown adipocytes were treated with either vehicle, 100 nM UK5099 or 1 µM norepinephrine (NE) for 24 h from n=4 individual experiments.(F,G) Contribution of lipolysis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 40 µM Atglistatin or a combination of Atglistatin with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, or Atglistatin. (F) Quantification of non-stimulated OCR from n=4 individual experiments. (G) Quantification of ATP-linked respiration from n=4 individual experiments. Data were normalized to vehicle for each individual experiment.(H,I) Contribution of TAG synthesis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 1 µM DGAT1 and 1 µM DGAT2 or a combination of DGAT1/2 inhibitors with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, DGAT1/2 inhibitors, or UK5099 + DGAT1/2 inhibitors. (H) Quantification of non-stimulated OCR from n=6 individual experiments. (I) Quantification of ATP-linked OCR from n=6 individual experiments. Data were normalized to vehicle for each individual experiment.(J,K) Contribution of ACS to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 5 µM Triacsin C or a combination of 5 µM Triacsin C with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, Triacsin C, or UK5099 + Triacsin C. (J) Quantification of non-stimulated OCR from n=4 individual experiments. (K) Quantification of ATP-linked OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment.


Figure 1 ( A ) Dually labeled D . discoideum amoebae producing MCS components fused to GFP , and either calnexin ( CnxA ) - mCherry ( pAW012 ) , P4C - mCherry ( pWS032 ) , or AmtA - mCherry were infected ( MOI 5 , 2 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) , fixed with 4 % PFA , and imaged by confocal fluorescence microscopy . Merged images are shown . Scale bars : 3 μm ( insets : 1 μm ) . ( B - D ) The Pearson ' s correlation coefficients were generated using Coloc 2 from Fiji ( ImageJ ) and are shown for MCS components fused to GFP with respect to ( B ) CnxA - mCherry ( n = 146 ) , ( C ) P4C - mCherry ( n = 138 ) , or ( D ) AmtA - mCherry ( n = 159 ) . Data represent mean and SEM of three independent biological replicates ( statistics refer to JR32 - infected D . discoideum producing GFP - Sac1 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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 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

Figure 1(A) Dually labeled D. discoideum amoebae producing MCS components fused to GFP, and either calnexin (CnxA)-mCherry (pAW012), P4C-mCherry (pWS032), or AmtA-mCherry were infected (MOI 5, 2 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed with 4 % PFA, and imaged by confocal fluorescence microscopy. Merged images are shown. Scale bars: 3 μm (insets: 1 μm).(B-D) The Pearson's correlation coefficients were generated using Coloc 2 from Fiji (ImageJ) and are shown for MCS components fused to GFP with respect to (B) CnxA-mCherry (n=146), (C) P4C-mCherry (n=138), or (D) AmtA-mCherry (n=159). Data represent mean and SEM of three independent biological replicates (statistics refer to JR32-infected D. discoideum producing GFP-Sac1; *P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


Figure 2 ( A ) Dually labeled D . discoideum Ax3 producing mCherry - Vap ( pSV048 ) and OSBP8 - GFP ( pMIB89 ) or GFP - OSBP11 ( pMIB39 ) were infected ( MOI 5 , 1 - 8 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm ( insets : 1 μm ) . ( B ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for OSBP8 or OSBP11 fused to GFP with respect to mCherry - Vap ( n = 100 - 150 each per time point ) . Data represent mean and SEM of three independent biological replicates ( * * P < 0 . 01 , Student ` s t - test ) .

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

Figure 2(A) Dually labeled D. discoideum Ax3 producing mCherry-Vap (pSV048) and OSBP8-GFP (pMIB89) or GFP-OSBP11 (pMIB39) were infected (MOI 5, 1-8 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm (insets: 1 μm).(B) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for OSBP8 or OSBP11 fused to GFP with respect to mCherry-Vap (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (**P<0.01, Student`s t-test).


Figure 3 ( A ) D . discoideum Ax3 , or the ∆ vap , ∆ osbG , ∆ osbH , or ∆ osbK mutant strains producing GFP ( pDM317 ) or GFP fusions of the MCS components were infected ( MOI 1 ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to JR32 infected D . discoideum Ax3 producing GFP ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) . To improve clarity , the different mutant strains are shown in separate graphs , each depicting the same data for Ax3 / GFP . ( B ) D . discoideum Ax3 producing CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , GFP - Sac1 _ C383S ( pSV015 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 1 ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to JR32 infected D . discoideum Ax3 producing GFP - Sac1 ; * * * P < 0 . 001 , Student ` s t - test ) . ( C ) RAW 264 . 7 macrophages were treated with increasing concentrations of OSW - 1 ( 0 - 100 nM , 1 - 60 h ) , infected ( MOI 1 ) with GFP - producing L . pneumophila JR32 ( pNT28 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to infected cells treated with 0 nM OSW - 1 ; * P < 0 . 05 ; * * * P < 0 . 001 , Student ` s t - test ) .

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 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 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

Figure 3(A) D. discoideum Ax3, or the ∆vap, ∆osbG, ∆osbH, or ∆osbK mutant strains producing GFP (pDM317) or GFP fusions of the MCS components were infected (MOI 1) with mCherry-producing L. pneumophila JR32 (pNP102), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to JR32 infected D. discoideum Ax3 producing GFP; *P<0.05; **P<0.01; ***P<0.001, Student`s t-test). To improve clarity, the different mutant strains are shown in separate graphs, each depicting the same data for Ax3/GFP.(B) D. discoideum Ax3 producing CnxA-GFP (pAW016), GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 1) with mCherry-producing L. pneumophila JR32 (pNP102), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to JR32 infected D. discoideum Ax3 producing GFP-Sac1; ***P<0.001, Student`s t-test).(C) RAW 264.7 macrophages were treated with increasing concentrations of OSW-1 (0-100 nM, 1-60 h), infected (MOI 1) with GFP-producing L. pneumophila JR32 (pNT28), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to infected cells treated with 0 nM OSW-1; *P<0.05; ***P<0.001, Student`s t-test).


Figure 4 ( A ) Dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and CnxA - GFP ( pAW016 ) , or Ax3 producing P4C - mCherry and either GFP - Sac1 ( pLS037 ) , GFP - Sac1 _ C383S ( pSV015 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 5 , 2 - 16 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B ) LCV area was measured using ImageJ ( n = 100 - 200 per condition from 3 independent biological replicates ) . Means and SEM of single cells are shown ( * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

Figure 4(A) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and CnxA-GFP (pAW016), or Ax3 producing P4C-mCherry and either GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 2-16 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B) LCV area was measured using ImageJ (n=100-200 per condition from 3 independent biological replicates). Means and SEM of single cells are shown (**P<0.01; ***P<0.001, Student`s t-test).


Figure 5 ( A ) Dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and GFP - Sac1 ( pLS037 ) were infected ( MOI 5 , 2 - 16 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B , C ) Imaging flow cytometry ( IFC ) analysis of dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and either GFP - Sac1 ( pLS037 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) , infected ( MOI 5 , 2 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Data information ( B , C , : Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) . ( D ) Dually labeled D . discoideum Ax3 producing GFP fusions of MCS components and either mCherry - Sac1 ( pSV044 ) or mCherry - Sac1 _ ΔTMD ( pSV045 ) were infected ( MOI 5 , 2 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( E ) IFC analysis of dually labeled D . discoideum Ax3 producing GFP fusions of MCS components and either mCherry - Sac1 ( pSV044 ) or mCherry - Sac1 _ ΔTMD ( pSV045 ) , infected ( MOI 5 , 2 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Quantification of ( B ) P4C - mCherry ( C ) Sac1 - GFP or ( E ) GFP fusions of MCS components localizing to LCVs at 2 h p . i . Due to the lower resolution of IFC , " LCVs " designates the LCV limiting membrane and tightly attached ER . Number of events per sample , n = 5000 . Data information E ) : Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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 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

Figure 5(A) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and GFP-Sac1 (pLS037) were infected (MOI 5, 2-16 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B, C) Imaging flow cytometry (IFC) analysis of dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and either GFP-Sac1 (pLS037) or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Data information (B, C, : Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).(D) Dually labeled D. discoideum Ax3 producing GFP fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045) were infected (MOI 5, 2 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (E) IFC analysis of dually labeled D. discoideum Ax3 producing GFP fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Quantification of (B) P4C-mCherry (C) Sac1-GFP or (E) GFP fusions of MCS components localizing to LCVs at 2 h p.i. Due to the lower resolution of IFC, "LCVs" designates the LCV limiting membrane and tightly attached ER. Number of events per sample, n = 5000. Data information E): Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


Figure 6 ( A ) Dually labeled D . discoideum Ax3 producing GFP - D4H * ( pSV046 ) or P4C - GFP ( pWS034 ) and P4C - mCherry ( pWS032 ) , CnxA - mCherry ( pAW012 ) , or AmtA - mCherry were infected ( MOI 5 , 15 - 120 min ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B - C ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for ( B ) AmtA - mCherry with respect to P4C - GFP or GFP - D4H * ( n = 100 - 150 per time point ) , and ( C ) GFP - D4H * with respect to P4C - mCherry or CnxA - mCherry ( n = 100 - 150 each per time point ) . Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 , Student ` s t - test ) . ( D ) Single labeled D . discoideum Ax3 producing either CnxA - GFP ( pAW016 ) or P4C - GFP ( pWS034 ) were infected ( MOI 5 , 15 - 120 min ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , fixed with 4 % PFA and stained with filipin . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( E ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for filipin with respect to CnxA - GFP or P4C - GFP ( n = 80 - 90 each per time point ) . Data represent mean and SEM of the means of three independent biological replicates ( * * * P < 0 . 001 , Student ` s t - test ) .

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 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

Figure 6(A) Dually labeled D. discoideum Ax3 producing GFP-D4H* (pSV046) or P4C-GFP (pWS034) and P4C-mCherry (pWS032), CnxA-mCherry (pAW012), or AmtA-mCherry were infected (MOI 5, 15 - 120 min) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B-C) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for (B) AmtA-mCherry with respect to P4C-GFP or GFP-D4H* (n=100-150 per time point), and (C) GFP-D4H* with respect to P4C-mCherry or CnxA-mCherry (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (*P<0.05, Student`s t-test).(D) Single labeled D. discoideum Ax3 producing either CnxA-GFP (pAW016) or P4C-GFP (pWS034) were infected (MOI 5, 15-120 min) with mCherry-producing L. pneumophila JR32 (pNP102), fixed with 4 % PFA and stained with filipin. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (E) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for filipin with respect to CnxA-GFP or P4C-GFP (n=80-90 each per time point). Data represent mean and SEM of the means of three independent biological replicates (***P<0.001, Student`s t-test).


Figure 7 ( A ) Dually labeled D . discoideum Ax3 producing P4C - mCherry ( pWS032 ) and either CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 5 , 1 - 8 h ) with mCerulean - producing L . pneumophila JR32 , ΔlepB or ΔsidC ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( C ) Imaging flow cytometry ( IFC ) analysis of dually labeled D . discoideum Ax3 producing P4C - mCherry ( pWS032 ) and either CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , or GFP - Sac1 _ ΔTMD ( pSV034 ) , infected ( MOI 5 , 1 - 8 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Number of events per sample , n = 5000 . Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

Figure 7(A) Dually labeled D. discoideum Ax3 producing P4C-mCherry (pWS032) and either CnxA-GFP (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 1-8 h) with mCerulean-producing L. pneumophila JR32, ΔlepB or ΔsidC (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm.(C) Imaging flow cytometry (IFC) analysis of dually labeled D. discoideum Ax3 producing P4C-mCherry (pWS032) and either CnxA-GFP (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 1-8 h) with mPlum-producing L. pneumophila JR32 (pAW014). Number of events per sample, n = 5000. Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


Figure 1A U2OS cells were transfected with Flag - NAT10 or control vectors . Forty - eight hours later , cells were harvested and whole cell extracts were immunoprecipitated with Flag - antibody affinity resin . The NAT10 - binding proteins were resolved by SDS - PAGE and detected by silver staining . B - F U2OS , HCT116 p53 + / + or HCT116 p53 - / - cell lysates were immunoprecipitated with control IgG , anti - NAT10 ( B and F ) , anti - p53 ( C ) and anti - Mdm2 ( D and E ) antibodies . The immunoprecipitates were subsequently immunoblotted with the indicated antibodies . G Purified NAT10 was incubated with GST , GST - p53 or GST - Mdm2 proteins coupled to Glutathione Sepharose 4B . Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated . The amount of GST fusion proteins are shown in the lower panel . H Full - length GST - p53 fusion protein , its deletion mutants or GST protein was used to pull - down experiments with purified NAT10 protein . The levels of the GST fusion proteins are shown in the left panel . I GST pull - down assay was performed using purified GST - NAT10 deletion mutants or GST protein and overexpressed Flag - p53 or Mdm2 protein in HEK293T cells . Schematic diagram represents the constructs of GST - NAT10 deletion mutants ( right panel ) .

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

Figure 1A U2OS cells were transfected with Flag-NAT10 or control vectors. Forty-eight hours later, cells were harvested and whole cell extracts were immunoprecipitated with Flag-antibody affinity resin. The NAT10-binding proteins were resolved by SDS-PAGE and detected by silver staining.B-F U2OS, HCT116 p53+/+ or HCT116 p53-/- cell lysates were immunoprecipitated with control IgG, anti-NAT10 (B and F), anti-p53 (C) and anti-Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies.G Purified NAT10 was incubated with GST, GST-p53 or GST-Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel.H Full-length GST-p53 fusion protein, its deletion mutants or GST protein was used to pull-down experiments with purified NAT10 protein. The levels of the GST fusion proteins are shown in the left panel.I GST pull-down assay was performed using purified GST-NAT10 deletion mutants or GST protein and overexpressed Flag-p53 or Mdm2 protein in HEK293T cells. Schematic diagram represents the constructs of GST-NAT10 deletion mutants (right panel).


Figure 2A Recombinant His - p53 was incubated with or without purified Flag - NAT10 as described in Experimental Procedures . The reaction products were resolved by SDS - PAGE and immunoblotted using anti - acetyl - lysine antibodies . Flag - PCAF was used as a positive control . B In vitro acetylation was performed and the acetylated p53 was detected as in A . C In vitro acetylation assay was performed as described in A . The reaction products were resolved by SDS - PAGE and acetylated p53 was purified from the SDS - PAGE and subjected to mass spectrometry analysis . D His - p53 or His - p53 - K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures . Reaction mixtures were subjected to western blot using the site specific monoclonal anti - Ac - p53 - K120 antibody . E H1299 cells were transfected with the indicated vectors . Total proteins and the anti - Flag antibody M2 - specific immunoprecipitates were analyzed by western blot using the indicated antibodies .

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Figure 2A Recombinant His-p53 was incubated with or without purified Flag-NAT10 as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE and immunoblotted using anti-acetyl-lysine antibodies. Flag-PCAF was used as a positive control.B In vitro acetylation was performed and the acetylated p53 was detected as in A.C In vitro acetylation assay was performed as described in A. The reaction products were resolved by SDS-PAGE and acetylated p53 was purified from the SDS-PAGE and subjected to mass spectrometry analysis.D His-p53 or His-p53-K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures. Reaction mixtures were subjected to western blot using the site specific monoclonal anti-Ac-p53-K120 antibody.E H1299 cells were transfected with the indicated vectors. Total proteins and the anti-Flag antibody M2-specific immunoprecipitates were analyzed by western blot using the indicated antibodies.


Figure 3A U2OS cells were transfected with the indicated siRNAs . Seventy - two hours later , cell lysates were prepared and proteins from lysates were subjected to western blot using the indicated antibodies . B U2OS cells were transfected as in A . Seventy - two hours later , total mRNAs were extracted and subjected to RT - qPCR for the indicated genes . C U2OS cells were transfected with increasing amount of plasmid DNA expressing NAT10 . Proteins from whole cell extracts were subjected to western blot using the indicated antibodies . D U2OS cells were transfected with control or NAT10 siRNAs . Seventy - two hours later , cells were treated with cycloheximide ( CHX ) and harvested at the indicated time points . Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 ( upper panels ) . Beta - actin was evaluated as a loading control ( lower panel ) . p53 half - life ( t1 / 2 ) obtained from three independent experiments was presented as mean ± SEM at the bottom . E U2OS cells were treated with MG132 ( 10μM ) for 4 hours after transfected with the indicated siRNAs . Proteins from cell extracts were detected by western blot for the indicated proteins . F HCT116 p53 + / + cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest . p53 was immunoprecipitated with anti - p53 polyclonal antibody and immunoblotted with anti - Ub antibody . G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest . Proteins from whole cell extracts were subjected to western blot using the indicated antibodies . H U2OS cells were transfected with vectors as indicated . Forty - eight hours later , cells were harvested and proteins from whole cell extracts were subjected to western blot using the indicated antibodies .

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Figure 3A U2OS cells were transfected with the indicated siRNAs. Seventy-two hours later, cell lysates were prepared and proteins from lysates were subjected to western blot using the indicated antibodies.B U2OS cells were transfected as in A. Seventy-two hours later, total mRNAs were extracted and subjected to RT-qPCR for the indicated genes.C U2OS cells were transfected with increasing amount of plasmid DNA expressing NAT10. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.D U2OS cells were transfected with control or NAT10 siRNAs. Seventy-two hours later, cells were treated with cycloheximide (CHX) and harvested at the indicated time points. Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 (upper panels). Beta-actin was evaluated as a loading control (lower panel). p53 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.E U2OS cells were treated with MG132 (10μM) for 4 hours after transfected with the indicated siRNAs. Proteins from cell extracts were detected by western blot for the indicated proteins.F HCT116 p53+/+ cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibody and immunoblotted with anti-Ub antibody.G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.H U2OS cells were transfected with vectors as indicated. Forty-eight hours later, cells were harvested and proteins from whole cell extracts were subjected to western blot using the indicated antibodies.


Figure 4A HCT116 cells were transfected with increasing amount of DNA expressing NAT10 . Forty - eight hours later , whole cell lysates were prepared and proteins from lysates were analyzed by western blot using the indicated antibodies . B HCT116 cells were transfected with Flag - NAT10 and harvested at indicated time points after treated with CHX . Proteins from cell lysates were immunoblotted with the antibodies as indicated ( left panel ) . Relative Mdm2 protein levels standardized by β - actin are shown in the right . Mdm2 half - life ( t1 / 2 ) obtained from three independent experiments was presented as mean ± SEM at the bottom . C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours . The Mdm2 protein was immunoprecipitated with anti - Mdm2 antibody and immunoblotted with anti - HA antibody . D In vitro ubiquitination was performed with recombinant GST - Mdm2 ( left ) or GST - Mdm2 C464A mutant ( right ) and Flag - NAT10 or Flag - NAT10 GE in the presence of E1 , E2 and Ub in vitro as described in Experimental Procedures . The reaction products were resolved by SDS - PAGE , followed by western blot for evaluation of the indicated proteins . E Mdm2 is ubiquitinated by NAT10 in a dose - dependent manner . In vitro ubiquitination assay were carried out with Mdm2 and different doses of purified Flag - NAT10 as described in D . G U2OS cells transfected with NAT10 siRNA - 3 ( targeting 3 ' - UTR of NAT10 mRNA ) or control siRNA were co - expressed different NAT10 deletion mutants . Cell lysates were extracted and proteins from lysates were subjected to western blot using the indicated antibodies . Relative p53 protein levels standardized by β - actin are shown in the lower . H In vitro ubiquitination reaction was conducted using recombinant GST - Mdm2 C464A and full - length Flag - NAT10 or Flag - NAT10 - D5 . Ubiquitinated Mdm2 was detected as in D . I HCT116 cells were transfected with the indicated constructs . Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared . Mdm2 protein levels were evaluated by western blot . Relative Mdm2 protein levels standardized by β - actin are shown in the right .

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

Figure 4A HCT116 cells were transfected with increasing amount of DNA expressing NAT10. Forty-eight hours later, whole cell lysates were prepared and proteins from lysates were analyzed by western blot using the indicated antibodies.B HCT116 cells were transfected with Flag-NAT10 and harvested at indicated time points after treated with CHX. Proteins from cell lysates were immunoblotted with the antibodies as indicated (left panel). Relative Mdm2 protein levels standardized by β-actin are shown in the right. Mdm2 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours. The Mdm2 protein was immunoprecipitated with anti-Mdm2 antibody and immunoblotted with anti-HA antibody.D In vitro ubiquitination was performed with recombinant GST-Mdm2 (left) or GST-Mdm2 C464A mutant (right) and Flag-NAT10 or Flag-NAT10 GE in the presence of E1, E2 and Ub in vitro as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE, followed by western blot for evaluation of the indicated proteins.E Mdm2 is ubiquitinated by NAT10 in a dose-dependent manner. In vitro ubiquitination assay were carried out with Mdm2 and different doses of purified Flag-NAT10 as described in D.G U2OS cells transfected with NAT10 siRNA-3 (targeting 3'-UTR of NAT10 mRNA) or control siRNA were co-expressed different NAT10 deletion mutants. Cell lysates were extracted and proteins from lysates were subjected to western blot using the indicated antibodies. Relative p53 protein levels standardized by β-actin are shown in the lower.H In vitro ubiquitination reaction was conducted using recombinant GST-Mdm2 C464A and full-length Flag-NAT10 or Flag-NAT10-D5. Ubiquitinated Mdm2 was detected as in D.I HCT116 cells were transfected with the indicated constructs. Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared. Mdm2 protein levels were evaluated by western blot. Relative Mdm2 protein levels standardized by β-actin are shown in the right.


Figure 5A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points . Proteins from extracts were analyzed by western blot using the indicated antibodies . B U2OS and H1299 cells were treated with actinomycin D for 12 hours . Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies . C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points . Proteins from cell lysates were subjected to western blot . D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin ( Dox ) for 12 hours . Cells were harvested and total proteins were immunoprecipitated with anti - NAT10 antibody . Immunoprecipitates were analyzed by western blot for the indicated proteins . E HCT116 cells were treated with actinomycin D for the indicated times . Cells were fixed and immuno - stained as indicated . Immunofluorescence images were taken by confocal microscopy . Scale bar represents 10 μM . F H1299 cells were transfected with the indicated vectors . After treated with actinomycin D , cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies . G H1299 cells were transfected with GFP - NAT10 vectors . After treated with actinomycin D for 12 hours , cells were fixed and nuclei were stained with DAPI . Bar represents 10μM . H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin . Western blot was performed with total cell extracts and Mdm2 - specific immunoprecipitates for the indicated proteins . I HCT116 cells were transfected with the indicated constructs . Cells were harvested after treatment with actinomycin D . p53 protein was immunoprecipitated with an anti - p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody ( DO - 1 ) .

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Figure 5A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points. Proteins from extracts were analyzed by western blot using the indicated antibodies.B U2OS and H1299 cells were treated with actinomycin D for 12 hours. Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies.C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Proteins from cell lysates were subjected to western blot.D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin (Dox) for 12 hours. Cells were harvested and total proteins were immunoprecipitated with anti-NAT10 antibody. Immunoprecipitates were analyzed by western blot for the indicated proteins.E HCT116 cells were treated with actinomycin D for the indicated times. Cells were fixed and immuno-stained as indicated. Immunofluorescence images were taken by confocal microscopy. Scale bar represents 10 μM.F H1299 cells were transfected with the indicated vectors. After treated with actinomycin D, cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies.G H1299 cells were transfected with GFP-NAT10 vectors. After treated with actinomycin D for 12 hours, cells were fixed and nuclei were stained with DAPI. Bar represents 10μM.H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin. Western blot was performed with total cell extracts and Mdm2-specific immunoprecipitates for the indicated proteins.I HCT116 cells were transfected with the indicated constructs. Cells were harvested after treatment with actinomycin D. p53 protein was immunoprecipitated with an anti-p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody (DO-1).


Figure 6A p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells . Luciferase activity was determined as described in Experimental Procedure . B U2OS cells were transfected with siRNAs as indicated . Seventy - two hours later , cells were harvested after treated with actinomycin D for 12 hours . Relative mRNA levels for the indicated genes were analyzed by RT - qPCR . C - E U2OS cells ( C and D ) and H1299 cells ( D ) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points . Cell cycle profiles were determined by FACS . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test ) F U2OS cells were transfected with the indicated siRNAs . Cells were irradiated with 50 J / m2 UV . Forty - eight hours later , apoptotic cells were determined . Quantification of apoptotic cells is presented . ( two - tailed Student ' s t test ) G U2OS cells transfected with the indicated siRNAs were plated . Cell numbers were determined as described in Experimental Procedures at the indicated time points . H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs . The right panel shows the quantification of formed colonies . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t test )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 6A p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells. Luciferase activity was determined as described in Experimental Procedure.B U2OS cells were transfected with siRNAs as indicated. Seventy-two hours later, cells were harvested after treated with actinomycin D for 12 hours. Relative mRNA levels for the indicated genes were analyzed by RT-qPCR.C-E U2OS cells (C and D) and H1299 cells (D) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)F U2OS cells were transfected with the indicated siRNAs. Cells were irradiated with 50 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Quantification of apoptotic cells is presented. (two-tailed Student's t test)G U2OS cells transfected with the indicated siRNAs were plated. Cell numbers were determined as described in Experimental Procedures at the indicated time points.H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs. The right panel shows the quantification of formed colonies. **p<0.01, ***p<0.001 (two-tailed Student's t test)