Global - transcript correlation for each sample , grouped by . RPKM iBAQ values were used to estimate transcript from matched - seq TMT - proteomics data obtained from same . An ANOVA was performed to evaluate significance among groups ( mean correlation at 5 wph : 0 . 48 ; at 12 wph : 0 . 43 ; at 39 wph : 0 . 33 ; p = 3 . 05e - 07 , n = 5 per group ) . In boxplots , horizontal represents median , bottom top of box 25th 75th percentile , respectively , whiskers extend 1 . 5 folds interquartile range .

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Global protein-transcript correlation for each sample, grouped by age. RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA-seq and TMT-based proteomics data obtained from the same animal. An ANOVA test was performed to evaluate significance among the age groups (mean correlation at 5 wph: 0.48; at 12 wph: 0.43; and at 39 wph: 0.33; p = 3.05e-07, n=5 per age group). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Scatter plot of log2 fold changes for genes differentially expressed both at transcript ( adj . p < 0 . 05 ) . color gradients indicate gene density in regions where points overlap . Numbers of genes in each quadrant value of Pearson ' s coefficient of correlation , r , are reported for each graph . Solid lines represent a spline fit ( r = 0 . 505 for genes significantly affected at both transcript , p < 2 . 2 x 10 - 16 , D ) ( r = 0 . 126 , p = 0 . 007 , E ) .

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Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels (adj. p < 0.05). The color gradients indicate gene density in the regions where individual points overlap. Numbers of genes in each quadrant and the value of Pearson's coefficient of correlation, r, are reported for each graph. Solid lines represent a spline fit (r = 0.505 for genes significantly affected at both transcript and protein levels, p < 2.2 x 10-16, D) (r = 0.126, p = 0.007, E).


Mechanisms affecting abundance during aging . Bar plots are on affected in either one of comparisons ( adj . p < 0 . 05 ) . were divided in following five groups : ( i ) transcripts with significant consistent changes ( brown ) , ( ii ) with significant changes , with consistent changes of transcripts ( brown ) , ( iii ) with no transcripts detected ( gray ) , ( iv ) with transcripts whose translation is potentially regulated by miRNAs ( gray ) , as assessed by workflow displayed in ( Figure EV2D ) , ( v ) remaining that we classified as regulated by other post - transcriptional mechanisms ( violet ) . pgs = groups .

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Mechanisms affecting protein abundance during aging. Bar plots are based on all the proteins affected in either one of the age comparisons (adj. p < 0.05). Proteins were divided in the following five groups: (i) proteins and transcripts with significant and consistent changes (dark brown), (ii) proteins with significant changes, and with consistent changes of the transcripts (light brown), (iii) proteins with no transcripts detected (dark gray), (iv) proteins with transcripts whose translation is potentially regulated by miRNAs (light gray), as assessed by the workflow displayed in (Figure EV2D), (v) all the remaining proteins that we classified as regulated by other post-transcriptional mechanisms (violet). pgs = protein groups.


Abundance changes of ribosomal their transcripts during aging . ribosomal of large small subunits are displayed separately changes are shown for both comparisons as box plots . Transcripts are displayed as blue as blue boxes . Changes of are displayed as dots ; orange dots identify significant cases ( adj . p < 0 . 05 , n = 4 per group for proteome n = 5 per group for transcriptome ) . In boxplots , horizontal represents median , bottom top of box 25th 75th percentile , respectively , whiskers extend 1 . 5 folds interquartile range .

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Abundance changes of ribosomal proteins and their transcripts during aging. Cytoplasmic ribosomal proteins of large and small subunits are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots; orange dots identify significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Brains from young ( 5 wph ) old ( 39 wph ) were homogenized clarified lysates separated by - exclusion chromatography ( SEC ) . For each group , two pools of brains were processed separately . For each experiment ( 4 in total ) , 39 fractions were collected along chromatogram , digested into peptides analysed by Data Independent Acquisition ( DIA ) quantitative mass spectrometry .

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

Brains from young (5 wph) and old (39 wph) were homogenized and clarified lysates separated by size-exclusion chromatography (SEC). For each age group, two pools of brains were processed separately. For each experiment (4 in total), 39 fractions were collected along the chromatogram, digested into peptides and analysed by Data Independent Acquisition (DIA) quantitative mass spectrometry.


Co - elution profiles for selected complexes . For each , median abundance of quantified subunits was used to generate profile across fractions ( Dataset EV5 ) . profiles are scaled to max value ( set to 1 ) to make profiles comparable across experiments . estimated molecular of displayed complexes is indicated in brackets .

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

Co-elution profiles for selected protein complexes. For each complex, the median abundance of all the quantified subunits was used to generate the complex profile across fractions (Dataset EV5). All the complex profiles are scaled to the max value (set to 1) to make profiles comparable across experiments. The estimated molecular weight of the displayed complexes is indicated in brackets.


Statistics of complexes undergoing stoichiometry changes with aging . Only complexes that had at least 5 members quantified were considered for each comparison . Complexes were considered affected if at least two members showed significant stoichiometry change ( adj . p < 0 . 05 absolute log2 fold change > 0 . 5 ) . complete list of stoichiometry changes is available in Dataset EV6 .

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

Statistics of protein complexes undergoing stoichiometry changes with aging. Only protein complexes that had at least 5 members quantified were considered for each comparison. Complexes were considered affected if at least two members showed significant stoichiometry change (adj. p < 0.05 and absolute log2 fold change > 0.5). The complete list of stoichiometry changes is available in Dataset EV6.


Violin plots depicting interquartile ranges ( IQRs ) of members of complexes during aging . IQR for each considered in G was calculated using transcript ( total dataset , blue ) or ( blue ) log2 fold changes between two groups ( n = 4 per group for proteome n = 5 per group for transcriptome ) . * p < 0 . 05 , Wilcoxon Rank Sum . central of violin plots indicates median value .

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Violin plots depicting interquartile ranges (IQRs) of individual members of protein complexes during aging. The IQR for each protein complex considered in G was calculated using transcript (total RNA dataset, light blue) or protein (dark blue) log2 fold changes between two age groups (n=4 per age group for proteome and n=5 per age group for transcriptome). * p < 0.05, Wilcoxon Rank Sum test. The central line of the violin plots indicates the median value.


Heat map showing relative fold changes for members of selected complexes affected by aging . Names of significantly affected members in 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 absolute log2 fold change > 0 . 5 ) are highlighted in bold with a star . Related to Figure EV3 Dataset EV5 EV6 .

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

Heat map showing relative protein fold changes for members of selected complexes affected by aging. Names of significantly affected members in the 39 wph vs. 12 wph comparison (adj. p < 0.05 and absolute log2 fold change > 0.5) are highlighted in bold with a star. Related to Figure EV3 and Dataset EV5 and EV6.


Representative Coomassie - stained SDS - PAGE gel showing isolation of SDS insoluble from mouse brain lysates . TH = total homogenate , SUP = supernatant , W1 = SDS - soluble fraction , Pellet = formic - acid soluble fraction ( see Figure EV4A ) . of yield of SDS - insoluble from young old brain lysates was on densitometry of Coomassie - stained gel bands obtained from different , n = 5 per group ( Figure EV4B ) ; * * p < 0 . 01 , unpaired t - . In boxplots , horizontal represents median , bottom top of box 25th 75th percentile , respectively , whiskers extend 1 . 5 folds interquartile range .

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

Representative Coomassie-stained SDS-PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates. TH = total homogenate, SUP = supernatant, W1 = SDS-soluble fraction, Pellet = formic-acid soluble fraction (see Figure EV4A). Quantification of the yield of SDS-insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie-stained gel bands obtained from different animals, n=5 per age group (Figure EV4B); ** p < 0.01, unpaired t-test. In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Volcano plot on by label - free mass spectrometry depicting enrichment of specific in . x - axis indicates log2 between abundance in ( Pellet ) starting total homogenate ( TH ) . horizontal dashed indicates a p value cut - off of 0 . 05 vertical lines a log2 fold change cut off of ± 0 . 5 . Selected are highlighted as colored dots as indicated in figure legend . was on samples obtained from 3 independent isolations .

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Volcano plot based on protein quantification by label-free mass spectrometry depicting the enrichment of specific proteins in protein aggregates. The x-axis indicates the log2 ratio between protein abundance in aggregates (Pellet) and starting total homogenate (TH). The horizontal dashed line indicates a p value cut-off of 0.05 and vertical lines a log2 fold change cut off of ± 0.5. Selected proteins are highlighted as colored dots as indicated in the figure legend. Protein quantification was based on samples obtained from 3 independent isolations.


Double - labelling of telencephalic sections of N . furzeri with anti - RPS6 ( green ) as ribosomal marker Proteostat as a marker for aggregated ( red ) . counterstaining was performed with DAPI ( blue ) . Scale bar = 10 μm .

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

Double-labelling of telencephalic sections of N. furzeri with anti-RPS6 (green) as ribosomal marker and Proteostat as a marker for aggregated proteins (red). Nuclear counterstaining was performed with DAPI (blue). Scale bar = 10 μm.


Abundance changes of proteasome their transcripts during aging . Members of 19S 20S are displayed separately changes are shown for both comparisons as box plots . Transcripts are displayed as blue as blue boxes . Changes of are displayed as dots , orange dots represent significant cases ( adj . p < 0 . 05 , n = 4 per group for proteome n = 5 per group for transcriptome ) . In boxplots , horizontal represents median , bottom top of box 25th 75th percentile , respectively , whiskers extend 1 . 5 folds interquartile range .

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Abundance changes of proteasome proteins and their transcripts during aging. Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots, orange dots represent significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Heat map showing transcript fold changes for members of 26 proteasome ( 19S 12S complexes ) . Genes are annotated according to significance of their changes at level of transcript ( adj . p < 0 . 05 : black , adj . p > 0 . 05 : ) or ( adj . p < 0 . 05 : green , adj . p > 0 . 05 : ) .

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

Heat map showing transcript and protein fold changes for members of the 26 proteasome (19S and 12S complexes). Genes are annotated according to significance of their changes at the level of transcript (adj. p < 0.05: black, adj. p > 0.05: white) or protein (adj. p < 0.05: green, adj. p > 0.05: white).


In - gel proteasome following native gel electrophoresis ( top ) immunoblotting of proteasome complexes ( 30S , 26S 20S ) ( bottom ) in young ( 5wph ) , adult ( 12 wph ) old ( 39 wph ) killifish brains . For additional samples low exposure pictures see Figure EV5B EV5C .

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In-gel proteasome assay following native gel electrophoresis (top) and immunoblotting of proteasome complexes (30S, 26S and 20S) (bottom) in young (5wph), adult (12 wph) and old (39 wph) killifish brains. For additional samples and low exposure pictures see Figure EV5B and EV5C.


Bar plots depicting of chymotrypsin - like ( CT - L ) activity from native gels calculated for doubly capped ( 30S ) or singly capped ( 26S ) proteasomes . n > = 5 per sample group ; error bars indicate standard error of mean . * p < 0 . 05 , * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison . For each sample group , mean value of activity in young samples ( 5 wph ) was set to 100 % .

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

Bar plots depicting the quantification of chymotrypsin-like (CT-L) activity from native gels calculated for doubly capped (30S) or singly capped (26S) proteasomes. n >=5 per sample group; error bars indicate standard error of the mean. * p < 0.05, ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of activity in young samples (5 wph) was set to 100%.


( % ) of chymotrypsin - like ( CT - L ) , trypsin - like ( T - L ) peptidylglutamyl peptide hydrolysing or caspase - like ( PGPH ) proteasome activities in brain extracts of killifish of different ages . n > = 6 per sample group ; error bars indicate standard error of mean . * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison . For each sample group , mean value of each activity in young samples ( 5 wph ) was set to 100 % .

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

Percentage (%) of chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing or caspase-like (PGPH) proteasome activities in brain extracts of killifish of different ages. n >=6 per sample group; error bars indicate standard error of the mean. ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of each activity in young samples (5 wph) was set to 100%.


- related changes of involved in ubiquitin cycle . displayed showed significant level changes in 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 ) .

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

Age-related changes of proteins involved in the ubiquitin cycle. All the displayed proteins showed significant protein level changes in the 39 wph vs. 12 wph comparison (adj. p < 0.05).


Chymotrypsin - like ( CT - L ) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors . activity was measured at day 4 after beginning of treatment . n = 8 per sample group ; error bars indicate standard deviation . * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA followed by Holm - Sidak ' s multiple comparisons .

Chymotrypsin-like (CT-L) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors. The activity was measured at day 4 after the beginning of treatment. n=8 per sample group; error bars indicate standard deviation. ** p < 0.01, **** p < 0.0001, one-way ANOVA followed by Holm-Sidak's multiple comparisons test.


Proteasome inhibition induces proteasome activators ( PSME4 ) , assembly factors ( POMP ) ubiquitin ligases ( UBE3C ) , mediators of autophagosome formation ( ATG2B ) , heat shock chaperones in killifish brain . abundances were quantified by DIA mass spectrometry they are shown relative to mean value of vehicle control samples ( DMSO ) set to 1 , n = 10 per sample group . Adj . p < 0 . 05 for displayed . Error bars indicate mean + / - SD .

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

Proteasome inhibition induces proteasome activators (PSME4), assembly factors (POMP) ubiquitin ligases (UBE3C), mediators of autophagosome formation (ATG2B), heat shock proteins and chaperones in killifish brain. Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples (DMSO) set to 1, n=10 per sample group. Adj. p < 0.05 for all the displayed proteins. Error bars indicate mean +/-SD.


Bortezomib treatment affects stoichiometry of a subset of complexes . number of affected ( adj . p < 0 . 25 ) for each is indicated . Only complexes that had at least two members affected are shown .

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

Bortezomib treatment affects the stoichiometry of a subset of protein complexes. The number of affected proteins (adj. p < 0.25) for each protein complex is indicated. Only protein complexes that had at least two members affected are shown.


Members of mitochondrial ribosomes affected by Bortezomib treatment . Relative abundances ( normalized to mean of to which they belong ) are shown . mean value of vehicle control samples ( DMSO ) was set to 1 . n = 10 per sample group . Adj . p < 0 . 25 for displayed . Error bars indicate mean + / - SD .

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

Members of the mitochondrial and cytoplasmic ribosomes affected by Bortezomib treatment. Relative protein abundances (normalized to the mean of the protein complex to which they belong) are shown. The mean value of vehicle control samples (DMSO) was set to 1. n=10 per sample group. Adj. p < 0.25 for all the displayed proteins. Error bars indicate mean +/-SD.


Distribution of change in expression for proteasome transcripts across entire cohort of 159 fish . Lifespan was compared among fish that showed extreme changes in proteasome between 10 20 wph ( 32 fish showing most pronounced decreases , shown in black vs . 32 fish showing most pronounce increases ) . * * * p < 0 . 001 Log - rank .

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

Distribution of change in expression for proteasome transcripts across the entire cohort of 159 fish. Lifespan was compared among fish that showed extreme changes in proteasome levels between 10 and 20 wph (32 fish showing the most pronounced decreases, shown in black vs. 32 fish showing the most pronounce increases). *** p<0.001 Log-rank test.


HeLa co - transfected with EMTB - CFP - FRB YFP - FKBP were treated with 100 nM rapamycin ( Rapa ) . Addition of rapamycin rapidly translocated YFP - FKBP onto EMTB - CFP - FRB - labeled microtubules increased FRET signal . scale for FRET / CFP is shown . Scale bar , 10 μm . normalized of FRET / CFP in before after rapamycin ( blue ) 0 . 1 % DMSO ( control ; red ) treatment . n = 6 10 in rapamycin DMSO groups , respectively , from three independent experiments . Data are shown as mean ± S . E . M .

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

HeLa cells co-transfected with EMTB-CFP-FRB and YFP-FKBP were treated with 100 nM rapamycin (Rapa). Addition of rapamycin rapidly translocated YFP-FKBP onto EMTB-CFP-FRB-labeled microtubules and increased the FRET signal. The scale for the FRET/CFP intensity ratio is shown. Scale bar, 10 μm. The normalized intensity of FRET/CFP in cells before and after rapamycin (blue) and 0.1% DMSO (control; red) treatment. n = 6 and 10 cells in the rapamycin and DMSO groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.


HeLa co - transfected with indicated constructs were treated with nocodazole ( Noc , 3 . 3 µM ; left ) or rapamycin ( Rapa , 100 nM ; right ) for 0 , 30 , 60 min . Transfected were then fixed labeled with anti - acetylated tubulin ( red ) . Dotted lines indicate boundary of transfected . Scale bars , 10 μm . of acetylated tubulin in transfected with EMTB - CFP - FRB ( orange ) or cotransfected with EMTB - CFP - FRB dNSpastin3Q - YFP - FKBP ( blue ) after addition of nocodazole ( Noc , 3 . 3 µM ) or rapamycin ( Rapa , 100 nM ) for indicated times . n = 123 81 in nocodazole dNSpastin3Q groups , respectively , from three independent experiments . Data are shown as mean ± S . E . M . Student ' s t - were performed , with p - values indicated .

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

HeLa cells co-transfected with the indicated constructs were treated with nocodazole (Noc, 3.3 µM; left) or rapamycin (Rapa, 100 nM; right) for 0, 30, and 60 min. Transfected cells were then fixed and labeled with anti-acetylated tubulin (red). Dotted lines indicate the boundary of transfected cells. Scale bars, 10 μm. The intensity of acetylated tubulin in cells transfected with EMTB-CFP-FRB (orange) or cotransfected with EMTB-CFP-FRB and dNSpastin3Q-YFP-FKBP (blue) after addition of nocodazole (Noc, 3.3 µM) or rapamycin (Rapa, 100 nM) for the indicated times. n = 123 and 81 cells in the nocodazole and dNSpastin3Q groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M. Student's t-tests were performed, with p-values indicated.


NIH3T3 fibroblasts co - transfected with 5HT6 - mCherry ( 5HT6 - mCh ; a ciliary marker ; red ) , CFP - FRB - MAP4m ( blue ) , dNSpastin3Q - YFP - FKBP ( green ) were serum - starved for 24 hr to induce ciliogenesis . Ciliated were then treated with rapamycin ( Rapa , 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to axonemal microtubules . Dotted lines indicate boundaries . Scale bar , 5 μm . normalized of axonemes ( blue ) , cilia ( red ) , dNSpastin3Q - YFP - FKBP in cilia ( green ) upon rapamycin treatment . n = 6 from three independent experiments . Data are shown as mean ± S . E . M .

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

NIH3T3 fibroblasts co-transfected with 5HT6-mCherry (5HT6-mCh; a ciliary membrane marker; red), CFP-FRB-MAP4m (blue), and dNSpastin3Q-YFP-FKBP (green) were serum-starved for 24 hr to induce ciliogenesis. Ciliated cells were then treated with rapamycin (Rapa, 100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to axonemal microtubules. Dotted lines indicate the cell boundaries. Scale bar, 5 μm. The normalized length of axonemes (blue), primary cilia (red), and dNSpastin3Q-YFP-FKBP in cilia (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.


HeLa co - transfected with H2B - mCherry ( H2B - mCh ; a chromosome marker ; red ) , CFP - FRB - MAP4m ( a marker of mitotic spindles ; blue ) , dNSpastin3Q - YFP - FKBP ( green ) were synchronized in metaphase treated with rapamycin ( 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to mitotic spindles . Dotted lines indicate boundaries . Scale bar , 10 μm . normalized of mitotic spindle ( blue ) of dNSpastin3Q - YFP - FKBP in mitotic spindle ( green ) upon rapamycin treatment . n = 6 from three independent experiments . Data are shown as mean ± S . E . M .

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

HeLa cells co-transfected with H2B-mCherry (H2B-mCh; a chromosome marker; red), CFP-FRB-MAP4m (a marker of mitotic spindles; blue), and dNSpastin3Q-YFP-FKBP (green) were synchronized in metaphase and treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to mitotic spindles. Dotted lines indicate the cell boundaries. Scale bar, 10 μm. The normalized area of the mitotic spindle (blue) and intensity of dNSpastin3Q-YFP-FKBP in the mitotic spindle (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.


HeLa co - transfected with H2B - mCherry ( red ) , CFP - FRB - MAP4m ( intercellular bridges ; blue ) , dNSpastin3Q - YFP - FKBP ( green ) were treated with rapamycin ( 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to intercellular bridges . Arrows indicate intercellular bridges . Dotted lines indicate boundaries . Scale bar , 10 μm . normalized of intercellular bridges ( blue ) of dNSpastin3Q - YFP - FKBP at intercellular bridges ( green ) upon rapamycin treatment . n = 5 from three independent experiments . Data are shown as mean ± S . E . M .

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

HeLa cells co-transfected with H2B-mCherry (red), CFP-FRB-MAP4m (intercellular bridges; blue), and dNSpastin3Q-YFP-FKBP (green) were treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to intercellular bridges. Arrows indicate the intercellular bridges. Dotted lines indicate the cell boundaries. Scale bar, 10 μm.   The normalized area of intercellular bridges (blue) and intensity of dNSpastin3Q-YFP-FKBP at intercellular bridges (green) upon rapamycin treatment. n = 5 cells from three independent experiments. Data are shown as the mean ± S.E.M.


COS7 transfected with TagRFP - FRB - A1AY1 ( red ) were labeled by anti - α - tubulin antibody ( green ; upper panel ) , anti - tyrosinated tubulin antibody ( green ; middle panel ) , or anti - detyrosinated tubulin antibody ( green ; lower panel ) , respectively . Scale bar , 10 µm . profiles of TagRFP - FRB - A1AY1 ( red ) tubulin with indicated PTMs ( green ) along dotted lines drawn in ( A ) . Pearson ' s correlation coefficients for TagRFP - FRB - A1AY1 indicated tubulin ( tub ) subtypes were calculated , data are shown as mean ± S . E . M . n = 16 , 13 , 15 from left to right , respectively ; three independent experiments .

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

COS7 cells transfected with TagRFP-FRB-A1AY1 (red) were labeled by anti-α-tubulin antibody (green; upper panel), anti-tyrosinated tubulin antibody (green; middle panel), or anti-detyrosinated tubulin antibody (green; lower panel), respectively. Scale bar, 10 µm. The intensity profiles of TagRFP-FRB-A1AY1 (red) and tubulin with the indicated PTMs (green) along dotted lines drawn in (A). The Pearson's correlation coefficients for TagRFP-FRB-A1AY1 and indicated tubulin (tub) subtypes were calculated, and data are shown as the mean ± S.E.M. n = 16, 13, and 15 cells from left to right, respectively; three independent experiments.


COS7 co - transfected with TagRFP - FRB - A1AY1 ( red ) TagCFP - FKBP ( green ) were treated with rapamycin ( 100 nM ) . addition of rapamycin rapidly recruits TagCFP - FKBP from cytosol onto A1AY1 - labeled microtubules ( arrows ) . Scale bar , 10 µm . normalized of TagCFP - FKBP at A1AY1 - labeled microtubules upon rapamycin treatments . n = 7 from three independent experiments . Data are shown as mean ± S . E . M .

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

COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and TagCFP-FKBP (green) were treated with rapamycin (100 nM). The addition of rapamycin rapidly recruits TagCFP-FKBP from cytosol onto A1AY1-labeled microtubules (arrows). Scale bar, 10 µm. The normalized intensity of TagCFP-FKBP at A1AY1-labeled microtubules upon rapamycin treatments. n=7 cells from three independent experiments. Data are shown as the mean ± S.E.M.


COS7 co - transfected with TagRFP - FRB - A1AY1 ( red ) dNSpastin3Q - TagCFP - FKBP ( cyan ) were treated with 0 . 1 % DMSO or rapamycin ( 100 nM ) for 1 hr followed by immunostaining with anti - tyrosinated tubulin antibody ( green ) . Dotted lines highlight transfected . Scale bar , 20 μm . normalized of tyrosinated microtubules in TagRFP - FRB - A1AY1 dNSpastin3Q - TagCFP - FKBP co - transfected upon 0 . 1 % DMSO or rapamycin treatment for 1 hr . n = 31 26 in DMSO rapamycin treated groups , respectively , from three independent experiments . Data ( blue ) represent as mean ± S . E . M . Student ' s t - were performed with p values indicated .

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

COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and dNSpastin3Q-TagCFP-FKBP (cyan) were treated with 0.1% DMSO or rapamycin (100 nM) for 1 hr and followed by immunostaining with anti-tyrosinated tubulin antibody (green). Dotted lines highlight the transfected cells. Scale bar, 20 μm. The normalized intensity of tyrosinated microtubules in TagRFP-FRB-A1AY1 and dNSpastin3Q-TagCFP-FKBP co-transfected cells upon 0.1% DMSO or rapamycin treatment for 1 hr. n=31 and 26 cells in DMSO and rapamycin treated groups, respectively, from three independent experiments. Data (blue) represent as mean ± S.E.M. Student's t-tests were performed with p values indicated.


COS7 co - transfected with EMTB - YFP - CIBN mCh - Cry2 ( red ) were incubated with SPY650 - tubulin ( SPY650 - tub ) to visualize microtubules . were illuminated by blue within a specified region ( indicated by dotted circle ) for indicated time period . Scale bar , 10 μm . normalized of mCh - Cry2 at microtubules in illuminated regions ( red ) non - illuminated regions ( green ) . n = 6 from three independent experiments . Data are shown as mean ± S . E . M .

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

COS7 cells co-transfected with EMTB-YFP-CIBN and mCh-Cry2 (red) were incubated with SPY650-tubulin (SPY650-tub) to visualize microtubules. The cells were illuminated by blue light within a specified region (indicated by the dotted circle) for the indicated time period. Scale bar, 10 μm. The normalized intensity of mCh-Cry2 at microtubules in illuminated regions (red) and non-illuminated regions (green). n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.


COS7 co - transfected with EMTB - YFP - CIBN dNSpastin3Q - mCh - Cry2 were incubated with SPY650 - tubulin to visualize microtubules . were illuminated by blue as in ( A ) . Scale bar , 10 μm . normalized of dNSpastin3Q - mCh - Cry2 mCh - Cry2 at microtubules normalized of SPY650 - tubulin in illuminated regions non - illuminated regions . n = 6 6 in dNSpastin3Q - mCh - Cry2 mCh - Cry2 groups , respectively , from three independent experiments . Data are shown as mean ± S . E . M .

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

COS7 cells co-transfected with EMTB-YFP-CIBN and dNSpastin3Q-mCh-Cry2 were incubated with SPY650-tubulin to visualize microtubules. The cells were illuminated by blue light as in (A). Scale bar, 10 μm. The normalized intensity of dNSpastin3Q-mCh-Cry2 and mCh-Cry2 at microtubules and the normalized area of SPY650-tubulin in illuminated regions and non-illuminated regions. n = 6 and 6 cells in dNSpastin3Q-mCh-Cry2 and mCh-Cry2 groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.


E - F . COS7 co - transfected with EMTB - CFP - FRB , dNSpastin3Q - mCh - FKBP , Tom20 - Neon were treated with rapamycin ( 100 nM ) for acute microtubule disruption . of mitochondria at different of microtubule disruption is shown . Scale bar , 10 µm . of long axis to short axis of each mitochondrion microtubule filament was determined . n = 4 different , three independent experiments . Data are shown as mean ± S . E . M . Data information : Student ' s t - were performed with p values indicated .

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

E-F. COS7 cells co-transfected with EMTB-CFP-FRB, dNSpastin3Q-mCh-FKBP, and Tom20-Neon were treated with rapamycin (100 nM) for acute microtubule disruption. The morphology of mitochondria at different levels of microtubule disruption is shown. Scale bar, 10 µm. The ratio of the long axis to the short axis of each mitochondrion and microtubule filament area was determined. n= 4 different cells, three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.


G - H . normalized of MitoTracker Red in non - transfected ( Mock ) or co - transfected with EMTB - CFRP - FRB dNSpastin3Q - YFP - FKBP or dNSpastin3QED - YFP - FKBP after rapamycin ( Rapa , 100 nM ) treatment for indicated time . Scale bar , 10 µm . n = 41 , 6 , 18 from three independent experiments . Data are shown as mean ± S . E . M . Data information : Student ' s t - were performed with p values indicated .

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

G-H. The normalized intensity of MitoTracker Red in non-transfected cells (Mock) or cells co-transfected with EMTB-CFRP-FRB and dNSpastin3Q-YFP-FKBP or dNSpastin3QED-YFP-FKBP after rapamycin (Rapa, 100 nM) treatment for the indicated time. Scale bar, 10 µm. n = 41, 6, and 18 cells from three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.


COS7 co - transfected with EMTB - CFP - FRB ( blue ) , dNSpastin3Q - mCh - FKBP ( red ) , Lifeact - Neon ( green ) were treated with rapamycin ( Rapa , 100 nM ) to induce microtubule disassembly . Arrows arrowheads indicate lamellipodia stress , respectively . Insets show of lamellipodia from regions corresponding to dotted squares with overexposed CFP mCherry signals . Scale bar , 10 µm . normalized level of microtubule , lamellipodia , filopodia density of COS - 7 treated as in ( A ) . n = 4 from three independent experiments . Data are shown as mean ± S . E . M .

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

COS7 cells co-transfected with EMTB-CFP-FRB (blue), dNSpastin3Q-mCh-FKBP (red), and Lifeact-Neon (heatmap and green) were treated with rapamycin (Rapa, 100 nM) to induce microtubule disassembly. Arrows and arrowheads indicate lamellipodia and stress fibers, respectively. Insets show images of lamellipodia morphology from regions corresponding to the dotted squares with overexposed CFP and mCherry signals. Scale bar, 10 µm. The normalized level of microtubule area, lamellipodia length, and filopodia density of COS-7 cells treated as in (A). n = 4 cells from three independent experiments. Data are shown as the mean ± S.E.M.


B . Carfilzomib , Bortezomib MLN9708 activate luciferase activity in M2 macrophages derived from Il - 1β - luciferase transgenic mice . BMDMs from IL - 1β - luciferase transgenic mice were treated by IL - 4 ( 20 ng / mL ) for 24 hours to differentiate into mature M2 macrophages , then used for screening of FDA compounds ( 5 μM ) . X axis stands for code of drug .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Carfilzomib, Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs from IL-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours to differentiate into mature M2 macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug.


F , G . Carfilzomib , Bortezomib MLN9708 promote mRNA expression of Il - 1β in M2 macrophages . BMDMs ( F ) or Raw264 . 7 ( G ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then treated by DMSO , Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) , or MLN9708 ( 2 μM ) . was extracted from BMDMs or Raw264 . 7 6 hours after stimulation expression of Il - 1β were quantified through RT - qPCR . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * * * p < 0 . 001 ( student ' s t - ) . H , I . Carfilzomib , Bortezomib MLN9708 promote secretion of inflammatory cytokine IL - 1β in M2 macrophages . BMDMs ( H ) or Raw264 . 7 ( I ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO , Carfilzomib ( 500 nM ) , Bortezomib ( 500 nM ) or MLN9708 ( 500 nM ) . Secretion of IL - 1β were measured through ELISA 24 hours later . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

F, G. Carfilzomib, Bortezomib and MLN9708 promote the mRNA expression of Il-1β in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from BMDMs or Raw264.7 cells 6 hours after stimulation and the expression of Il-1β were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test). H, I. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL-1β in M2 macrophages. BMDMs (H) or Raw264.7 cells (I) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO, Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). Secretion of IL-1β were measured through ELISA 24 hours later. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).


A . most effective one among candidate compounds , Carfilzomib , significantly promotes expression of M1 macrophage markers , as well as reduces expression of M2 macrophage markers . BMDMs were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 1 μM ) . was extracted from 6 hours after stimulation expression of M1 ( Il - 6 / Inos ) or M2 ( Cd206 / Arg1 ) macrophage markers were quantified through RT - qPCR . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * * * p < 0 . 001 ( student ' s t - ) .

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

A. The most effective one among the candidate compounds, Carfilzomib, significantly promotes the expression of M1 macrophage markers, as well as reduces the expression of M2 macrophage markers. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). RNA was extracted from cells 6 hours after stimulation and the expression of M1 (Il-6/Inos) or M2 (Cd206/Arg1) macrophage markers were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test).


D . Statistics for proportion of CD86 , CD80 , MHC - II or CD206 positive in BMDMs . Data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - ) .

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

D. Statistics for proportion of CD86, CD80, MHC-II or CD206 positive cells in BMDMs. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).


E . Carfilzomib promotes phagocytosis of macrophages . BMDMs were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 500 nM ) for 12 hours . After starving for 2 hours stained with Red dye , BMDMs ( red ) were incubated with L1210 - GFP ( green ) in serum - free medium for another 2 hours . Phagocytosis effect was observed photographed under microscope . Scale bars , 50 μm ( up ) , 20 μm ( down ) . yellow arrows indicated L1210 that is phagocytosed by macrophages . F . Statistics represent number of phagocytosis L1210 in 100 macrophages . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - ) .

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

E. Carfilzomib promotes the phagocytosis of macrophages. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (500 nM) for 12 hours. After starving for 2 hours and stained with Red membrane dye, BMDMs (red) were incubated with L1210-GFP cells (green) in serum-free medium for another 2 hours. Phagocytosis effect was observed and photographed under fluorescence microscope. Scale bars, 50 μm (up), 20 μm (down). The yellow arrows indicated L1210 that is phagocytosed by macrophages. F. Statistics represent the number of phagocytosis L1210 in 100 macrophages. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).


M . Carfilzomib promotes M1 - polarization of associated macrophages in vitro . BMDMs were cultured with culture supernatant ( TSN ) produced by L1210 , followed by activation with DMSO or Carfilzomib ( 1 μM ) . was extracted from expression of Il - 1β , Il - 6 , Inos were quantified through RT - qPCR 6 hours after stimulation . data are means ± SD of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

M. Carfilzomib promotes the M1-polarization of Tumor associated macrophages in vitro. BMDMs were cultured with tumor culture supernatant (TSN) produced by L1210 cells, followed by activation with DMSO or Carfilzomib (1 μM). RNA was extracted from cells and the expression of Il-1β, Il-6, Inos were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).


A , B . Carfilzomib proteasome inhibitor MG132 activate Il - 1β luciferase in macrophages . ( A ) BMDMs were treated with IL - 4 ( 20 ng / mL ) for 24 hours then stimulated by DMSO , Carfilzomib ( 1 μM ) , or MG132 ( 5 μM ) . ( B ) BMDMs were stimulated by DMSO , Carfilzomib ( 1 μM ) or MG132 ( 5 μM ) ) for 1 hour , then induced with IL - 4 ( 20 ng / mL ) . Luciferase assays were monitored 12 hours after stimulation . data are means ± SD of three independent experiments . * * * p < 0 . 001 ( student ' s t - ) .

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

A, B. Carfilzomib and proteasome inhibitor MG132 activate Il-1β luciferase in macrophages. (A) BMDMs were treated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, Carfilzomib (1 μM), or MG132 (5 μM). (B) BMDMs were stimulated by DMSO, Carfilzomib (1 μM) or MG132 (5 μM)) for 1 hour, then induced with IL-4 (20 ng/mL). Luciferase assays were monitored 12 hours after stimulation. The data are means ± SD of three independent experiments. ***p < 0.001 (student's t-test).


E . Carfilzomib , Bortezomib , MLN9708 MG132 induce ER stress response . BMDMs were pretreated with IL - 4 ( 20 ng / mL ) for 24 hours then stimulated by DMSO or Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) , MLN9708 ( 2 μM ) , MG132 ( 5 μM ) . was extracted from 3 hours or 6 hours after stimulation expression of ER stress related genes ( Bip , Chop ) were detected through RT - qPCR . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

E. Carfilzomib, Bortezomib, MLN9708 and MG132 induce ER stress response. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO or Carfilzomib (1 μM), Bortezomib (1 μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 3 hours or 6 hours after stimulation and the expression of ER stress related genes (Bip, Chop) were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


F . ER stress agonists , TUN TG , promote expression of M1 macrophage markers , as well as reduce expression of M2 macrophage markers . BMDMs were pretreated with IL - 4 ( 20 ng / mL ) for 24 hours then stimulated by DMSO , TUN ( 500 nM ) or TG ( 500 nM ) . was extracted from 6 hours after stimulation indicated genes were quantified through RT - qPCR . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

F. ER stress agonists, TUN and TG, promote the expression of M1 macrophage markers, as well as reduce the expression of M2 macrophage markers. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, TUN (500 nM) or TG (500 nM). RNA was extracted from cells 6 hours after stimulation and indicated genes were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


G . Inhibition of ER stress impairs ability of Carfilzomib , Bortezomib , MLN9708 MG132 to reprogram M2 towards M1 - like macrophages . BMDMs were induced by IL - 4 ( 20 ng / mL ) for 24 hours , then pretreated with 4 - PBA ( 5 mM ) for 1 hour stimulated by Carfilzomib ( 1μM ) , Bortezomib ( 1μM ) , MLN9708 ( 2 μM ) , MG132 ( 5 μM ) . was extracted from 6 hours after stimulation expression of Il - 1β , Il - 6 Inos were detected through RT - qPCR . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

G. Inhibition of ER stress impairs the ability of Carfilzomib, Bortezomib, MLN9708 and MG132 to reprogram M2 towards M1-like macrophages. BMDMs were induced by IL-4 (20 ng/mL) for 24 hours, then pretreated with 4-PBA (5 mM) for 1 hour and stimulated by Carfilzomib (1μM), Bortezomib (1μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 6 hours after stimulation and the expression of Il-1β, Il-6 and Inos were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


A , B Deficiency of Ern1 reduces expression of inflammatory related genes . Wild - type Ern1 - / - Raw264 . 7 ( A ) or BMDMs ( B ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 1 μM ) . mRNA of Il - 1β Il - 6 were quantified through RT - qPCR 6 hours after stimulation . data are means ± SD of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

A, B Deficiency of Ern1 reduces the expression of inflammatory related genes. Wild-type and Ern1-/- Raw264.7 cells (A) or BMDMs (B) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). The mRNA of Il-1β and Il-6 were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).


E . Kira6 inhibits expression of ER stress related genes inflammatory related genes . BMDMs was treated by IL - 4 ( 20 ng / mL ) for 24 hours , treated with Kira6 ( 100 nM ) for 1 hour , then stimulated by Carfilzomib ( 1 μM ) for 6 hours . was extracted from expression of Bip , Chop , Il - 1β Il - 6 were quantified through RT - qPCR . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - ) . F , G . Carfilzomib has no effect on splicing of XBP1 in M2 macrophages . BMDMs ( F ) or Raw264 . 7 ( G ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated with DMSO , Carfilzomib ( 1 μM ) or TUN ( 500 nM ) . mRNA expression of sXBP1 unXBP1 were detected through RT - qPCR 6 hours after stimulation . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

E. Kira6 inhibits the expression of ER stress related genes and inflammatory related genes. BMDMs was treated by IL-4 (20 ng/mL) for 24 hours, treated with Kira6 (100 nM) for 1 hour, and then stimulated by Carfilzomib (1 μM) for 6 hours. RNA was extracted from cells and the expression of Bip, Chop, Il-1β and Il-6 were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test). F, G. Carfilzomib has no effect on splicing of XBP1 in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated with DMSO, Carfilzomib (1 μM) or TUN (500 nM). The mRNA expression of sXBP1 and unXBP1 were detected through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


J . Carfilzomib promotes association between IRE1α TRAF2 in M2 macrophages . Raw264 . 7 were pretreated by IL - 4 ( 20ng / mL ) for 24 hours , then stimulated by Carfilzomib ( 1 μM ) for 2 or 4 hours . Coimmunoprecipitation ( Co - IP ) IB were performed with indicated antibodies .

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

J. Carfilzomib promotes the association between IRE1α and TRAF2 in M2 macrophages. Raw264.7 cells were pretreated by IL-4 (20ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for 2 or 4 hours. Coimmunoprecipitation (Co-IP) and IB were performed with indicated antibodies.


L . Carfilzomib activates NF - κB signaling pathway through IRE1α in M2 macrophages . Wild - type Ern1 - / - Raw264 . 7 were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by Carfilzomib ( 1 μM ) for indicated time points . WCL were analyzed by IB with indicated antibodies .

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

L. Carfilzomib activates NF-κB signaling pathway through IRE1α in M2 macrophages. Wild-type and Ern1-/- Raw264.7 cells were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for indicated time points. WCL were analyzed by IB with indicated antibodies.


C . Representative of Hematoxylin eosin ( H & E ) of lung tissue obtained from different treatment groups . Expression of Ki - 67 in lung tissues was detected by immunohistochemistry method . D . Statistics of number of Ki67 positive in sections of lung tissue . Data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

C. Representative images of Hematoxylin and eosin (H&E) staining of the lung tissue obtained from different treatment groups. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. D. Statistics of the tumor area and the number of Ki67 positive cells in sections of lung tissue. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


E . Carfilzomib treatment promotes infiltration of M1 macrophages . bearing TD mice were treated as indicated in ( A ) euthanized . Lung tissues were dissected for evaluating M1 / M2 populations through analyze expression of CD80 CD206 by flow cytometry . F . Statistics represent proportion of CD80 or CD206 positive in lung tissue . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - ) . G . Carfilzomib treatment promotes infiltration activation of CD8 + T . Lung tissues of ( E ) were used for evaluating infiltration of CD8 + T their expression of CD69 by flow cytometry . H . Statistics about proportion of CD8 + or CD69 + positive in lung tissue . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - ) .

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

E. Carfilzomib treatment promotes the infiltration of M1 macrophages. Tumor bearing TD mice were treated as indicated in (A) and euthanized. Lung tissues were dissected for evaluating M1/M2 populations through analyze the expression of CD80 and CD206 by flow cytometry. F. Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test). G. Carfilzomib treatment promotes the infiltration and activation of CD8+ T cells. Lung tissues of (E) were used for evaluating the infiltration of CD8+ T cells and their expression of CD69 by flow cytometry. H. Statistics about the proportion of CD8+ or CD69+ positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).


K , L . Carfilzomib shrinks through modulating microenvironment . EG7 were inoculated in wild - type or LyzM - cre ; lsl - dTA to allow xenograft to reach a volume of around 80 mm3 . Mice were randomized for treatment ( n = 5 ) by saline or Carfilzomib for two . xenografts were dissected to photograph ( K ) or weigh ( L ) after treatment . Data are shown as mean ± SD n indicates number of biological replicates . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s t - ) .

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

K, L. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or LyzM-cre;lsl-dTA to allow tumor xenograft to reach a volume of around 80 mm3. Mice were randomized for treatment (n=5) by saline or Carfilzomib for two weeks. The xenografts were dissected to photograph (K) or weigh (L) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).


D . CD4 + CD8 + T play a role in mediating cancer - shrinking effect of Carfilzomib . Lung - cancer - bearing TD mice were treated by saline , Carfilzomib , anti - mouse CD4 antibody ( ip . ) , anti - mouse CD8 antibody ( ip . ) , or Carfilzomib plus anti - mouse CD4 / 8 antibody . CT scanning was performed after two - week treatment . E . Representative of H & E of treated lung tissues . Expression of Ki - 67 in lung tissues was detected by immunohistochemistry method . F . Carfilzomib synergized with PD1 antibody in shrinking lung cancers . Lung cancer bearing TD mice were treated by saline , anti - PD1 antibody ( ip ) , Carfilzomib , or Carfilzomib plus anti - PD1 ( ip ) . CT scanning was performed after two - week treatment . G . Representative of H & E of lung tissues . Expression of Ki - 67 in lung tissue was detected by immunohistochemistry method .

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

D. CD4+ and CD8+ T cells play a role in mediating cancer-shrinking effect of Carfilzomib. Lung-cancer-bearing TD mice were treated by saline, Carfilzomib, anti-mouse CD4 antibody (ip.), anti-mouse CD8 antibody (ip.), or Carfilzomib plus anti-mouse CD4/8 antibody. CT scanning was performed after two-week treatment. E. Representative images of H&E staining of the treated lung tissues. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. F. Carfilzomib synergized with PD1 antibody in shrinking lung cancers. Lung cancer bearing TD mice were treated by saline, anti-PD1 antibody (ip), Carfilzomib, or Carfilzomib plus anti-PD1 (ip). CT scanning was performed after two-week treatment. G. Representative images of H&E staining of the lung tissues. Expression of Ki-67 in lung tissue was detected by immunohistochemistry method.


A . Carfilzomib , Bortezomib MLN9708 promote expression of M1 macrophage markers ( Il - 1β , Il - 6 , Tnfα ) reduce expression of M2 macrophage markers ( Il - 10 , Tgfβ ) in macrophages derived from PBMCs . After induced by IL - 4 ( 20 ng / ml ) for 24 hours to differentiate into mature M2 macrophages , PBMCs derived macrophages were stimulated by Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) or MLN9708 ( 2 μM ) . was extracted from 6 hours after stimulation expression of mRNA were quantified through RT - qPCR . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

A. Carfilzomib, Bortezomib and MLN9708 promote the expression of M1 macrophage markers (Il-1β, Il-6, Tnfα) and reduce the expression of M2 macrophage markers (Il-10, Tgfβ) in macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (1 μM), Bortezomib (1 μM) or MLN9708 (2 μM). RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


C - F . Carfilzomib , Bortezomib MLN9708 increase expression of CD80 ( C ) reduce expression of CD206 ( E ) in M2 macrophages derived from PBMCs . After activated by IL - 4 ( 20 ng / ml ) for 24 hours to differentiate into mature M2 macrophages , PBMCs derived macrophages were stimulated by Carfilzomib ( 500 nM ) , Bortezomib ( 500 nM ) or MLN9708 ( 500 nM ) . representative histogram of CD80 CD206 expression was analyzed by flow cytometry 12 hours after stimulation . ( D , F ) Statistics represent proportion of CD80 ( D ) or CD206 ( F ) positive in macrophages derived from PBMCs . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - ) .

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

C-F. Carfilzomib, Bortezomib and MLN9708 increase the expression of CD80 (C) and reduce the expression of CD206 (E) in M2 macrophages derived from PBMCs. After activated by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation. (D, F) Statistics represent the proportion of CD80 (D) or CD206 (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01 (student's t-test).


( A , B ) Effect of MPC inhibition on lipid droplet content . ( A ) Live - super - resolution confocal imaging of brown adipocytes pre - stained overnight with fatty acid tracer BODIPY C12 558 / 568 ( Bodipy C12 , red ) . were stained with mitotracker green ( MTG , green ) prior to imaging . were imaged before 120 minutes after treatment with either vehicle ( DMSO ) or 100 nM UK5099 . As a positive control , were imaged 30 min after treatment with 1 µM norepinephrine . LD , lipid droplet ; N , nucleus . Scale bar = 10 µm . ( B ) of changes in lipid droplet cross - sectional with indicated treatments from shown in ( A ) . Data are represented as change in LD at time = 120 min compared to time = 0 min .

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

(A,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 incorporation into triacylglycerides ( TAGs ) ( C ) Representative thin - layer chromatography ( TLC ) plate of lipids extracted from brown adipocytes culture media . were incubated with Bodipy C12 in presence of vehicle ( V ) , 100 nM UK5099 ( UK ) or 1 µM norepinephrine ( NE ) for 24 h triacylglyceride ( TAG ) synthesis ( Lipids in ) fatty acid uptake ( Lipids in media ) were detected . relative polarity of lipid species determines motility , with nonpolar TAG migrating furthest . ( D ) of C12 uptake from media , TAG in TAG per amount of free fatty acid uptake shown in ( C ) , n = 5 experiments . UK5099 treatment decreases amount of synthesized TAG per free fatty acid uptake , similarly to NE .

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

(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 energy expenditure . Fully differentiated 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 3 mM glutamine in presence of vehicle or UK - 5099 . ( E ) of non - stimulated OCR from n = 3 - 7 experiments . Data were normalized to vehicle for each 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 ) antimycin A ( AA ; 4 µM ) were injected where indicated . Representative OCR traces averaging 6 technical replicates . ( G ) of non - stimulated OCR , etomoxir sensitive , etomoxir insensitive OCR from n = 4 experiments . Data were normalized to vehicle for each experiment . *

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

(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 brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h . Data shows mole enrichment ( MPE ) of isotope labeled substrate into respective metabolite .

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(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.


( A , B ) Effect of UK5099 treatment on norepinephrine - stimulated energy expenditure . Fully differentiated 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 3 mM glutamine in presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 4 technical replicates . ( B ) of non - stimulated OCR , NE - stimulated OCR , ATP - linked OCR , mitochondrial proton leak Eto - sensitive OCR as shown in ( A ) from n = 9 experiments . effects of UK5099 treatment were normalized to vehicle for each experiment .

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(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.


brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble ( Scramble ) . ( C ) Representative Western Blot of MPC1 , MPC2 actin . ( D ) of MPC1 MPC2 expression normalized to actin from Western Blots in ( C ) from n = 3 experiments .

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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.


brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble ( Scramble ) . ( E ) mRNA of MPC1 MPC2 from n = 3 experiments .

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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.


brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble ( Scramble ) . ( F ) Representative OCR traces of differentiated brown adipocytes averaging 6 technical replicates . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( G ) of basal , NE - stimulated Eto - sensitive OCR as shown in ( F ) from n = 7 experiments . Data were normalized to Scramble for each experiment . ( H ) of basal OCR in response to 100 nM UK5099 treatment in Scramble of MPC1 siRNA transfected from n = 5 experiments . Data were normalized to vehicle for each experiment .

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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.


( A , B ) Requirement for glutamine in UK5099 - induced energy expenditure . 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 3 mM glutamine or 5 mM glucose alone . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 6 technical replicates . ( B ) of non - stimulated OCR , NE - stimulated OCR , ATP - linked OCR , mitochondrial proton leak etomoxir - sensitive OCR after vehicle or UK5099 treatment as shown in ( A ) from n = 4 experiments .

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(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 . 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 3 mM glutamine in presence of vehicle , UK5099 or UK5099 + CB839 from n = 5 experiments . Data were normalized to vehicle for each experiment .

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(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 measurement . Glutamate / aspartate was quantified using GC - MS as a measure of glutamine catabolism . brown adipocytes were treated for 24 h with vehicle ( DMSO ) or 10 µM UK5099 from n = 6 experiments . * *

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(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. **


( 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 AOA for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of tested compounds . Data show of non - stimulated OCR from n = 5 experiments .

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(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 . brown adipocytes were transduced with Scramble ( Scramble ) or shOGC1 ( OGC1 KD ) adenovirus . 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 3 mM glutamine in presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM , ) oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( C ) Representative OCR traces averaging 4 technical replicates . ( D ) of non - stimulated OCR as measured in ( C ) from n = 6 experiments . ( E ) of Eto - sensitive OCR as measured in ( C ) from n = 6 experiments . Data were normalized to vehicle for each experiment .

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(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 . brown adipocytes were transfected with Scramble ( Scramble ) or siRNA for Aralar1 ( Aralar1 KD ) . 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 3 mM glutamine in presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM ) , oligomycin a ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin a ( AA ; 4 µM ) were injected where indicated . ( F ) Representative OCR traces averaging 4 technical replicates . ( G ) of non - stimulated OCR as measured ( G ) from n = 4 experiments . Data were normalized to vehicle for each experiment . ( H ) of Eto - sensitive OCR as measured in ( F ) from n = 4 experiments .

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(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 or Aralar1 siRNA were treated for 24 hours with vehicle ( DMSO ) or 10 µM UK5099 . Data shows of of aspartate to glutamate abundance as measured by GC - MS from n = 3 experiments .

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(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.


( A , B ) Effect of UK5099 on energy expenditure contributed by ATP demand by mitochondrial proton leak in non - stimulated brown adipocytes . 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 3 mM glutamine in presence of vehicle or UK5099 . Oligomycin A ( Oligo ; 4 µM ) , mitochondrial uncoupler FCCP ( 2 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 6 technical replicates . ( B ) of mitochondrial proton leak ATP - linked OCR ( oligomycin sensitive ) as measured in ( A ) from n = 9 experiments . Data were normalized to vehicle for each experiment .

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(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 contribution of ATP demand to increased energy expenditure under UK5099 were permeabilized ADP concentrations were clamped . OCR were measured in presence of 5 mM pyruvate , 3 mM malate , 0 . 1 mM palmitoyl - CoA , 0 . 5 mM carnitine 5 mM ADP . were treated with either 100 nM , 1 µM or 10 µM UK5099 . Data shows maximal state 3 OCR normalized to maximal 4 activity from n = 3 experiments .

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(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 ) 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 experiments .

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(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 . 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 3 mM glutamine in presence of vehicle , UK5099 , or Atglistatin . ( F ) of non - stimulated OCR from n = 4 experiments . ( G ) of ATP - linked respiration from n = 4 experiments . Data were normalized to vehicle for each experiment .

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(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 . brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 1 µM DGAT1 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 3 mM glutamine in presence of vehicle , UK5099 , DGAT1 / 2 inhibitors , or UK5099 + DGAT1 / 2 inhibitors . ( H ) of non - stimulated OCR from n = 6 experiments . ( I ) of ATP - linked OCR from n = 6 experiments . Data were normalized to vehicle for each experiment .

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(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.