D . Time course showing - induced EYFP - Parkin accumulation in irradiated of neuronal soma of rat cortical neurons . were transfected with CFP - mito EYFP - Parkin visualized using a confocal microscope . Mitochondria were irradiated in a 0 . 9 µm x 0 . 9 µm - frame using a 405 - nm ( 25 iterations using 100 % power , pixel time 164 µs )

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D. Time course showing laser-induced EYFP-Parkin accumulation in the irradiated area of the neuronal soma of primary rat cortical neurons. Cells were transfected with CFP-mito and EYFP-Parkin and visualized using a confocal microscope. Mitochondria were irradiated in a 0.9 µm x 0.9 µm-frame using a 405-nm laser line (25 iterations using 100% laser power, pixel time 164 µs)


E , F Miro1 - EF delays reduces irradiation - induced EYFP - Parkin translocation . ( E ) time course of translocation strength in control Miro1 - EF - expressing neurons . Difference between curves P = 0 . 0016 , two - way ANOVA . ( F ) Comparison at 60 - min time point ( * P < 0 . 05 , n = 6 dishes , at least 9 neurons per dish , t - ) .

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E, F Miro1-EF delays and reduces irradiation-induced EYFP-Parkin translocation. (E) The time course of translocation strength in control and Miro1-EF-expressing neurons. Difference between the curves P = 0.0016, two-way ANOVA. (F) Comparison at the 60-min time point (*P < 0.05, n = 6 dishes, at least 9 neurons per dish, t-test).


A . EYFP - Parkin translocation requires Ca2 + . PC6 plated in separated compartments of same dish maintained during experiment in Krebs containing 1 mM Ca2 + or in Ca2 + free Krebs containing 5 μM BAPTA - AM , were treated with A & O ( both 10 μM ) . ( left panels ) demonstrate that EYFP - Parkin translocation was significantly weaker at zero Ca2 + at 195 min after A & O treatment ( compared with 15 min ; note spatial heterogeneity values shown in photos ) . Spatial heterogeneity ( right panel ) demonstrated that Parkin translocation was significantly decelerated in zero Ca2 + conditions ( n = 25 - 27 ; difference between curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


B . EYFP - Parkin translocation accelerates in presence of L - type Ca2 + channel agonist Bay K 8644 ( 5 μM ; n = 19 - 21 ; difference between curves p < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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B. EYFP-Parkin translocation accelerates in the presence of the L-type Ca2+ channel agonist Bay K 8644 (5 μM; n = 19-21 cells; difference between the curves p < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into cytosol , visualised by increased GCaMP7b

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Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol, visualised by increased cytosolic GCaMP7b fluorescence


Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into cytosol Glutamate - induced Ca2 + entry was not affected by Miro1 WT or Miro1 EF overexpression ; n = 31 - 48 neurons per group ; ns , Kruskal - Wallis )

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Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol Glutamate-induced Ca2+ entry was not affected by Miro1 WT or Miro1 EF overexpression; n = 31-48 neurons per group; ns, Kruskal-Wallis test)


E , F Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) led to appearance of a few Parkin - positive strong co - localising with mitochondrial marker ( arrow ) , imaged 2 . 5 h after treatment ( E ) . Further demonstrated that expression of Miro1 - EF mutant decreased Miro1 WT increased number of neurons with EYFP - Parkin - positive mitochondria following glutamate treatment ( F ; n = 6 - 10 ; * * P < 0 . 01 * * * * P < 0 . 0001 , one - way ANOVA )

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E, F Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) led to the appearance of a few Parkin-positive strong puncta co-localising with mitochondrial marker (white arrow), imaged 2.5 h after treatment (E). Further analysis demonstrated that the expression of Miro1-EF mutant decreased and Miro1 WT increased the number of neurons with EYFP-Parkin-positive mitochondria following glutamate treatment (F; n = 6-10; **P < 0.01 and ****P < 0.0001, one-way ANOVA)


G . Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) was associated with an increased number of mitochondrial co - localisation with autophagosome marker EGFP - LC3B in neuronal soma at 5 - 6 h later , which was not observed in Miro1 - EF - expressing neurons ( n = 8 dishes ; * P < 0 . 05 , one - way ANOVA )

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G. Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) was associated with an increased number of mitochondrial co-localisation with the autophagosome marker EGFP-LC3B in neuronal soma at 5-6 h later, which was not observed in Miro1-EF-expressing neurons (n = 8 dishes; *P < 0.05, one-way ANOVA)


H . Glutamate treatment ( 30 μM glutamate 1 μM glycine for 10 min ) was also associated with a significant loss of mitochondrial density in axons at 6 h later , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 60 - 90 axons from 6 - 9 dishes ; * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , Kruskal - Wallis )

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H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test)


I . Glutamate treatment ( 100 μM glutamate 10 μM glycine for 10 min ) led to a decline , at 6 - 8 h , in ATP measured using ATP / ADP sensor Perceval HR , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 59 - 60 neurons from 6 dishes ; * * P < 0 . 01 , Welch " s

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

I. Glutamate treatment (100 μM glutamate and 10 μM glycine for 10 min) led to a decline, at 6-8 h, in cytosolic ATP measured using ATP/ADP sensor Perceval HR, which was reversed by Miro1-EF but not by Miro1 WT (n = 59-60 neurons from 6 dishes; **P < 0.01, Welch"s A


J . Glutamate ( 100 μM glutamate 10 μM glycine for 10 min ) induced excitotoxicity ( 24 h after treatment ) that was partially reversed by Miro1 - EF but not by Miro1 WT . Data is presented as a of control neuron ( n = 10 dishes ; * * * P < 0 . 001 , repeated measures ANOVA ) .

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J. Glutamate (100 μM glutamate and 10 μM glycine for 10 min) induced excitotoxicity (24 h after treatment) that was partially reversed by Miro1-EF but not by Miro1 WT. Data is presented as a percentage of control neuron count (n = 10 dishes; ***P < 0.001, repeated measures ANOVA).


A microfluidic device for quantitative recovery time measurements . Each PDMS - fabricated chip consists of six independent channels , one of which is shown , from top view ( top row ) . Green circles indicate media inflow ; black circle indicates outflow . are manually loaded in inflow ports are trapped in culturing chambers ( gray circles ) , height of which ensures that are imaged in a monolayer ( bottom row ) . Growth conditions ( e . g . antibiotic dose profile ) are controlled via programmable syringe pumps . ( bottom row ) show representative growth of a monolayer of E . coli BW25113 constitutively expressing GFP over four hours .

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A microfluidic device for quantitative recovery time measurements. Each PDMS-fabricated chip consists of six independent channels, one of which is shown, from top view (top row). Green circles indicate media inflow; black circle indicates outflow. Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers (gray circles), the height of which ensures that bacteria are imaged in a monolayer (bottom row). Growth conditions (e.g. antibiotic dose profile) are controlled via programmable syringe pumps. Fluorescent images (bottom row) show representative growth of a monolayer of E. coli BW25113 cells constitutively expressing GFP over four hours.


Representative time series data showing dose - dependent population recovery in response to transient streptomycin treatment . Here , time zero corresponds to end of treatment ( 120 minutes ) . Trajectories show mean standard deviation for five replicates ; colors indicate increasing antibiotic ( 2 , 4 , 6 , 8 , 10 , 12 μg / mL ) . values are normalized to those at time zero . Dotted indicates a 2 - fold increase , corresponding to recovery time for each population .

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Representative time series fluorescence data showing dose-dependent population recovery in response to transient streptomycin treatment. Here, time zero corresponds to the end of treatment (120 minutes). Trajectories show mean and standard deviation for five replicates; colors indicate increasing antibiotic concentration (2, 4, 6, 8, 10, 12 μg/mL). Fluorescence values are normalized to those at time zero. Dotted line indicates a 2-fold increase, corresponding to the recovery time for each population.


Recovery time increases with total antibiotic exposure . Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations ( as in panel C ) . When plotted against total antibiotic exposure ( calculated as ) , recovery time values collapse onto a single exponential function ( black , R2 = 0 . 83 ) . Recovery time was measured in response to streptomycin concencentrations of 0 , 2 , 4 , 6 , 8 , 10 , 12 μg / mL , treatment durations 30 , 60 , 90 , 120 minutes .

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Recovery time increases with total antibiotic exposure. Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations (as in panel C). When plotted against total antibiotic exposure (calculated as ), all recovery time values collapse onto a single exponential function (black line, R2 = 0.83). Recovery time was measured in response to streptomycin concencentrations of 0, 2, 4, 6, 8, 10, and 12 μg/mL, and treatment durations 30, 60, 90, and 120 minutes.


Streptomycin treatment ( closed data points , R2 = 0 . 83 ) results in significantly longer recovery times than chloramphenicol treatment ( open data points ) . Here , difference between two responses is statistically significant ( p < 0 . 05 by ANOVA ) .

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Streptomycin treatment (closed data points, R2 = 0.83) results in significantly longer recovery times than chloramphenicol treatment (open data points). Here, the difference between two responses is statistically significant (p<0.05 by ANOVA).


addition of efflux pump inhibitor ( CCCP ) ( closed data points , R2 = 0 . 91 ) increases population recovery time in response to chloramphenicol treatment ( open data points , R2 = 0 . 80 ) . Here , CCCP was added at subinhibitory concentrations ( 3μg / mL ) ; in absence of antibiotic treatment , CCCP alone did not inhibit population recovery . CCCP - mediated increase in recovery time is statistically significant ( p < 0 . 01 ) .

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The addition of efflux pump inhibitor (CCCP) (closed data points, R2 = 0.91) increases population recovery time in response to chloramphenicol treatment (open data points, R2 = 0.80). Here, CCCP was added at subinhibitory concentrations (3μg/mL); in the absence of antibiotic treatment, CCCP alone did not inhibit population recovery. The CCCP-mediated increase in recovery time is statistically significant (p < 0.01).


ribosome synthesis rate was increased by increasing of casamino in media from 0 . 01 % w / v ( closed data points ) to 0 . 05 % w / v ( open data points ) . Faster synthesis resulted in lower recovery times ( p < 0 . 001 ) in response to streptomycin treatment . In each of these panels , color scheme indicates increasing antibiotic , as in Figure 1C .

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The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0.01% w/v (closed data points) to 0.05% w/v (open data points). Faster synthesis resulted in lower recovery times (p<0.001) in response to streptomycin treatment. In each of these panels, the color scheme indicates increasing antibiotic concentration, as in Figure 1C.


Inhibiting antibiotic efflux with CCCP significantly increased recovery time for that induced rapid target degradation involved a positive feedback loop . are as follows : penicillin G ( PenG ) , spectinomycin ( Spec ) , gentamicin ( Gent ) , streptomycin ( Str ) , chloramphenicol ( Cm ) , tetracyclin ( Tet ) , carbenicillin ( Carb ) . Antibiotic concentrations used have been scaled by their respective IC50 values ( solid points open circles show response with no efflux inhibition 2μg / mL CCCP , respectively ) . Colors correspond to motifs of action shown in Figure 4A .

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Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop. Antibiotics are as follows: penicillin G (PenG), spectinomycin (Spec), gentamicin (Gent), streptomycin (Str), chloramphenicol (Cm), tetracyclin (Tet), and carbenicillin (Carb). Antibiotic concentrations used have been scaled by their respective IC50 values (solid points and open circles show response with no efflux inhibition and 2μg/mL CCCP, respectively). Colors correspond to the motifs of action shown in Figure 4A.


a , Silver of LC3 complexes purified from U2OS expressing ZZ - Flag - LC3 in normal medium or Earle ' s balanced salt solution ( EBSS ) for 2 h . Asterisks mark centriolar satellite .

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a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ-Flag-LC3 in normal medium or Earle's balanced salt solution (EBSS) for 2 h. Asterisks mark centriolar satellite proteins.


b - d , Co - immunoprecipitation ( IP ) of OFD1 with LC3 , LC3 with PCM1 , or OFD1 with PCM1 in HEK293T . HA , haemagglutinin .

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b-d, Co-immunoprecipitation (IP) of OFD1 with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, haemagglutinin.


e , Western blotting of PCM1 OFD1 in control or PCM1 knockdown ( KD ) HEK293T .

e, Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown (KD) HEK293T cells.


f , Co - immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T . IP efficiency is calculated as of immunoprecipitated OFD1 / LC3 .

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f, Co-immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3.


g , h , Western blotting of of indicated in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation ( SS ) . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

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g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation (SS). Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


g , h , Western blotting of of indicated in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation ( SS ) , 50 nM bafilomycin A1 ( Baf ) , 20 µM chloroquine ( CQ ) or 1 µM MG132 . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

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g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


a , Representative confocal of endogenous OFD1 with cilium marker acetylated tubulin ( Ac - tubulin ) , or centriole marker γ - tubulin in hTERT - RPE1 in normal medium ( Un ) or subjected to 24 hserum starvation ( SS ) .

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a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker γ-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 hserum starvation (SS).


b , Representative confocal of endogenous OFD1 acetylated tubulin in Atg5 + / + Atg5 − / − MEFs subjected to 24 hserum starvation . a , b , Data shown represent mean ± s . d . of with centriolar satelliteOFD1 for 100 per well in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - . Similar results were observed in three independent experiments .

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b, Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5+/+ and Atg5−/− MEFs subjected to 24 hserum starvation. a, b, Data shown represent mean ± s.d. percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments.


a , b , Representative confocal of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation . Data shown represent mean ± s . d . of with cilia or of cilia for 500 or 100 per well , respectively , in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - . Similar results were observed in three independent experiments . Un , untreated ; SS , serum - starved .

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a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate samples. ***P  0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments. Un, untreated; SS, serum-starved.


a , Western blotting of OFD1 in control or OFD1 knockdown MCF7 ( C - 19 ) , quantified OFD1 level was normalized with alpha - tubulin .

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a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with alpha-tubulin.


b , of of with cilia in MCF7 or C19 subjected to 72 h serum starvation . Data shown represent mean ± s . d . of with cilia for 500 per well in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - .

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b, Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia for 500 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test.


c , of of cilia in C19 subject to 48 h or 72 h serum starvation .

c, Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation.


d , e , Representative confocal of cilia with variable formed in MCF7 or C19 subject to 72 h serum starvation . Data shown represent 100 per well in triplicate samples .

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d, e, Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. Data shown represent 100 cells per well in triplicate samples.


d , e , Representative confocal of cilia with variable formed in C19 subject to 72 hserum starvation . Data shown represent 100 per well in triplicate samples .

d, e, Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation. Data shown represent 100 cells per well in triplicate samples.


f , Scanning electron microscope of cilia ( marked by arrows ) formed in C - 19 subject to 72 h serum starvation cycling Atg5 + / + MEFs in normal medium for 24 h . Similar results were observed in three independent experiments .

f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.


( A ) PI3Kγ is expressed by human hepatocytes infiltrating immune in biopsies from with minimal to mild inflammatory activity . Triangles point to immune ( clusters ) , including some neutrophils known to express PI3Kγ highly . In negative control , antibody was replaced by an equal volume buffer . number of included , gender , diagnosis are summarized

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(A) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity. Triangles point to immune cells (clusters), including some neutrophils known to express PI3Kγ highly. In the negative control, the primary antibody was replaced by an equal volume buffer. The number of included patients, gender, and diagnosis are summarized


( A ) PI3Kγ is expressed by human hepatocytes infiltrating immune in biopsies from with minimal to mild inflammatory activity . Triangles point to immune ( clusters ) , including some neutrophils known to express PI3Kγ highly . In negative control , antibody was replaced by an equal volume buffer . number of included , gender , diagnosis are

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( B ) PI3Kγ expression in human hepatocytes from 20 ( ♂ ) or ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . characterization of donor pools is summarized

(B) PI3Kγ expression in human primary hepatocytes from 20 male (♂) or female (♀) donor pools (HEP, DP20), but not non-parenchymal cells (NPC). Isolated human leukocytes from healthy volunteers (LEU) served as positive controls. The characterization of the donor pools is summarized


( B ) PI3Kγ expression in human hepatocytes from 20 ( ♂ ) or ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . characterization of donor pools is

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( D ) Survival of WT , PI3Kγ null ( left ) liver - specific PI3Kγ knockout mice ( PI3Kγ floxtg / tg x AlbCre ( tg ) / tg , middle ) or systemic application of PI3Kγ inhibitor , AS605240 ( right ) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension . numbers : B6 : 86 ( % : 52 , % : 48 ) , B6 - Pi3kcgko / ko : 59 ( % : 51 , % 49 ) , B6 - Pi3kcgflox / flox x AlbCre ( tg ) / tg : 25 ( % : 68 , % 32 ) , FVB / N Vehicle : 25 ( % : 44 , % 56 ) , FVB / N AS : 43 ( % : 44 , % 56 )

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(D) Survival of WT, PI3Kγ null (left) and liver-specific PI3Kγ knockout mice (PI3Kγ floxtg/tg x AlbCre(tg)/tg, middle) or systemic application of the PI3Kγ inhibitor, AS605240 (right) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension. Animal numbers: B6: 86 (%male: 52, %female: 48), B6-Pi3kcgko/ko: 59 (%male: 51, %female 49), B6-Pi3kcgflox/flox x AlbCre(tg)/tg: 25 (%male: 68, %female 32), FVB/N Vehicle: 25 (%male: 44, %female 56), FVB/N AS: 43 (%male: 44, %female 56)


( C ) Uptake characteristics of T - LipoAS by human hepatocytes demonstrate competitive inhibition by cyclosporin A - energy dependence , confirming active uptake by organic anion transporters ( OATPs ) . data points are depicted mean ± SD from two batches of human hepatocytes in quadruples .

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(C) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples.


( F ) T - LipoAS effectively inhibits fMLP - stimulated PI3Kγ , reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes . mean pAKT / AKT standard deviation ( SD ) were calculated for experiments separately .

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(F) T-LipoAS effectively inhibits fMLP-stimulated PI3Kγ, reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes. The mean pAKT/AKT ratio and standard deviation (SD) were calculated for all experiments separately.


( G ) Episcopic imaging of mice treated with T - LipoAS for 15 or 60 min relative of targeting moiety DY - 635 in different compartments .

(G) Episcopic imaging of mice treated with T-LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY-635 in the different compartments.


( B Pro - anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination infection ( PCI ) sham . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

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(B Pro- anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


C ) anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination infection ( PCI ) sham . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

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C) anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


( F , G ) of ( F ) neutrophils ( G ) monocytes in 2 mL peritoneal lavage .

(F,G) Concentration of (F) neutrophils and (G) monocytes in 2 mL peritoneal lavage.


( A ) Intravital microscopy of elimination of CDFDA , a substrate subject to excretory elimination by hepatocytes via Mrp - 2 , allows direct visualization of excretory function in sham or septic ( PCI ) treated with vehicle or a hepatocyte - directed inhibitor of PI3Kγ ( T - LipoAS ) .

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(A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS).


( A ) GFP - PAQR3 transfected HeLa were fixed for immunofluorescence with indicated antibodies before or after glucose starvation ( GS ) for 4 h . nuclei were stained with Hoechst 33342 . Scale bar : 10 μm .

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(A) GFP-PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation (GS) for 4 h. The nuclei were stained with Hoechst 33342. Scale bar: 10 μm.


( B ) Immunoblotting ( IB ) of wild type ( WT ) PAQR3 - deficient MEFs after GS for different time as indicated .

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(B) Immunoblotting (IB) analysis of wild type (WT) and PAQR3-deficient MEFs after GS for different time as indicated.


( C ) WT PAQR3 - deficient MEFs were treated with 80 mM chloroquine ( CQ ) for 2 h to induce LC3 - II accumulation . Whole - lysates were harvested for IB of LC3 - II .

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(C) WT and PAQR3-deficient MEFs were treated with 80 mM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole-cell lysates were harvested for IB analysis of LC3-II.


( D ) Autophagosome - like structures in WT PAQR3 - deleted MEFs were detected after GS for 4 h by transmission electron microscopy . * indicates autophagosome - like structures . N , nucleus . ( E ) Quantitative of autophagic vacuoles in transmission electron microscopy . Thirty were quantified from each independent experiment ， which was repeated for three times with similar results . Values are presented as mean ± SD , * * * p < 0 . 001 .

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(D) Autophagosome-like structures in WT and PAQR3-deleted MEFs were detected after GS for 4 h by transmission electron microscopy. * indicates autophagosome-like structures. N, nucleus.(E) Quantitative analysis of autophagic vacuoles in transmission electron microscopy images. Thirty cells were quantified from each independent experiment，which was repeated for three times with similar results. Values are presented as mean ± SD, ***p < 0.001.


( F ) Immunofluorescence of LC3 in WT or PAQR3 - deficient MEFs after GS for 4 h . arrowhead indicates representative LC3 - positive . Scale bar : 10 μm .

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(F) Immunofluorescence staining of LC3 in WT or PAQR3-deficient MEFs after GS for 4 h. The arrowhead indicates representative LC3-positive puncta. Scale bar: 10 μm.


( G , H ) HeLa were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control . After GS for 1 h , lysates were harvested for IB .

(G, H) HeLa cells were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control virus. After GS for 1 h, the cell lysates were harvested for IB.


( A ~ C ) HeLa stably expressing GFP - DFCP1 were transfected with or without PAQR3 - specific shRNA . GFP signals were observed in these before or after glucose starvation ( GS , shown in A ) . knockdown efficiency of PAQR3 is shown in B statistics of GFP - positive per is illustrated in C . Values are presented as mean ± SD , * * * P < 0 . 001 . Scale bar : 10 μm .

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(A~C) HeLa cells stably expressing GFP-DFCP1 were transfected with or without PAQR3-specific shRNA. GFP signals were observed in these cells before or after glucose starvation (GS, shown in A). The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP-positive puncta per cell is illustrated in C. Values are presented as mean ± SD, *** P < 0.001. Scale bar: 10 μm.


( D ) GFP - WIPI1 was transfected into WT or PAQR3 - deficient MEFs . 24 h later , were incubated under GS for 4 h , followed by observation . Scale bar : 10 μm .

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(D) GFP-WIPI1 was transfected into WT or PAQR3-deficient MEFs. 24 h later, the cells were incubated under GS for 4 h, followed by fluorescence observation. Scale bar: 10 μm.


( E ) Different VPS34 complexes of WT or PAQR3 knockout HeLa were immunoprecipitated by ATG14L UVRAG antibodies respectively in normal medium ( NM ) or GS . Then PI ( 3 ) P level was determined by a quantitative ELISA . PI ( 3 ) P level was normalized to amount of ATG14L or UVRAG used in ( n = 5 ; * * * p < 0 . 001 ; ns , not significant ) .

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(E) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium (NM) or GS. Then PI(3)P level was determined by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L or UVRAG used in the assay (n = 5; ***p < 0.001; ns, not significant).


( F ) HeLa infected with control or PAQR3 - overexpressed lentivirus were treated with GS for 4 h . PI ( 3 ) P were determined as in Figure 2E ( n = 5 ; * * p < 0 . 01 ; ns , not significant ) .

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(F) HeLa cells infected with control or PAQR3-overexpressed lentivirus were treated with GS for 4 h. PI(3)P levels were determined as in Figure 2E(n = 5; **p < 0.01; ns, not significant).


( G , H ) PAQR3 knockout HeLa were transfected with plasmids of multiple PAQR3 deletion mutants . At 24 h after transfection , lysates were used to immunoprecipitate VPS34 by ATG14L antibody , followed by immunoblotting . activity of ATG14L - linked VPS34 activity was detected by a quantitative ELISA . PI ( 3 ) P level was normalized to amount of ATG14L ( n = 5 ; * * p < 0 . 01 ) .

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(G, H) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants. At 24 h after the transfection, the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody, followed by immunoblotting. The activity of ATG14L-linked VPS34 activity was detected by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; **p < 0.01).


( A ) Interaction between endogenous PAQR3 components of VPS34 in HEK293T . lysates were used for immunoprecipitation ( IP ) immunoblotting ( IB ) with antibodies as indicated .

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(A) Interaction between endogenous PAQR3 and the components of VPS34 complex in HEK293T cells. The cell lysates were used for immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.


( B ) Cytosol Golgi fractionations were isolated from livers of PAQR3 - deleted mice their littermate controls . Equal amounts of cytosol Golgi fractions were subjected to IB .

(B) Cytosol and Golgi fractionations were isolated from the livers of PAQR3-deleted mice and their littermate controls. Equal protein amounts of cytosol and Golgi fractions were subjected to IB.


( C ) HEK293T were transiently transfected with Myc - tagged PAQR3 . At 24 h after transfection , were treated with normal medium ( NM ) ， acid starvation ( AS ) or glucose starvation ( GS ) , HBSS solution , or rapamycin ( Rapa , 50 nM ) for 4 h respectively . lysates were then used in IP IB .

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(C) HEK293T cells were transiently transfected with Myc-tagged PAQR3. At 24 h after transfection, the cells were treated with normal medium (NM)，amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h respectively. The cell lysates were then used in IP and IB.


( D ) Four VPS34 complexes of MEFs were immunopurified using indicated antibodies under NM , AS or GS conditions . relative abundance of VPS34 - binding partners was determined by IB . Each IP was normalized to amount of VPS34 .

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(D) Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM, AS or GS conditions. The relative abundance of VPS34-binding partners was determined by IB. Each IP was normalized to the amount of VPS34.


( E ) HEK293T were transfected with plasmids as indicated lysates were used in IP IB .

(E) HEK293T cells were transfected with the plasmids as indicated and the cell lysates were used in IP and IB.


( F ) lysates of WT or PAQR3 knockout MEFs were used in IP IB .

(F) The lysates of WT or PAQR3 knockout MEFs were used in IP and IB.


( G ) MEFs were infected with control or PAQR3 - expressing lentivirus . Then a quantitative immunodepletion was performed with increasing amounts of indicated antibodies . Supernatants of MEF lysates after immunodepletion ( Post - IP ) were examined to determine level of VPS34 .

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(G) MEFs were infected with control or PAQR3-expressing lentivirus. Then a quantitative immunodepletion assay was performed with increasing amounts of the indicated antibodies. Supernatants of MEF lysates after immunodepletion (Post-IP) were examined to determine the level of VPS34 complex proteins.


( A ) A two - step co - IP in HEK293T to determine ternary containing PAQR3 , Beclin1 ATG14L . procedures of two - step co - IP are outlined in top . samples were subjected to immunoprecipitation ( IP ) immunoblotting ( IB ) with antibodies as indicated .

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(A) A two-step co-IP assay in HEK293T cells to determine the ternary complex containing PAQR3, Beclin1 and ATG14L. The procedures of the two-step co-IP are outlined in the top. The samples were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.


( B ) HeLa were transiently transfected with plasmids as indicated . 24 h after transfection , were treated with or without glucose starvation ( GS ) for 4 h . lysates were used in IP IB .

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(B) HeLa cells were transiently transfected with the plasmids as indicated. 24 h after transfection, the cells were treated with or without glucose starvation (GS) for 4 h. The cell lysates were used in IP and IB.


( C , D ) Different truncations of ATG14L ( C ) were co - transfected with Myc - tagged PAQR3 into HEK293T as indicated , followed by IP IB .

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(C, D) Different truncations of ATG14L (C) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.


( C , D ) Different truncations of Beclin1 ( D ) were co - transfected with Myc - tagged PAQR3 into HEK293T as indicated , followed by IP IB .

(C, D) Different truncations of Beclin1 (D) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.


( E ) Purified GST or GST - fused PAQR3 ( 1 - 71 ) ( GST fused NH2 - terminal 71aa of PAQR3 ) on glutathione agarose beads was mixed with purified His tagged as indicated . After incubation at 4 ° C for 3 h , samples were washed extensively subjected to IB .

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(E) Purified GST or GST-fused PAQR3 (1-71) (GST fused NH2-terminal 71aa of PAQR3) on glutathione agarose beads was mixed with purified His tagged proteins as indicated. After incubation at 4 °C for 3 h, the samples were washed extensively and subjected to IB.


( F , G ) Different PAQR3 deletion constructs were co - transfected with Flag - tagged Beclin1 ( F ) into HEK293T . were then subjected to IP IB .

(F, G) Different PAQR3 deletion constructs were co-transfected with Flag-tagged Beclin1 (F) into HEK293T cells. The cells were then subjected to IP and IB.


( F , G ) Different PAQR3 deletion constructs were co - transfected with GFP - fused ATG14L ( G ) into HEK293T . were then subjected to IP IB .

(F, G) Different PAQR3 deletion constructs were co-transfected with GFP-fused ATG14L (G) into HEK293T cells. The cells were then subjected to IP and IB.


( A ) HeLa were cultured in normal medium ( NM ) or glucose starvation ( GS ) for 4 h . starved lysates were treated with calf intestinal phosphatase ( CIP ) to remove phosphate groups analyzed by immunoblotting ( IB ) with Phos - tag gel or regular SDS - PAGE .

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(A) HeLa cells were cultured in normal medium (NM) or glucose starvation (GS) for 4 h. The starved lysates were treated with calf intestinal phosphatase (CIP) to remove phosphate groups and analyzed by immunoblotting (IB) with Phos-tag gel or regular SDS-PAGE.


( B ) lysates from HeLa were collected analyzed after GS for indicated time ( lane 1 ~ 5 ) , or GS for 2 h followed by 2 h chase in NM ( lane 6 ) . lysates were subjected to Phos - tag gel regular SDS PAGE respectively .

(B) The cell lysates from HeLa cells were collected and analyzed after GS for indicated time (lane 1~5), or GS for 2 h followed by 2 h chase in NM (lane 6). The cell lysates were subjected to Phos-tag gel and regular SDS PAGE respectively.


( C ) WT or AMPKa1 / 2 double knockout MEFs were incubated under NM or GS for 4 h , followed by IB with Phos - tag gel or regular SDS - PAGE .

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(C) WT or AMPKa1/2 double knockout MEFs were incubated under NM or GS for 4 h, followed by IB with Phos-tag gel or regular SDS-PAGE.


( D ) HeLa were incubated with compound C ( 20 mM , 30 min ) with or without GS , followed by IB with Phos - tag gel or regular SDS - PAGE .

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(D) HeLa cells were incubated with compound C (20 mM, 30 min) with or without GS, followed by IB with Phos-tag gel or regular SDS-PAGE.


( E ) purified His - tagged NH2 - terminal 71 acid of PAQR3 was incubated with or without purified Flag - tagged ATG14L . Then complexes were treated with WT AMPK or DN - AMPK ( dominant negative AMPK ) for indicated time in vitro . PAQR3 phosphorylation was examined by Phos - tag gel .

(E) Bacterial purified His-tagged NH2-terminal 71 amino acid of PAQR3 was incubated with or without purified Flag-tagged ATG14L. Then the complexes were treated with WT AMPK or DN-AMPK (dominant negative AMPK) for the indicated time in vitro. PAQR3 phosphorylation was examined by Phos-tag gel.


( F ) HeLa infected with control or ATG14L - expressing lentivirus were incubated under GS for different time as indicated . Whole lysates were analyzed by both Phos - tag gel regular SDS - PAGE .

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(F) HeLa cells infected with control or ATG14L-expressing lentivirus were incubated under GS for different time as indicated. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.


( G ) HeLa were infected by lentivirus expressing control shRNA or ATG14L - specific shRNA . Then ATG14L knockdown were stably re - expressed with shRNA - resistant ATG14L ( rATG14L ) or Beclin1 incubated under NM or GS for 4 h . Whole lysates were analyzed by both Phos - tag gel regular SDS - PAGE .

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(G) HeLa cells were infected by lentivirus expressing control shRNA or ATG14L-specific shRNA. Then the ATG14L knockdown cells were stably re-expressed with shRNA-resistant ATG14L (rATG14L) or Beclin1 and incubated under NM or GS for 4 h. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.


( A ) purified His - tagged WT or T32A NH2 - terminal 71aa of PAQR3 was incubated with or without Flag - tagged ATG14L purified from HEK293T . Then complexes were incubated with AMPK for indicated time subjected to Phos - tag gel or regular SDS - PAGE .

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(A) Bacterial purified His-tagged WT or T32A NH2-terminal 71aa of PAQR3 was incubated with or without Flag-tagged ATG14L purified from HEK293T cells. Then the complexes were incubated with AMPK for the indicated time and subjected to Phos-tag gel or regular SDS-PAGE.


( B ) PAQR3 - deficient HeLa were transfected with WT or T32A mutants of PAQR3 plasmids . 24 h later , were incubated with normal medium ( NM ) or glucose starvation ( GS ) for 4 h , followed by immunoblotting ( IB ) in Phos - tag gel or regular SDS - PAGE .

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(B) PAQR3-deficient HeLa cells were transfected with WT or T32A mutants of PAQR3 plasmids. 24 h later, the cells were incubated with normal medium (NM) or glucose starvation (GS) for 4 h, followed by immunoblotting (IB) in Phos-tag gel or regular SDS-PAGE.


( C ) HeLa were incubated with NM or GS for 1 h or treated with compound C ( C . C , 20 mM , 30 min ) prior to GS for 1 h . In parallel , 25 mM 2 - DG or 1 mM AICAR was added in NM for 1 h . lysates were analyzed by IB .

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(C) HeLa Cells were incubated with NM or GS for 1 h or treated with compound C (C.C, 20 mM, 30 min) prior to GS for 1 h. In parallel, 25 mM 2-DG or 1 mM AICAR was added in NM for 1 h. Cell lysates were analyzed by IB.


( D ) HEK293T were transfected with Myc - tagged PAQR3 . 24 h after transfection , were treated with GS for 4 h . Then lysates were used in IB immunoprecipitation ( IP ) with antibodies as indicated . immunoprecipitates were treated with CIP to remove phosphate groups analyzed by IB in Phos - tag gel or regular SDS - PAGE .

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(D) HEK293T cells were transfected with Myc-tagged PAQR3. 24 h after transfection, the cells were treated with GS for 4 h. Then the cell lysates were used in IB and immunoprecipitation (IP) with the antibodies as indicated. The immunoprecipitates were treated with CIP to remove phosphate groups and analyzed by IB in Phos-tag gel or regular SDS-PAGE.


( E ) WT , phosphorylation defective ( T32A ) , or phospho - mimetic ( T32E ) PAQR3 were transfected in presence or absence of constitutively active AMPK ( CA - AMPK ) into PAQR3 - deficient HeLa as indicated . 24 h later , without AMPK transfection were treated with GS for 4 h . 10 % of lysates were subjected to IB to detect PAQR3 expression level ( shown in Appendix Fig S4A ) , remaining lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody , followed by VPS34 activity measurement by a quantitative PI ( 3 ) P ELISA . PI ( 3 ) P level was normalized to amount of ATG14L ( n = 5 ; * p < 0 . 05 ; * * p < 0 . 01 as compared to first group ) .

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

(E) WT, phosphorylation defective (T32A), or phospho-mimetic (T32E) PAQR3 were transfected in the presence or absence of constitutively active AMPK (CA-AMPK) into PAQR3-deficient HeLa cells as indicated. 24 h later, the cells without AMPK transfection were treated with GS for 4 h. 10% of the cell lysates were subjected to IB to detect PAQR3 expression level (shown in Appendix Fig S4A), the remaining cell lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody, followed by VPS34 activity measurement by a quantitative PI(3)P ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; *p < 0.05; **p < 0.01 as compared to the first group).


( F ) PAQR3 - deficient HeLa were infected with lentivirus expressing WT or T32A PAQR3 . Then PAQR3 expression were examined by IB .

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(F) PAQR3-deficient HeLa cells were infected with lentivirus expressing WT or T32A PAQR3. Then PAQR3 expression levels were examined by IB.


( G ) Both WT HeLa PAQR3 knockout HeLa infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively . lysates were analyzed by IB .

(G) Both WT HeLa cells and PAQR3 knockout HeLa cells infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively. Cell lysates were analyzed by IB.


( A , B ) Representative immunohistochemical of LC3 in skeletal muscle ( A ) liver ( B ) after 80 min exercise in WT PAQR3 - deleted mice . Scale bar in ( A ) : 20 μm . Scale bar in ( B ) : 50 μm .

(A, B) Representative immunohistochemical images of LC3 staining in skeletal muscle (A) and liver (B) after 80 min exercise in WT and PAQR3-deleted mice. Scale bar in (A): 20 μm. Scale bar in (B): 50 μm.


( C , D ) Immunoblotting of skeletal muscle ( C ) liver ( D ) from WT or PAQR3 knockout mice at or after 80 min exercise .

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(C, D) Immunoblotting analysis of skeletal muscle (C) and liver (D) from WT or PAQR3 knockout mice at rest or after 80 min exercise.


( E ) Abnormal limb - clasping of PAQR3 knockout mice when suspended by its tail . scoring of limb clasping phenotype is shown on right ( n = 7 for each group ; * * p < 0 . 01 ) .

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(E) Abnormal limb-clasping of PAQR3 knockout mice when suspended by its tail. Quantification scoring of the limb clasping phenotype is shown on the right (n = 7 for each group; **p < 0.01).


( F ) Motor performance was measured using an accelerating rotarod apparatus , time of each mouse spent on rotating rod until it fell off was recorded . Average time on rod for each group is shown ( n = 7 for each group ; * * p < 0 . 01 ) .

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(F) Motor performance was measured using an accelerating rotarod apparatus, and the time of each mouse spent on the rotating rod until it fell off was recorded. Average time on the rod for each group is shown (n = 7 for each group; **p < 0.01).


( G ) Co - immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype . Immunoprecipitation ( IP ) immunoblotting ( IB ) were performed using antibodies as indicated .

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(G) Co-immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype. Immunoprecipitation (IP) and immunoblotting (IB) were performed using the antibodies as indicated.


( H ) supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration , followed by IB .

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(H) The supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration analysis, followed by IB.


( I ) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI ( 3 ) P ELISA ( n = 5 ; * * p < 0 . 01 ) .

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(I) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI(3)P ELISA assay (n = 5; **p < 0.01).


A . Labeling of non - permeabilized ( top ) permeabilized ( bottom ) demonstrate that surface labeling does not label internal . GluA2 labeling ( green ) is visible both on surface internally , while DARPP32 labeling ( red ) is only visible internally .

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A. Labeling of non-permeabilized (top) and permeabilized (bottom) cells demonstrate that surface labeling does not label internal proteins. GluA2 labeling (green) is visible both on the surface and internally, while DARPP32 labeling (red) is only visible internally.


B . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no treatment or 20 min 2APB dantrolene . Bottom . Quantified expression of GluA2 under both conditions , n = 9 replicates of 10 each , 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 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

B. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no treatment or 20 min 2APB and dantrolene. Bottom. Quantified expression of GluA2 under both conditions, n = 9 replicates of 10 cells each, Student's t test.


C . Representative of surface GluA1 DARPP32 expression in striatal neurons from co - cultures with either no treatment , 30 min DHPG , or 2APB dantrolene pretreatment followed by DHPG . D . Quantified expression of GluA1 under each condition , n = 9 replicates of 10 , p < 0 . 001 , ANOVA . E . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures under same conditions as in C . F . Quantified expression of GluA2 under each condition , n = 12 replicates of 10 each , p < 0 . 001 , ANOVA . G . Representative of surface myc - GluA2 , internal myc - GluA2 , DARPP32 expression in striatal neurons from co - cultures under same conditions as in C . H . Quantified expression of surface myc : total myc under each condition , n = 4 replicates of 10 each , p < 0 . 05 , ANOVA . error bars represent s . e . m . Abbr . A1 = GluA1 , A2 = GluA2 , D = DARPP32 , s - myc = surface myc , i - myc = internal myc , m = merge .

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 I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 surface GluA1 and DARPP32 expression in striatal neurons from co-cultures with either no treatment, 30 min DHPG, or 2APB and dantrolene pretreatment followed by DHPG. D. Quantified expression of GluA1 under each condition, n = 9 replicates of 10 cells, p < 0.001, ANOVA. E. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. F. Quantified expression of GluA2 under each condition, n = 12 replicates of 10 cells each, p < 0.001, ANOVA. G. Representative images of surface myc-GluA2, internal myc-GluA2, and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. H. Quantified expression of surface myc:total myc under each condition, n = 4 replicates of 10 cells each, p < 0.05, ANOVA. All error bars represent s.e.m. Abbr. A1 = GluA1, A2 = GluA2, D = DARPP32, s-myc = surface myc, i-myc = internal myc, m = merge.


A . Left . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures treated either for 30 min with DHPG ( DHPG ) or for 30 min with DHPG pretreated with Xestospungin C S107 ( XeC / S107 + DHPG ) . Right . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 or 20 each , p < 0 . 0001 , 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

A. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG) or for 30 min with DHPG and pretreated with Xestospungin C and S107 (XeC/S107 + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 or 20 cells each, p < 0.0001, Student's t test.


B . Left . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures treated either for 30 min with DHPG ( DHPG ) , or for 30 min with DHPG pretreated with S107 ( S107 + DHPG ) , or for 30 min with DHPG pretreated with Xestospungin C ( XeC + DHPG ) . Right . Quantified expression of surface GluA2 under each condition , n = 4 replicates of 10 each , ANOVA .

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

B. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG), or for 30 min with DHPG and pretreated with S107 (S107 + DHPG), or for 30 min with DHPG and pretreated with Xestospungin C (XeC + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, ANOVA.


C . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures under control or 10 min Thapsigargin ( TG ) treatment . Bottom . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 each , Student ' s t . D . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with treatment with either no DHPG or with DHPG for 30 min , after 15 min of Thapsigargin . Bottom . Quantified expression of surface GluA2 under each condition , n = 4 replicates of 10 each , Student ' s t . E . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no DHPG or with DHPG for 30 min , after 5 min of BAPTA . Bottom . Quantified expression of GluA2 under each condition , n = 3 replicates of 10 each , p < 0 . 001 , Student ' s t . F . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA - AM . Bottom . Quantified expression of GluA2 under each condition , n = 3 replicates of 10 each , Student ' s t . error bars represent 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 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-CONTROLLED_VAR 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 O O O O O O O

C. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under control or 10 min Thapsigargin (TG) treatment. Bottom. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. D. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with treatment with either no DHPG or with DHPG for 30 min, after 15 min of Thapsigargin. Bottom. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, Student's t test. E. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min, after 5 min of BAPTA. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, p < 0.001, Student's t test. F. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA-AM. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.


A . Top . Representative image of a radiograph of mature immature bands of GluA2 after no treatment , DHPG , or 2APB / dan followed by DHPG incubated with Endo H . Bottom . Quantified graph of of mature , endo H insensitive GluA2 band to total GluA2 for each condition . For DHPG , n = 16 ; for DHPG + 2APB / dan , n = 11 , p < 0 . 05 , ANOVA .

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

A. Top. Representative image of a radiograph of mature and immature bands of GluA2 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA2 band to the total GluA2 for each condition. For DHPG, n = 16; for DHPG + 2APB/dan, n = 11, p < 0.05, ANOVA.


B . Top . Representative image of a radiograph of mature immature bands of GluA1 after no treatment , DHPG , or 2APB / dan followed by DHPG incubated with Endo H . Bottom . Quantified graph of of mature , endo H insensitive GluA1 band to total GluA1 for each condition , For DHPG , n = 4 ; for DHPG + 2APB / dan , n = 4 , ANOVA .

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

B. Top. Representative image of a radiograph of mature and immature bands of GluA1 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA1 band to the total GluA1 for each condition, For DHPG, n = 4; for DHPG + 2APB/dan, n = 4, ANOVA.


C . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment . D . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 each , Student ' s t . error bars represent s . e . m .

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C. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment. D. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.


A Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 GluA2 after no treatment , 10 min DHPG , 2APB / dantrolene followed by 10 min DHPG . Bottom . of Sec23 normalized to GluA2 under each of these conditions . For 10 min DHPG , n = 8 ; for 10 min DHPG + 2APB / dan , n = 6 , p < 0 . 05 , ANOVA .

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A Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and 2APB/dantrolene followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 10 min DHPG, n = 8; for 10 min DHPG + 2APB/dan, n = 6, p < 0.05, ANOVA.


B . Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 GluA2 after no treatment , 10 min DHPG , cycloheximide followed by 10 min DHPG . Bottom . of Sec23 normalized to GluA2 under each of these conditions , For 10 min DHPG , n = 8 ; for 10 min DHPG + CHX , n = 5 , p < 0 . 05 , ANOVA .

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B. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and and cycloheximide followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions, For 10 min DHPG, n = 8; for 10 min DHPG + CHX, n = 5, p < 0.05, ANOVA.


C . Top . Representative immunoblots from GluA2 co - IPs probed for Sec23 GluA2 after no treatment , 30 min DHPG , 2APB / dantrolene followed by 30 min DHPG . Bottom . of Sec23 normalized to GluA2 under each of these conditions . For 30 min DHPG , n = 17 ; For 30 min DHPG + 2APB / dan , n = 14 , p < 0 . 05 , ANOVA .

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C. Top. Representative immunoblots from GluA2 co-IPs probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and 2APB/dantrolene followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 17; For 30 min DHPG + 2APB/dan, n = 14, p < 0.05, ANOVA.


D . Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 GluA2 after no treatment , 30 min DHPG , cycloheximide followed by 30 min DHPG . Bottom . of Sec23 normalized to GluA2 under each of these conditions . For 30 min DHPG , n = 12 ; For 30 min DHPG + CHX , n = 11 p < 0 . 01 , ANOVA .

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D. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and cycloheximide followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 12; For 30 min DHPG + CHX, n = 11 p < 0.01, ANOVA.


E . Top . Representative immunoblot demonstrating IgG GluA2 coIP after no treatment 30 min DHPG . Bottom . of Sec23 after subtracting non - specific signal , n = 3 , p < 0 . 01 , Student ' s t .

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E. Top. Representative immunoblot demonstrating IgG and GluA2 coIP after no treatment and 30 min DHPG. Bottom. Quantification of Sec23 after subtracting the non-specific signal, n = 3, p < 0.01, Student's t test.


F . Representative immunoblot of a control coIP that demonstrates specificity of GluA2 coIP . error bars represent s . e . m .

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F. Representative immunoblot of a control coIP that demonstrates the specificity of the GluA2 coIP. All error bars represent s.e.m.


A . Representative immunoblots from GluA2 coIP probed for Sec23 , Sec13 , GluA2 after no DHPG , 10 , 30 , 60 minutes of treatment with DHPG . B . Graph depicting kinetics of association between GluA2 Sec23 as well as GluA2 Sec13 after treatment with DHPG for same points in time as in A . For Sec23 , 10 min DHPG , n = 16 ; 30 min DHPG , n = 15 ; 60 min DHPG , n = 7 , p < 0 . 0001 , ANOVA . For Sec13 , 10 min DHPG , n = 7 ; 30 min DHPG , n = 14 ; 60 min DHPG , n = 6 , p < 0 . 01 , ANOVA .

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A. Representative immunoblots from GluA2 coIP probed for Sec23, Sec13, and GluA2 after no DHPG, 10, 30, and 60 minutes of treatment with DHPG. B. Graph depicting the kinetics of the association between GluA2 and Sec23 as well as GluA2 and Sec13 after treatment with DHPG for the same points in time as in A. For Sec23, 10 min DHPG, n = 16; 30 min DHPG, n = 15; 60 min DHPG, n = 7, p < 0.0001, ANOVA. For Sec13, 10 min DHPG, n = 7; 30 min DHPG, n = 14; 60 min DHPG, n = 6, p < 0.01, ANOVA.


C . Representative of total expression of Sec23 ( top ) Sec13 ( bottom ) . Expression do not change in presence of DHPG . error bars represent s . e . m .

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C. Representative images of total expression levels of Sec23 (top) and Sec13 (bottom). Expression levels do not change in the presence of DHPG. All error bars represent s.e.m.


A . traces of mEPSCs of both control naspm treated neurons after TTX , TTX + DHPG , TTX + DHPG + 2APB / dan treatment .

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A. Individual traces of mEPSCs of both control and naspm treated neurons after TTX, TTX + DHPG, and TTX + DHPG + 2APB/dan treatment.


B . Averaged mEPSCs an enlarged time scale from under same conditions as A . Traces from that were not treated with naspm are in black naspm treated traces are in grey .

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B. Averaged mEPSCs and an enlarged time scale from cells under the same conditions as A. Traces from cells that were not treated with naspm are in black and naspm treated traces are in grey.


C . Average amplitude of mEPSCs ( pA ) for both control ( bars ) naspm treated ( black bars ) neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . TTX , n = 26 ; TTX + naspm , n = 17 ; TTX + DHPG , n = 18 ; TTX + DHPG + naspm , n = 23 ; TTX + DHPG + 2APB / dan , n = 32 ; TTX + DHPG + 2APB / dan + naspm , n = 36 , p < 0 . 005 , ANOVA . D . Average frequency of mEPSC ( Hz ) for both control ( bars ) naspm treated ( black bars ) neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . TTX , n = 26 ; TTX + naspm , n = 12 ; TTX + DHPG , n = 18 ; TTX + DHPG + naspm , n = 23 ; TTX + DHPG + 2APB / dan , n = 32 ; TTX + DHPG + 2APB / dan + naspm , n = 36 , p < 0 . 0001 , ANOVA .

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

C. Average amplitude of mEPSCs (pA) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 17; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.005, ANOVA. D. Average frequency of mEPSC (Hz) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 12; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.0001, ANOVA.


E . Average amplitude of mEPSCs ( pA ) for only naspm treated neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . F . Average frequency of mEPSC ( Hz ) for only naspm treated neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene .

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

E. Average amplitude of mEPSCs (pA) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. F. Average frequency of mEPSC (Hz) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene.


G . Average rise time of mEPSC ( ms ) for both control ( bars ) naspm treated ( black bars ) neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . H . Average decay time of mEPSC ( ms ) for both control ( bars ) naspm treated ( black bars ) neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . error bars represent s . e . m .

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

G. Average rise time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. H. Average decay time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. All error bars represent s.e.m.


Gata6 - WT Gata6 - KOmye peritoneal macrophages ( pMɸ ) were unstimulated ( - ) or stimulated with TLR2L Pam3CSK4 ( 500 ng . ml - 1 ) , TLR3L Poly I : C ( 1 µg . ml - 1 ) , TLR4L purified LPS ( 100 ng . ml - 1 ) , TLR5L Flagellin ( 100 ng . ml - 1 ) , TLR7 8L R848 ( 1 µg . ml - 1 ) or TLR9L CpG ODN1826 ( 5 µM ) . Culture supernatants were collected 24 h after start of stimulation IL - 1β TNF ELISA were performed . n = 5 , two - way ANOVA with Tukeys multiple comparison post - .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 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 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

Gata6-WT and Gata6-KOmye peritoneal macrophages (pMɸ) were unstimulated (-) or stimulated with TLR2L Pam3CSK4 (500 ng.ml-1), TLR3L Poly I:C (1 µg.ml-1), TLR4L purified LPS (100 ng.ml-1), TLR5L Flagellin (100 ng.ml-1), TLR7 and 8L R848 (1 µg.ml-1) or TLR9L CpG ODN1826 (5 µM). Culture supernatants were collected 24 h after the start of stimulation and IL-1β and TNF ELISA were performed. n=5, two-way ANOVA analysis with Tukeys multiple comparison post-test.


IL - 1β ( C ) TNF ( D ) ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated for 24 h with indicated LPS concentrations . n = 3 , two - way ANOVA with Sidak ' s multiple comparison post - .

O 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

IL-1β (C) and TNF (D) ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 24 h with the indicated LPS concentrations. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.


IL - 1β ( E ) TNF ( F ) ELISA from Gata6 - WT Gata6 - KOmye pMɸ stimulated with LPS ( 100 ng . ml - 1 ) for indicated times . n = 3 , two - way ANOVA with Sidak ' s multiple comparison post - .

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

IL-1β (E) and TNF (F) ELISA from Gata6-WT and Gata6-KOmye pMɸ stimulated with LPS (100 ng.ml-1) for the indicated times. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.


IL - 1β ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated for 18 h with 100 ng . ml - 1 LPS or recombinant IL - 1 receptor antagonist ( rIL - 1ra ) , n = 4 to 5 , two - way ANOVA with Tukey ' s multiple comparison post - .

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

IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or recombinant IL-1 receptor antagonist (rIL-1ra), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.


IL - 1β ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated for 18 h with 100 ng . ml - 1 LPS or 100 ng . ml - 1 recombinant TNF ( recTNF ) , n = 4 to 5 , two - way ANOVA with Tukey ' s multiple comparison post - .

IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or 100 ng.ml-1 recombinant TNF (recTNF), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.


Normalised expression of Tlr Cd14 genes following microarray performed on unstimulated - sorted Gata6 - WT Gata6 - KOmye pMɸ . Data are shown as mean + / - from three biological replicates . Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by Benjamin - Hochberg procedure . n . d = non detectable .

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

Normalised expression of Tlr and Cd14 genes following microarray analysis performed on unstimulated cell-sorted Gata6-WT and Gata6-KOmye pMɸ. Data are shown as mean +/- SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin-Hochberg procedure. n.d = non detectable.


Mean ( MFI ) of extracellular TLR2 , TLR4 CD14 expression on naïve Gata6 - WT Gata6 - KOmye pMɸ . n = 4 to 7 mice per group .

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

Mean fluorescence intensity (MFI) of extracellular TLR2, TLR4 and CD14 expression on naïve Gata6-WT and Gata6-KOmye pMɸ. n = 4 to 7 individual mice per group.


Il1b Tnf mRNA relative expression of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h 6 h respectively . Data shown are representative of at least 3 independent experiments .

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

Il1b and Tnf mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments.


IL - 1β Western blot ( D ) of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h 6 h respectively . Data shown are representative of at least 3 independent experiments . Western blot was performed on whole lysates .

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

IL-1β Western blot protein analysis (D) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.


Representative dot plot , mean ( MFI ) of pro - IL - 1β + Gata6 - WT Gata6 - KOmye pMФ flow cytometry 3 h after stimulation with 100 ng . ml - 1 LPS . n = at least 3 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 B-CONTROLLED_VAR O O O O O O O O O

Representative dot plot, percentage and mean fluorescence intensity (MFI) analysis of pro-IL-1β+ Gata6-WT and Gata6-KOmye pMФ flow cytometry analysis 3 h after stimulation with 100 ng.ml-1 LPS. n = at least 3 independent experiments.


Nlrp3 mRNA relative expression of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h . Data shown are pooled from 3 independent experiments .

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

Nlrp3 mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h. Data shown are pooled from 3 independent experiments.


Western blot of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 6 h . Data shown are representative of at least 3 independent experiments . Western blot was performed on whole lysates .

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

Western blot protein analysis of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 6 h. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.


IL - 1β ELISA of supernatants collected from Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS either vehicle control ( Vh , DMSO ) or 10 µM MCC950 for 24 h ( n = 5 ) . Data shown in ( H ) are pooled from 5 independent replicates .

O O O O O O O O O O O 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

IL-1β ELISA of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5). Data shown in (H) are pooled from 5 independent replicates.


Western blot ( I ) of supernatants collected from Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS either vehicle control ( Vh , DMSO ) or 10 µM MCC950 for 24 h ( n = 5 ) .

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

Western blot protein analysis (I) of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5).


Caspase1 ( Casp1 ) mRNA relative expression of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h 6 h respectively . Data shown are pooled from 3 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Caspase1 (Casp1) mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.


Western blot ( K ) of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h 6 h respectively . Data shown are pooled from 3 independent experiments .

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

Western blot protein analysis (K) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.


IL - 1β ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS either vehicle control ( Vh , DMSO ) or Ac - YVAD - cmk for 24 h . Data shown are pooled of 5 independent replicates .

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

IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or Ac-YVAD-cmk for 24 h. Data shown are pooled of 5 independent replicates.


IL - 1β ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h , followed by a 30 min with either vehicle control ( Vh ) , 5 mM ATP or 20 µM Nigericin . Data shown are pooled of 5 independent replicates .

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

IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 µM Nigericin. Data shown are pooled of 5 independent replicates.


Representative picture of confocal immunofluorescence of Gata6 - WT Gata6 - KOmye pMФ stimulated with 100 ng . ml - 1 LPS for 3 h , followed by a 30 min with 5 mM ATP . arrows show ASC specks . 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 O O

Representative picture of confocal immunofluorescence analysis of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with 5 mM ATP. The white arrows show ASC specks. Scale bar = 10 µm.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ in monoculture or co - cultured ( 1 : 1 ) stimulated for 24 h with 100 ng . ml - 1 LPS . Data shown are pooled from 3 independent experiments . One - way ANOVA statistical with Tukey ' s multiple comparison was performed .

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

IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ in monoculture or co-cultured (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ co - cultured in same well ( co - culture ) or using Transwell system ( 1 : 1 ) stimulated for 24 h with 100 ng . ml - 1 LPS . Data shown are pooled from 3 independent experiments . One - way ANOVA statistical with Tukey ' s multiple comparison was performed .

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

IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ co-cultured in the same well (co-culture) or using Transwell system (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.


Microarray of Ptgis , Tbxas1 , Ptges2 Ptgs1 expression from Gata6 - WT Gata6 - KOmye pMɸ isolated from naïve mice . Data is shown as mean + / - from three biological replicates . Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by Benjamin - Hochberg procedure .

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

Microarray analysis of Ptgis, Tbxas1, Ptges2 and Ptgs1 expression from Gata6-WT and Gata6-KOmye pMɸ isolated from naïve mice. Data is shown as mean +/- SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin-Hochberg procedure.


Western blot of Ptgis level of unstimulated pMФ from Gata6 - WT Gata6 - KOmye mice .

Western blot analysis of Ptgis protein level of unstimulated pMФ from Gata6-WT and Gata6-KOmye mice.


Mass spectrometry of 6 - keto - PGF1α , TXB2 PGE2 content of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated for 3 h with 100 ng . ml - 1 LPS . n = 6 . Two - way ANOVA statistical with Tukey ' s multiple comparison post - was performed .

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

Mass spectrometry analysis of 6-keto-PGF1α, TXB2 and PGE2 content of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated for 3 h with 100 ng.ml-1 LPS. n = 6. Two-way ANOVA statistical analysis with Tukey's multiple comparison post-test was performed.


IL - 10 ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS . Data shown are representative of at least 5 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 B-CONTROLLED_VAR O O O O O O O O O O O O

IL-10 ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS. Data shown are representative of at least 5 independent experiments.


IL - 10 ( B ) IL - 1β ( C ) ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , beraprost ( 1 10 µM ) , cicaprost ( 1 10 µM ) , Iloprost ( 1 or 10 nM ) or vehicle control ( Vh , DMSO ) . Data shown are representative of at least 3 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

IL-10 (B) and IL-1β (C) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, beraprost (1 and 10 µM), cicaprost (1 and 10 µM), Iloprost (1 or 10 nM) or vehicle control (Vh, DMSO). Data shown are representative of at least 3 independent experiments.


IL - 10 ( D ) IL - 1β ( E ) ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 µM PGE2 , 10 µM U46619 , 100 µM picotamide or vehicle control ( Vh , methyl acetate ) . Data shown are representative of at least 3 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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

IL-10 (D) and IL-1β (E) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 10 µM PGE2, 10 µM U46619, 100 µM picotamide or vehicle control (Vh, methyl acetate). Data shown are representative of at least 3 independent experiments.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 ng . ml - 1 IL - 10 , 10 µM beraprost or vehicle control ( Vh ) . Data shown are representative of at least 3 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 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

IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10, 10 µM beraprost or vehicle control (Vh). Data shown are representative of at least 3 independent experiments.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM beraprost , 5 µg . ml - 1 αIL - 10R or isotype antibody . Data shown are representative of at least 3 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 B-CONTROLLED_VAR 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

IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 5 µg.ml-1 αIL-10R or isotype antibody. Data shown are representative of at least 3 independent experiments.


IL - 1β ( H ) IL - 10 ( I ) ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM MCC950 , 5 µg . ml - 1 αIL - 10R or isotype antibody stimulations were performed for 16 h . Data shown are representative of at least 3 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 B-CONTROLLED_VAR 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

IL-1β (H) and IL-10 (I) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM MCC950, 5 µg.ml-1 αIL-10R or isotype antibody All stimulations were performed for 16 h. Data shown are representative of at least 3 independent experiments.