( B ) Time ‐ lapse of TFEB ‐ and Rab7 ‐ positive lysosomes from the boxed region in ( A ) . Time intervals are in seconds .

O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O

(B) Time‐lapse of TFEB‐ and Rab7‐positive lysosomes from the boxed region in (A). Time intervals are in seconds.


( C ) Time ‐ lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB - GFP . Arrow indicates the time of Torin 1 addition . Yellow arrowheads indicate Torin 1 ‐ induced lysosomal accumulation of TFEB - GFP . Time intervals are in minutes .

O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

(C) Time‐lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB-GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1‐induced lysosomal accumulation of TFEB-GFP. Time intervals are in minutes.


( D ) Immunofluorescence of HEK ‐ 293T cells expressing TFEB - 3 × FLAG , treated with DMSO ( top ) or Torin1 ( bottom ) and stained with antibodies against FLAG and mTOR ( green and red in the merge , respectively ; DAPI is in blue ) .

O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O

(D) Immunofluorescence of HEK‐293T cells expressing TFEB-3 × FLAG, treated with DMSO (top) or Torin1 (bottom) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( E ) FRAP analysis of TFEB - GFP ‐ positive lysosomes from control MEFs ( blue ) or MEFs treated with Torin 1 ( red ) . Each data point represents mean ± s . d . from five independent spots .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O B-CELL_LINE O O O O B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(E) FRAP analysis of TFEB-GFP‐positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots.


( F ) Time ‐ lapse of photobleaching and fluorescence recovery of TFEB - GFP ‐ positive lysosomes from control ‐ treated MEFs ( top ) or MEFs treated with Torin 1 ( bottom ) . Red arrowheads indicate time of photobleaching . Time intervals are in seconds .

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(F) Time‐lapse of photobleaching and fluorescence recovery of TFEB-GFP‐positive lysosomes from control‐treated MEFs (top) or MEFs treated with Torin 1 (bottom). Red arrowheads indicate time of photobleaching. Time intervals are in seconds.


( G ) Torin 1 increases binding of TFEB to mTORC1 . HEK ‐ 293T cells that express TFEB - 3 × FLAG along with HAGST ‐ Rap2A or HAGST ‐ RagsDN were treated with vehicle or with Torin1 , lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor . FLAG - Metap2 served as negative control .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O

(G) Torin 1 increases binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG along with HAGST‐Rap2A or HAGST‐RagsDN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG-Metap2 served as negative control.


( H ) Immunofluorescence of HEK ‐ 293T cells that express TFEB - 3 × FLAG along with Rap2A ( top ) or the RagsDN mutants ( bottom ) , treated with Torin 1 and stained with antibodies against FLAG and LAMP2 ( green and red in the merge , respectively ; DAPI is in blue ) . In all images , scale bars represent 10 μm .

O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

(H) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (top) or the RagsDN mutants (bottom), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.


( A - C ) Immunofluorescence of HEK ‐ 293T cells that express TFEB - 3 × FLAG along with a control GTPase or the indicated Rag mutants . Cells were deprived of amino acids ( top ) or deprived and then stimulated ( bottom ) for the indicated times and stained for FLAG and mTOR ( green and red in the merge , respectively ; DAPI is in blue ) .

O O O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O

(A-C) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with a control GTPase or the indicated Rag mutants. Cells were deprived of amino acids (top) or deprived and then stimulated (bottom) for the indicated times and stained for FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( D ) Quantification of the number of cells with nuclear TFEB from each condition in ( A - C ) .

O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O

(D) Quantification of the number of cells with nuclear TFEB from each condition in (A-C).


( E , F ) Immunofluorescence of HEK ‐ 293T cells that express TFEB - 3 × FLAG along with Rap2A ( E ) or the RagsCA mutants ( F ) , subjected to the indicated treatments and stained with antibodies against FLAG and mTOR ( green and red in the merge , respectively ; DAPI is in blue ) .

O O O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O

(E, F) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (E) or the RagsCA mutants (F), subjected to the indicated treatments and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( G ) Quantification of the number of cells with nuclear TFEB from DMSO ‐ and CQ ‐ treated fields in ( E ) and ( F ) . In all fields , scale bars represent 10 μm . In all histograms , each value represents mean ± s . d . from three independent fields with N = 300 .

O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Quantification of the number of cells with nuclear TFEB from DMSO‐ and CQ‐treated fields in (E) and (F). In all fields, scale bars represent 10 μm. In all histograms, each value represents mean±s.d. from three independent fields with N=300.


( A ) Chloroquine treatment inhibits mTORC1 activity in primary hepatocytes . Primary hepatocytes isolated from 2 ‐ month ‐ old Tcfebflox / flox ( control ) and Tcfebflox / flox ; Alb ‐ Cre ( Tcfeb − / − ) mice were left untreated , or treated overnight with Torin 1 , U0126 , or Chloroquine . Subsequently , cells were lysed and protein extracts were immunoblotted with the indicated antibodies .

O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O

(A) Chloroquine treatment inhibits mTORC1 activity in primary hepatocytes. Primary hepatocytes isolated from 2‐month‐old Tcfebflox/flox (control) and Tcfebflox/flox;Alb‐Cre(Tcfeb−/−) mice were left untreated, or treated overnight with Torin 1, U0126, or Chloroquine. Subsequently, cells were lysed and protein extracts were immunoblotted with the indicated antibodies.


( B , C ) TFEB mediates the transcriptional response to chloroquine and Torin 1 . Quantitative PCR ( qPCR ) of TFEB target genes in primary hepatocytes from control ( flox / flox ) and Tcfeb − / − mice . Cells were treated with Chloroquine ( left ) or Torin 1 ( right ) . The graphs show the relative increased expression in the treated versus the corresponding untreated samples . Values represent means ± s . d . of three independent hepatocyte preparations ( three mice / genotype ) . Student ' s t ‐ test ( two tailed ) * P ‐ value ⩽ 0 . 05 .

O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

(B, C) TFEB mediates the transcriptional response to chloroquine and Torin 1. Quantitative PCR (qPCR) of TFEB target genes in primary hepatocytes from control (flox/flox) and Tcfeb−/− mice. Cells were treated with Chloroquine (left) or Torin 1 (right). The graphs show the relative increased expression in the treated versus the corresponding untreated samples. Values represent means±s.d. of three independent hepatocyte preparations (three mice/genotype). Student's t‐test (two tailed) *P‐value ⩽0.05.


( A - B ) Levels of miR - 122 in Huh7 cells and released EVs either untreated ( Fed ) or subjected to starvation for metabolites including amino acids for 16h ( Starved ) . miR - 122 signals were detected by Northern blotting and position of the 32P - labelled Oligos that served as size markers are shown in the lane ( A ) . U6 snRNA was used as loading control .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). miR-122 signals were detected by Northern blotting and position of the 32P-labelled Oligos that served as size markers are shown in the lane (A). U6 snRNA was used as loading control.


( A - B ) Levels of miR - 122 in Huh7 cells and released EVs either untreated ( Fed ) or subjected to starvation for metabolites including amino acids for 16h ( Starved ) . Cellular CAT - 1 levels were measured by qRT - PCR using GAPDH mRNA as control ( B ) . A scheme of experiment is shown in the top panel in B .

O O O O O O O O O O O B-CELL_LINE O O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). Cellular CAT-1 levels were measured by qRT-PCR using GAPDH mRNA as control (B). A scheme of experiment is shown in the top panel in B.


( C - D ) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells . Relative changes in cellular and extracellular levels ( EV associated ) of miR - 122 and miR - 24 in Fed or 8h Starved Huh7 cells were quantified by qRT - PCR and plotted ( mean + / - s . e . m . , n = 3 ) . Levels of individual miRNAs in Fed condition were taken as unit . Fold change ( with SD ) in the cellular level of all miRNAs and pre - miR - 122 measured were shown in the bottom panel ( C ) . Cellular miRNA levels were normalized against U6 snRNA .

O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_LINE O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O

(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Relative changes in cellular and extracellular levels (EV associated) of miR-122 and miR-24 in Fed or 8h Starved Huh7 cells were quantified by qRT-PCR and plotted (mean+/- s.e.m., n=3). Levels of individual miRNAs in Fed condition were taken as unit. Fold change (with SD) in the cellular level of all miRNAs and pre-miR-122 measured were shown in the bottom panel (C). Cellular miRNA levels were normalized against U6 snRNA.


( C - D ) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells . Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions . CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels . Mean of three independent measurements are plotted ( middle panel in D ) . Calnexin and GAPDH are not detected in EVs but visible in the cellular extract . EV associated HA - HuR levels for Fed and Starved Huh7 cells expressing HA - HuR were also detected in the bottom panel in D .

O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions. CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels. Mean of three independent measurements are plotted (middle panel in D). Calnexin and GAPDH are not detected in EVs but visible in the cellular extract. EV associated HA-HuR levels for Fed and Starved Huh7 cells expressing HA-HuR were also detected in the bottom panel in D.


( E ) Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells . Levels of miRNAs were measured by real - time quantification and normalized against U6 snRNA . Mean data are from three independent experiments . DMSO treatment was used as control for GW4869 treated cells .

O O O O O B-SMALL_MOLECULE 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-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O

(E) Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells. Levels of miRNAs were measured by real-time quantification and normalized against U6 snRNA. Mean data are from three independent experiments. DMSO treatment was used as control for GW4869 treated cells.


( F ) CAT - 1 and Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 cells . qRT - PCR techniques was adopted for quantification using 18S rRNA values for normalization ( mean + / - s . e . m . , n = 3 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(F) CAT-1 and Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 cells. qRT-PCR techniques was adopted for quantification using 18S rRNA values for normalization (mean +/- s.e.m., n=3).


( G ) Effect of siSMPD2 treatment on miR - 122 content of EVs from Fed and Starved Huh7 cells . EVs from Fed and Starved Huh7 cells , depleted for SMPD2 , were isolated and miR - 122 content were measured by qRT - PCR and normalized against protein content of EVs . Fold change in EV associated miR - 122 levels upon starvation both for control and siSMPD2 treated cells were shown above the respective bars .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O B-CELL_LINE O O B-SUBCELLULAR O O O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

(G) Effect of siSMPD2 treatment on miR-122 content of EVs from Fed and Starved Huh7 cells. EVs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 content were measured by qRT-PCR and normalized against protein content of EVs. Fold change in EV associated miR-122 levels upon starvation both for control and siSMPD2 treated cells were shown above the respective bars.


( H ) Effect of siSMPD2 treatment on CAT - 1 mRNA content ( left panel ) and miR - 122 levels ( right panel ) in Huh7 cells . RNAs , from Fed and Starved Huh7 cells , depleted for SMPD2 , were isolated and miR - 122 and CAT - 1 mRNA contents were measured by qRT - PCR . miR - 122 contents were normalized against U6 snRNA . GAPDH mRNA was used for normalization of CAT - 1 mRNA levels ( mean + / - s . e . m . , n = 3 ) . miR - 122 levels for siCon treated cells ( Fed and Starved ) were considered as units .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

(H) Effect of siSMPD2 treatment on CAT-1 mRNA content (left panel) and miR-122 levels (right panel) in Huh7 cells. RNAs, from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 and CAT-1 mRNA contents were measured by qRT-PCR. miR-122 contents were normalized against U6 snRNA. GAPDH mRNA was used for normalization of CAT-1 mRNA levels (mean +/- s.e.m., n=3). miR-122 levels for siCon treated cells (Fed and Starved) were considered as units.


( I - J ) A schematic representation of the experiment to separate Extracellular Vesicles ( EVs ) on OptiPrep @ density gradient ( left panel ) . Densities of fractions 1 - 10 are plotted and a best - fit curve is drawn ( right upper panel ) . CD63 levels in individual fractions of Fed and Starved cells were detected by western blots to confirm the presence of exosomes of Fed and Straved cells ( right lower panel ) . Mean Ct values of miR - 122 in RNAs isolated from Exosome enriched ( 8 - 9 ) vs Non - exosomal ( 1 - 3 ) fractions were analyzed and plotted ( J ) ( mean + / - s . e . m . , n = 3 ) .

O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O 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-J) A schematic representation of the experiment to separate Extracellular Vesicles (EVs) on OptiPrep@ density gradient (left panel). Densities of fractions 1-10 are plotted and a best-fit curve is drawn (right upper panel). CD63 levels in individual fractions of Fed and Starved cells were detected by western blots to confirm the presence of exosomes of Fed and Straved cells (right lower panel). Mean Ct values of miR-122 in RNAs isolated from Exosome enriched (8-9) vs Non-exosomal (1-3) fractions were analyzed and plotted (J) (mean +/- s.e.m., n=3).


( K ) Effect of Starvation on apoptic cell numbers in Huh7 cells . Western Blot detection of apoptosis marker Cytochrome C in cellular and in EV associated fractions of Fed and Starved Huh 7 cells . β - Actin and CD63 are used as loading control for cellular and EV associated fraction respectively ( left panel ) . TUNEL positive cells in Fed and Starved Huh7 cells . TUNEL assays of Fed and Starved Huh 7 cells were performed and TUNEL positive cells were quantified . Fed , but DNase treated , cells were used as positive control ( + ve control ) ( right panel ) .

O O O O O O O O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O B-CELL_LINE O O B-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(K) Effect of Starvation on apoptic cell numbers in Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C in cellular and in EV associated fractions of Fed and Starved Huh 7 cells. β-Actin and CD63 are used as loading control for cellular and EV associated fraction respectively (left panel). TUNEL positive cells in Fed and Starved Huh7 cells. TUNEL assays of Fed and Starved Huh 7 cells were performed and TUNEL positive cells were quantified. Fed, but DNase treated, cells were used as positive control (+ve control) (right panel).


( L ) Effect of thapsigargain ( TG ) treatment of Huh7 cells on cellular and EV associated miR - 122 level . A schematic representation of the experiment ( upper panel ) . miR - 122 levels were measured by qRT - PCR in total cellular RNA and in EVs released by the treated cells . Values were normalized either against U6 snRNA or protein content of EVs ( mean + / - s . e . m . , n = 3 ) ( lower panels ) for cellular and EV associated RNA respectively .

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O

(L) Effect of thapsigargain (TG) treatment of Huh7 cells on cellular and EV associated miR-122 level. A schematic representation of the experiment (upper panel). miR-122 levels were measured by qRT-PCR in total cellular RNA and in EVs released by the treated cells. Values were normalized either against U6 snRNA or protein content of EVs (mean+/- s.e.m., n=3) (lower panels) for cellular and EV associated RNA respectively.


( M ) Effect of TG on cellular level of eIF2 - a and its phosphorylated form , and Ago2 and CD63 in EVs . b - Actin was used as loading control for cellular samples .

O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(M) Effect of TG on cellular level of eIF2-a and its phosphorylated form, and Ago2 and CD63 in EVs. b-Actin was used as loading control for cellular samples.


( A ) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells . Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR - 122 and miR - 21 were done by quantitative RT - PCR ( mean + / - s . e . m . , n = 3 ) . GFP used as negative control to confer the specificity of the anti - HuR3A2 antibody in immunoprecipitating HuR . The miR - 122 content in the GFP - antibody immunoprecipitated materials was too low to get detected reliably using the qRT - PCR .

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(A) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells. Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR-122 and miR-21 were done by quantitative RT-PCR (mean +/- s.e.m., n=3). GFP used as negative control to confer the specificity of the anti-HuR3A2 antibody in immunoprecipitating HuR. The miR-122 content in the GFP-antibody immunoprecipitated materials was too low to get detected reliably using the qRT-PCR.


( B - C ) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells . Schemes of HuR depletion experiments in Huh7 cells ( B ) . In siHuR treated cells , depletion of HuR was monitored by western blotting ( mean + / - s . e . m . , n = 4 )

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(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Schemes of HuR depletion experiments in Huh7 cells (B). In siHuR treated cells, depletion of HuR was monitored by western blotting (mean+/- s.e.m., n=4)


( B - C ) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells . Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT - PCR . Cellular miRNA contents were normalized against U6 snRNA . Levels of each miRNA in control siRNA ( siCon ) treated set were taken as unit . ( C ) . CAT - 1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT - PCR and normalized against GAPDH mRNA .

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(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT-PCR. Cellular miRNA contents were normalized against U6 snRNA. Levels of each miRNA in control siRNA (siCon) treated set were taken as unit. (C). CAT-1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT-PCR and normalized against GAPDH mRNA.


( D ) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells . Relative levels were estimated by real time qPCR , normalized with respect to U6 snRNA and plotted in the left panel . Levels of miR - 122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT - PCR and plotted in the right panel ( mean + / - s . e . m . , n = 3 ) .

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(D) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells. Relative levels were estimated by real time qPCR, normalized with respect to U6 snRNA and plotted in the left panel. Levels of miR-122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT-PCR and plotted in the right panel (mean+/- s.e.m., n=3).


( E ) Level of eIF - 2a and its phosphorylated form , estimated by western blot using specific antibodies , in siCon or siHuR treated and starved Huh7 cell extracts . Western blot data of HuR confirm reduction of this protein upon siRNA treatment . CAT - 1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level . Values of siCon treated samples were taken as unit ( mean + / - s . e . m . , n = 3 ) . In the right panel the relative quantity of phosphorylated eIF - 2a measured by densitometry were plotted . Levels of p38 and phosphor - p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot .

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(E) Level of eIF-2a and its phosphorylated form, estimated by western blot using specific antibodies, in siCon or siHuR treated and starved Huh7 cell extracts. Western blot data of HuR confirm reduction of this protein upon siRNA treatment. CAT-1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level. Values of siCon treated samples were taken as unit (mean+/- s.e.m., n=3). In the right panel the relative quantity of phosphorylated eIF-2a measured by densitometry were plotted. Levels of p38 and phosphor-p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot.


( F ) Western blot analysis to detect the level of phosphorylated eIF - 2a and eIF - 4E - BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant - let - 7a or anti - miR - 122 oligos . miR - 122 inactivation was monitored by measuring cat - 1 level by qRT - PCR using GAPDH as control ( mean + / - s . e . m . , n = 3 ) . * denotes the phosphorylated eIF - 4E - BP1 . b - Actin was used as loading control . Relative levels of phosphorylated eIF - 2a were measured by densitometric estimation of three independent measurements .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

(F) Western blot analysis to detect the level of phosphorylated eIF-2a and eIF-4E-BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant-let-7a or anti-miR-122 oligos. miR-122 inactivation was monitored by measuring cat-1 level by qRT-PCR using GAPDH as control (mean+/- s.e.m., n=3). * denotes the phosphorylated eIF-4E-BP1. b-Actin was used as loading control. Relative levels of phosphorylated eIF-2a were measured by densitometric estimation of three independent measurements.


( A ) Northern blot of miR - 122 in Huh7 cells expressing the Control and HA - HuR expression plasmids . The U6 was used for loading control . Position of the 32P - labelled Oligos , blotted to the membrane used for Northern blotting , served as size markers are shown in the M lane .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(A) Northern blot of miR-122 in Huh7 cells expressing the Control and HA-HuR expression plasmids. The U6 was used for loading control. Position of the 32P-labelled Oligos, blotted to the membrane used for Northern blotting, served as size markers are shown in the M lane.


( B ) Schematic representation of experiments done in Huh7 cells expressing HA - HuR ( bottom ) . For control , pCIneo transfected Huh7 cells were used . Relative expression of HuR in transfected cells were analysed by western blot ( top ) . b - actin blots were used as loading controls .

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(B) Schematic representation of experiments done in Huh7 cells expressing HA-HuR (bottom). For control, pCIneo transfected Huh7 cells were used. Relative expression of HuR in transfected cells were analysed by western blot (top). b-actin blots were used as loading controls.


( C ) Cellular and EV - associated miRNA levels were measured in Huh7 cells either expressing the pCIneo or HA - HuR expressing vectors by qRT - PCR ( mean + / - s . e . m . , n = 5 ) . Cellular miRNA levels were normalized against U6 snRNA .

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(C) Cellular and EV-associated miRNA levels were measured in Huh7 cells either expressing the pCIneo or HA-HuR expressing vectors by qRT-PCR (mean+/- s.e.m., n=5). Cellular miRNA levels were normalized against U6 snRNA.


( E ) Effect of HA - HuR expression on miRNA levels on EV and non - EV fractions of the culture supernatant collected from control and HA - HuR encoding plasmid transfected Huh7 cells . For isolation of non - EV fraction , supernatant obtained after removal of EVs by ultracentrifugation was used for RNA preparation ( left panel ) ( mean + / - s . e . m . , n = 3 ) . Effect of HuR expression on CD63 , Alix and HuR levels in EVs . Extracts from EVs isolated from control or HA - HuR expressing Huh7 cells were western blotted for respective proteins ( right panel ) .

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(E) Effect of HA-HuR expression on miRNA levels on EV and non-EV fractions of the culture supernatant collected from control and HA-HuR encoding plasmid transfected Huh7 cells. For isolation of non-EV fraction, supernatant obtained after removal of EVs by ultracentrifugation was used for RNA preparation (left panel) (mean+/- s.e.m., n=3). Effect of HuR expression on CD63, Alix and HuR levels in EVs. Extracts from EVs isolated from control or HA-HuR expressing Huh7 cells were western blotted for respective proteins (right panel).


( F ) Relative miR - 122 levels in CD63 positive affinity purified EVs isolated from HA - HuR and pCIneo transfected Huh7 cells were quantified by qRT - PCR ( mean + / - s . e . m . , n = 3 ) . The levels of miR - 122 were normalized against CD63 content of EVs .

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(F) Relative miR-122 levels in CD63 positive affinity purified EVs isolated from HA-HuR and pCIneo transfected Huh7 cells were quantified by qRT-PCR (mean+/- s.e.m., n=3). The levels of miR-122 were normalized against CD63 content of EVs.


( G ) Effect of GW4869 on EV associated content of three different miRNAs in HA - HuR expressing Huh7 cells quantified by qRT - PCR ( mean + / - s . e . m . , n = 5 ) . EVs from DMSO treated cells were used as control .

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(G) Effect of GW4869 on EV associated content of three different miRNAs in HA-HuR expressing Huh7 cells quantified by qRT-PCR (mean+/- s.e.m., n=5). EVs from DMSO treated cells were used as control.


( H ) A schematic representation of the experiment done to characterize EVs isolated from Huh7 cells expressing HA - HuR or pCIneo respectively by running OptiPrepTM density gradient ( left panel ) and western blotting for CD63 ( right upper panel ) . Relative miR - 122 levels in fractions 1 - 2 and 8 - 9 were compared by qRT - PCR ( right lower panel ) .

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(H) A schematic representation of the experiment done to characterize EVs isolated from Huh7 cells expressing HA-HuR or pCIneo respectively by running OptiPrepTM density gradient (left panel) and western blotting for CD63 (right upper panel). Relative miR-122 levels in fractions 1-2 and 8-9 were compared by qRT-PCR (right lower panel).


( I ) Levels of apoptic cells in HA - HuR expressing Huh7 cells . Western Blot detection of apoptosis marker Cytochrome C was done ( top ) for EV associated and cellular fractions of HA - HuR and pCIneo ( control ) expressing Huh 7 cells respectively . Western blot for HA - HuR was done to analyse the expression of HA - HuR in EVs isolated from HA - HuR and pCIneo expressing Huh7 cells . CD63 and β - actin are used as loading control for EV associated fractions and cellular fractions respectively ( upper panels ) . TUNEL assay of HA - HuR and pCIneo expressing and non - transfected Huh 7 cells was performed and representative pictures of HA - HuR and pCIneo expressing cells along with picture of non - transfected but DNase treated ( positive control ) cells were compared ( lower panels ) . Scale bar 50 µM .

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(I) Levels of apoptic cells in HA-HuR expressing Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C was done (top) for EV associated and cellular fractions of HA-HuR and pCIneo (control) expressing Huh 7 cells respectively. Western blot for HA-HuR was done to analyse the expression of HA-HuR in EVs isolated from HA-HuR and pCIneo expressing Huh7 cells. CD63 and β-actin are used as loading control for EV associated fractions and cellular fractions respectively (upper panels). TUNEL assay of HA-HuR and pCIneo expressing and non-transfected Huh 7 cells was performed and representative pictures of HA-HuR and pCIneo expressing cells along with picture of non-transfected but DNase treated (positive control ) cells were compared (lower panels). Scale bar 50 µM.


( B ) Change in miR - 122 level in starved mouse liver . Endogenous miR - 122 ( left panel ) and CAT - 1 mRNA ( middle panel ) levels in mouse liver starved for 12h were quantified by qRT - PCR . miR - 122 levels in the blood serum of corresponding animals were also measured ( right panel ) . The Ct values of miR - 122 and CAT - 1 mRNA obtained for each individual animal liver and blood serum were plotted ( mean + / - s . e . m . , n = 5 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O

(B) Change in miR-122 level in starved mouse liver. Endogenous miR-122 (left panel) and CAT-1 mRNA (middle panel) levels in mouse liver starved for 12h were quantified by qRT-PCR. miR-122 levels in the blood serum of corresponding animals were also measured (right panel). The Ct values of miR-122 and CAT-1 mRNA obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).


( C ) Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots , β - actin was used as loading control .

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(C)Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots, β-actin was used as loading control .


( E ) Western Blot analysis to check expression of HA - HuR in liver samples of injected mice . β - Actin was used as loading control . HA - HuR expressing Huh7 cells lysate was used as positive control .

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(E) Western Blot analysis to check expression of HA-HuR in liver samples of injected mice. β-Actin was used as loading control. HA-HuR expressing Huh7 cells lysate was used as positive control.


( F ) . Change in miR - 122 level upon expression of HA - HuR in mouse liver . Endogenous miR - 122 level in HA - HuR expression plasmid injected mouse liver were quantified by RT - PCR and compared against liver RNA samples collected from control ( pCIneo injected ) group of animals ( mean + / - s . e . m . , n = 5 ) .

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(F). Change in miR-122 level upon expression of HA-HuR in mouse liver. Endogenous miR-122 level in HA-HuR expression plasmid injected mouse liver were quantified by RT-PCR and compared against liver RNA samples collected from control (pCIneo injected) group of animals (mean+/- s.e.m., n=5).


( G ) Endogenous miR - 122 target CAT - 1 ( left panel ) and Aldolase ( right panel ) mRNA levels in HA - HuR or pCIneo ( control ) injected mouse liver were quantified by RT - PCR ( mean + / - s . e . m . , n = 4 ) .

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(G) Endogenous miR-122 target CAT-1 (left panel) and Aldolase (right panel) mRNA levels in HA-HuR or pCIneo (control) injected mouse liver were quantified by RT-PCR (mean+/- s.e.m., n=4).


( H ) miR - 122 levels in the blood serum of pCIneo ( control ) or HA - HuR expression plasmid injected mice were measured . The Ct values of miR - 122 and CAT - 1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted ( mean + / - s . e . m . , n = 5 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O

(H) miR-122 levels in the blood serum of pCIneo (control) or HA-HuR expression plasmid injected mice were measured. The Ct values of miR-122 and CAT-1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).


( A ) Time course experiment of HuR binding of miR - 122 replaced from Ago2miRNPs . A schematic representation of in vitro miRNA binding assay of HuR ( left panel ) . Equal amounts of recombinant HuR ( rHuR ) , after indicated time of the miRNP interaction on FLAG - beads , were immunoprecipitated and HuR associated miR - 122 levels were detected by qRT - PCR ( right top panel ) . Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials . HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials , at different time points ( right bottom panel ) .

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(A) Time course experiment of HuR binding of miR-122 replaced from Ago2miRNPs. A schematic representation of in vitro miRNA binding assay of HuR (left panel). Equal amounts of recombinant HuR (rHuR), after indicated time of the miRNP interaction on FLAG-beads, were immunoprecipitated and HuR associated miR-122 levels were detected by qRT-PCR (right top panel). Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials. HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials, at different time points (right bottom panel).


( C ) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay . The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation . The recombinant P protein and N protein of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti - HuR antibody .

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(C) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay. The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation. The recombinant P protein and N protein of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti-HuR antibody.


( D ) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials . A representative domain picture of HuR and HuRΔIII are shown in the upper panel . Relative levels of HuR bound miR - 122 were determined by qRT - PCR ( mean + / - s . e . m . , n = 3 ) ( middle left panel ) . Amount of miR - 122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials . Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials ( bottom panel ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O

(D) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials. A representative domain picture of HuR and HuRΔIII are shown in the upper panel. Relative levels of HuR bound miR-122 were determined by qRT-PCR (mean+/- s.e.m., n=3) (middle left panel). Amount of miR-122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials. Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials (bottom panel).


( E ) RNA electrophoretic mobility shift assay done with 32P - labeled synthetic miR - 122 and recombinant HuR proteins . Radiolabelled miR - 122 ( 100 fmol ) was incubated with increasing concentration of FL - HuR ( 5 - 100nM ) or HuRΔIII ( 100 nM ) and gel shifting of miR - 122 were marked by arrowhead . Position of the free probes is marked by an arrow . In the right panel , 1pmol of unlabelled TNF - alpha AU - rich element encoding synthetic RNA was used to compete with miR - 122 binding of full length HuR . Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O

(E) RNA electrophoretic mobility shift assay done with 32P-labeled synthetic miR-122 and recombinant HuR proteins. Radiolabelled miR-122 (100 fmol) was incubated with increasing concentration of FL-HuR (5-100nM) or HuRΔIII (100 nM) and gel shifting of miR-122 were marked by arrowhead. Position of the free probes is marked by an arrow. In the right panel, 1pmol of unlabelled TNF-alpha AU-rich element encoding synthetic RNA was used to compete with miR-122 binding of full length HuR. Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page.


( F ) Association of miRNAs with recombinant HuR in vitro . Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA , recovered from immunoprecipitated HuR after the binding reaction , were quantified along with the input RNA used for binding . The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted ( mean + / - s . e . m . , n = 3 ) . The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot .

O O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O

(F) Association of miRNAs with recombinant HuR in vitro. Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA, recovered from immunoprecipitated HuR after the binding reaction, were quantified along with the input RNA used for binding. The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted (mean+/- s.e.m., n=3). The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot.


( A ) Subcellular distribution of HuR and Ago2 in Huh7 cells . Isotonic lysates of Huh7 cells transfected with HA - HuR expression or pCIneo control vector were analyzed on 3 - 30 % OptiprepTM gradients for separation of organelles and localization of Ago2 and HuR were determined by western blotting analysis . Alix and Calnexin were used as markers of late endosome / MVB and ER respectively . Positions of specific organelle containing fractions were marked above the panels . * denotes the position of the high molecular weight HuR bands detected in the MVB fractions .

O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O

(A) Subcellular distribution of HuR and Ago2 in Huh7 cells. Isotonic lysates of Huh7 cells transfected with HA-HuR expression or pCIneo control vector were analyzed on 3-30% OptiprepTM gradients for separation of organelles and localization of Ago2 and HuR were determined by western blotting analysis. Alix and Calnexin were used as markers of late endosome/MVB and ER respectively. Positions of specific organelle containing fractions were marked above the panels. * denotes the position of the high molecular weight HuR bands detected in the MVB fractions.


( B ) Detection of ubiquitinated HuR in Huh7 cells expressing HA - Ub . HuR was immunoprecipitated with anti - HuR antibody from cell lysate of Huh7 cells transfected with HA - Ub expression or pCIneo ( control ) plasmids and western blotted for HA or HuR ( left panel ) . In vitro ubiquitination assay was done with recombinant His - tagged HuR and cell extract of Huh7 cells expressing HA - Ub . The scheme of the experiment and western blot data for input and cell extract along with immunoprecipitated materials are shown . Specific bands of Ub - HuR are marked by arrows and HuR is marked by arrowhead .

O O O O O B-GENEPROD I-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O

(B) Detection of ubiquitinated HuR in Huh7 cells expressing HA-Ub. HuR was immunoprecipitated with anti-HuR antibody from cell lysate of Huh7 cells transfected with HA-Ub expression or pCIneo (control) plasmids and western blotted for HA or HuR (left panel). In vitro ubiquitination assay was done with recombinant His-tagged HuR and cell extract of Huh7 cells expressing HA-Ub. The scheme of the experiment and western blot data for input and cell extract along with immunoprecipitated materials are shown. Specific bands of Ub-HuR are marked by arrows and HuR is marked by arrowhead.


( C ) Changes in relative level of miR - 122 in the subcellular ER and MVB associated fractions of OptiprepTM gradients done with the cell lysates from pCIneo or HA - HuR expression vector transfected cells were measured by qRT - PCR ( mean + / - s . e . m . , n = 5 ) .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O

(C) Changes in relative level of miR-122 in the subcellular ER and MVB associated fractions of OptiprepTM gradients done with the cell lysates from pCIneo or HA-HuR expression vector transfected cells were measured by qRT-PCR (mean+/- s.e.m., n=5).


( D ) Detection of ubiquitinated HuR in HuR - immunoprecipitated materials obtained from ER and MVB enriched fractions of Huh7 cells . Organelles were separated on OptiprepTM gradients , and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for ubiquitin to detect ubiquitinated form of HuR . Whole cell lysates used for optiprep analysis were western blotted for ubiquitinated proteins . Positions of the high molecular weight bands of ubiquitinated HuR are marked by arrowheads . Levels of HuR in the immunoprecipitated materials were detected using anti - HuR antibody . Anti - GFP antibody immnoprecipitated materials were used as control .

O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O

(D) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of Huh7 cells. Organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for ubiquitin to detect ubiquitinated form of HuR. Whole cell lysates used for optiprep analysis were western blotted for ubiquitinated proteins. Positions of the high molecular weight bands of ubiquitinated HuR are marked by arrowheads. Levels of HuR in the immunoprecipitated materials were detected using anti-HuR antibody. Anti-GFP antibody immnoprecipitated materials were used as control.


( E ) Detection of ubiquitinated HuR in HuR - immunoprecipitated materials obtained from ER and MVB enriched fractions of cells co - expressing HA - Ubiquitin and FLAG - HuR . Cells were lysed and organelles were separated on OptiprepTM gradients , and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for HA to detect ubiquitinated form of HuR . HuR in the lysate of the FLAG - HuR and HA - Ub co expressing cells were detected by HuR specific antibody .

O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O

(E) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of cells co-expressing HA-Ubiquitin and FLAG-HuR. Cells were lysed and organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for HA to detect ubiquitinated form of HuR. HuR in the lysate of the FLAG-HuR and HA-Ub co expressing cells were detected by HuR specific antibody.


( F ) Loss of miRNA association of HuR on MVB . HA - HuR expressing Huh7 cell lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA - HuR and associated miRNA levels were measured by qRT - PCR and normalized against immunoprecipitated proteins . Levels of immunoprecipitated HA - HuR were detected by western blotting and quantified by densitometry . Data represented as mean + / - s . e . m . , n = 3 .

O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(F) Loss of miRNA association of HuR on MVB. HA-HuR expressing Huh7 cell lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA-HuR and associated miRNA levels were measured by qRT-PCR and normalized against immunoprecipitated proteins. Levels of immunoprecipitated HA-HuR were detected by western blotting and quantified by densitometry. Data represented as mean+/- s.e.m., n=3.


( G ) Loss of miRNA from ubiquitinated form of HuR . Scheme of the double immunoprecipitation experiment is shown in the left panel . Western blot analysis for HuR in the anti - HA and anti - HuRimmunoprecipitatedFLAG - HuR along with the input of FLAG - HuR used . Lysates of cell expressing the FLAG - HuR and used for FLAGHuR purification is also shown ( right upper panels ) . qRT - PCR based estimation of the miRNA per unit of HuRimmunoprecipitated were estimated for both HA and HuRimmunoprecipitated materials and normalized against respective protein levels quantified by densitometric quantification of western blots ( right lower panel ) . Data represented as mean + / - s . e . m . , n = 3 . Position of ubiquitinated band of FLAG - HuR is marked by arrow .

O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O 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-GENEPROD O B-GENEPROD O O O O O

(G) Loss of miRNA from ubiquitinated form of HuR. Scheme of the double immunoprecipitation experiment is shown in the left panel. Western blot analysis for HuR in the anti-HA and anti-HuRimmunoprecipitatedFLAG-HuR along with the input of FLAG-HuR used. Lysates of cell expressing the FLAG-HuR and used for FLAGHuR purification is also shown (right upper panels). qRT-PCR based estimation of the miRNA per unit of HuRimmunoprecipitated were estimated for both HA and HuRimmunoprecipitated materials and normalized against respective protein levels quantified by densitometric quantification of western blots (right lower panel). Data represented as mean+/- s.e.m., n=3. Position of ubiquitinated band of FLAG-HuR is marked by arrow.


( H ) Increase in ubiquitinated form of HuR in Starved Huh7 cells . Levels of ubiquitinated HuR in Fed and Starved ( 5h ) HA - Ub expressing Huh7 cells treated either with ethanol or MG132 ( 20µM ) for same duration . The lysates were immunoprecipitated with HuR and western blotted for HA and HuR . Position of ubiquitinated bands were marked by arrows . The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non - ubiquitinated form were measured and plotted ( mean + / - s . e . m . , n = 3 ) .

O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Increase in ubiquitinated form of HuR in Starved Huh7 cells. Levels of ubiquitinated HuR in Fed and Starved (5h) HA-Ub expressing Huh7 cells treated either with ethanol or MG132 (20µM) for same duration. The lysates were immunoprecipitated with HuR and western blotted for HA and HuR. Position of ubiquitinated bands were marked by arrows. The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non-ubiquitinated form were measured and plotted (mean+/- s.e.m., n=3).


( I ) Full length and a truncated version of HA - HuR without the hinge region ( graphical scheme in upper panel ) was used to score the effect of deletion of hinge region on EV associated miR - 122 content measured by qRT - PCR ( bottom panel ) . Western blots to check expression of HuR and its truncated variants are also shown . " * " denotes the HA - HuR band ( left lower panel ) . Data represented as mean + / - s . e . m . , n = 3 .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O 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) Full length and a truncated version of HA-HuR without the hinge region (graphical scheme in upper panel) was used to score the effect of deletion of hinge region on EV associated miR-122 content measured by qRT-PCR (bottom panel). Western blots to check expression of HuR and its truncated variants are also shown." * " denotes the HA-HuR band (left lower panel). Data represented as mean+/- s.e.m., n=3.


( J ) Ubiquitination status of full length and a truncated version of HA - HuR without the hinge region between RRMII and RRMIII in Huh7 cells . HuR ( full lenght or truncated version ) were immunoprecipitated from cell lysates of Huh7 cells expressing these proteins and the immunoprecipitated materials were western blotted for HA - HuR and ubiquitination . Ubiquinitated HuR bands are marked by arrows . postion of non - ubiquitinated proteins were marked by arrowheads ( upper panel ) . qRT - PCR based estimation of associated miRNA per unit of HA - HuR immunoprecipitated materials were estimated and normalized against respective protein levels quantified by densitometric quantification of western blots ( lower panel ) . Data represented as mean + / - s . e . m . , n = 3 . PCIneo transfected cells were used as control in immunoprecipitation reaction but no amplification of miR - 122 were detected for HA immunoprecipitated materials from pCIneo tranfected cells .

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O O O

(J) Ubiquitination status of full length and a truncated version of HA-HuR without the hinge region between RRMII and RRMIII in Huh7 cells. HuR (full lenght or truncated version) were immunoprecipitated from cell lysates of Huh7 cells expressing these proteins and the immunoprecipitated materials were western blotted for HA-HuR and ubiquitination. Ubiquinitated HuR bands are marked by arrows. postion of non-ubiquitinated proteins were marked by arrowheads (upper panel). qRT-PCR based estimation of associated miRNA per unit of HA-HuR immunoprecipitated materials were estimated and normalized against respective protein levels quantified by densitometric quantification of western blots (lower panel). Data represented as mean+/- s.e.m., n=3. PCIneo transfected cells were used as control in immunoprecipitation reaction but no amplification of miR-122 were detected for HA immunoprecipitated materials from pCIneo tranfected cells.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with ( A ) a murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( white bars ) . Graphs show mean ± SEM from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired t test ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.2) treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with a human macrophage cell line ( U937 ) , treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( white bars ) . Graphs show mean ± SEM from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired t test ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with a human macrophage cell line (U937), treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).


Heat map represents cytokine secretion by in vitro expanded ( C ) murine splenic iNKT cells co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . The heat map shows the average result of two pooled biological replicates .

O O O O O O O O O O O O B-ORGANISM B-TISSUE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O

Heat map represents cytokine secretion by in vitro expanded (C) murine splenic iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.


Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . The heat map shows the average result of two pooled biological replicates .

O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O O

Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.


Wild type C57BL / 6 mice were injected intravenously with thapsigargin - loaded PLGA nanoparticles or vehicle , and 12 h later , hepatic iNKT cells were analysed by flow cytometry . Histograms represent expression of intracellular IL - 4 and IFN - γ levels in hepatic iNKT cells from mice injected with thapsigargin - loaded PLGA nanoparticles ( red histogram ) or vehicle - treated ( grey histogram ) mice . Dot plots represents MFI for intracellular IL - 4 and IFN - γ expression in hepatic iNKT cells from thapsigargin - loaded PLGA nanoparticles ( red dots ) or vehicle - treated ( black dots ) mice . Data plots show mean ± SEM from n = 5 mice per group . * * P ≤ 0 . 001 ( Mann - Whitney U test ) .

O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE B-CELL_TYPE O O B-ORGANISM O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

Wild type C57BL/6 mice were injected intravenously with thapsigargin-loaded PLGA nanoparticles or vehicle, and 12 h later, hepatic iNKT cells were analysed by flow cytometry. Histograms represent expression of intracellular IL-4 and IFN-γ levels in hepatic iNKT cells from mice injected with thapsigargin-loaded PLGA nanoparticles (red histogram) or vehicle-treated (grey histogram) mice. Dot plots represents MFI for intracellular IL-4 and IFN-γ expression in hepatic iNKT cells from thapsigargin-loaded PLGA nanoparticles (red dots) or vehicle-treated (black dots) mice. Data plots show mean ± SEM from n = 5 mice per group. **P≤ 0.001 (Mann-Whitney U test).


Thapsigargin or αGalCer treated J774 . 2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d - specific antibody ( black circles ) or isotype ( red squares ) for 16 hours . The addition of an anti - CD1d antibody blocked the activation of 2C12 demonstrating that the NKT - cell activation was CD1d - specific . Graphs show mean ± SEM from n = 2 biological replicates .

B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O

Thapsigargin or αGalCer treated J774.2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d-specific antibody (black circles) or isotype (red squares) for 16 hours. The addition of an anti-CD1d antibody blocked the activation of 2C12 demonstrating that the NKT-cell activation was CD1d-specific. Graphs show mean ± SEM from n=2 biological replicates.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin - or tunicamycin - treated CD1d knockdown ( white bars ) or untransfected ( black bars ) . Knockdown of CD1d blocked IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O B-ORGANISM B-CELL_TYPE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured murine macrophage cell line (J774.2) treated either with thapsigargin- or tunicamycin-treated CD1d knockdown (white bars) or untransfected (black bars). Knockdown of CD1d blocked IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured either with thapsigargin - or tunicamycin - treated primary BMDM isolated from CD1d - / - ( white bars ) or wild type ( black bars ) mice . 2C12 cocultured with CD1d - / - BMDMs resulted in reduced IL - 2 secretion . Graphs show mean ± SEM from n = 3 biological replicates . * * * P < 0 . 001 ( Unpaired t test ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured either with thapsigargin- or tunicamycin-treated primary BMDM isolated from CD1d-/- (white bars) or wild type (black bars) mice. 2C12 cocultured with CD1d-/- BMDMs resulted in reduced IL-2 secretion. Graphs show mean ± SEM from n=3 biological replicates. ***P < 0.001 (Unpaired t test).


qPCR analyses showing the expression ( fold induction ) of UPR signature genes compared to DMSO in murine macrophage cell lines J774 . 2 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


qPCR analyses showing the expression ( fold induction ) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


Bar graph represents percentage of IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor ( 4μ8c ) ( black bar ) or PERK inhibitor ( GSK ) ( grey bar ) or ATF6α inhibitor ( Ceapin - a7 ) ( red bar ) respectively . Inhibition of IRE1α or PERK resulted in significant reduction in IL - 2 secretion by iNKT hybridoma compared to controls . In total , n = 2 biological replicates were performed ( two and four technical replicates per biological replicate respectively ) . Graphs show mean ± SEM of each biological replicate .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with murine macrophage cell line (J774.2) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor (4μ8c) (black bar) or PERK inhibitor (GSK) (grey bar) or ATF6α inhibitor (Ceapin-a7) (red bar) respectively. Inhibition of IRE1α or PERK resulted in significant reduction in IL-2 secretion by iNKT hybridoma compared to controls. In total, n=2 biological replicates were performed (two and four technical replicates per biological replicate respectively). Graphs show mean ± SEM of each biological replicate.


Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774 . 2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene .

B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O

Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774.2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene.


Bar graph represents percentage of IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with thapsigargin - treated murine IRE1α or PERK knockdown macrophage cell line ( J774 . 2 ) . Knockdown of IRE1α ( black bar ) or PERK ( grey bar ) resulted in significant reduction in IL - 2 secretion by iNKT hybridoma . In total n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with thapsigargin-treated murine IRE1α or PERK knockdown macrophage cell line (J774.2). Knockdown of IRE1α (black bar) or PERK (grey bar) resulted in significant reduction in IL-2 secretion by iNKT hybridoma. In total n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 6 ) were isolated from J774 . 2 macrophages stimulated with thapsigargin , tunicamycin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or tunicamycin - treated ( red bars ) J774 . 2 cells . Polar lipid fractions induce very little IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or tunicamycin-treated (red bars) J774.2 cells. Polar lipid fractions induce very little IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin - treated J774 . 2 at acidic pH and reduced mCD1d loading at neutral pH . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin-treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 5 ) , isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges , were co - cultured with purified NKT1 subsets . Bar graphs represent IFN - γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( white bars ) primary murine macrophages . Polar lipid fractions induce a minimal amount of IFN - γ secretion by the NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± SEM of each biological replicate .

O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-5), isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges, were co-cultured with purified NKT1 subsets. Bar graphs represent IFN-γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 5 ) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents IFN - γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( white bars ) human primary macrophages . Polar lipid fractions induce only minimal amounts of IFN - γ secretion by the NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± SEM of each biological replicate .

O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM B-TISSUE I-TISSUE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents IFN-γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL - 2 secretion by NKT cells . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL-2 secretion by NKT cells. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


qPCR analyses showing the expression ( fold induction ) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774 . 2 stimulated either with thapsigargin or DMSO for 8 hours . In total n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin or DMSO for 8 hours. In total n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells . IL - 2 secretion in UPR - induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin . Graphs show mean ± SEM from n = 3 biological replicates . n . s ( non - significant ) ( One - way ANOVA ) biological replicates .

B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells. IL-2 secretion in UPR-induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin. Graphs show mean ± SEM from n = 3 biological replicates. n.s (non-significant) (One-way ANOVA) biological replicates.


L363 antibody does not impact UPR - induced NKT cell activation . 2C12 NKT cells were co - cultured with ER - stressed macrophages J774 . 2 cells and treated with L363 antibody or isotype control . α - GalCer induced responses served as positive control . In total , n = 2 biological replicates were performed ( Four technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L363 antibody does not impact UPR-induced NKT cell activation. 2C12 NKT cells were co-cultured with ER-stressed macrophages J774.2 cells and treated with L363 antibody or isotype control. α-GalCer induced responses served as positive control. In total, n=2 biological replicates were performed (Four technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Representative confocal 3D images of LysoTracker ( red ) , DAPI ( blue ) , and BODIPY ( green ) stained ATM subsets obtained from eWAT of HFD ( 16 weeks ) treated mice ( n = 8 mice from two independent experiments ) . The merged figures are the result of the overlap of all three fluorochromes . Scale bar , 10 μm .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O B-ORGANISM O O 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 confocal 3D images of LysoTracker (red), DAPI (blue), and BODIPY (green) stained ATM subsets obtained from eWAT of HFD (16 weeks) treated mice (n = 8 mice from two independent experiments). The merged figures are the result of the overlap of all three fluorochromes. Scale bar, 10 μm.


Mito Stress assay of sorted ATM subsets ( red , MHCIIlow ; green , MHCIIhi ; and blue , CD11c + ) from 16 - week - HFD obese mice showing overall oxygen consumption rate ( OCR ) , maximal respiration , spare capacity respiration , basal respiration , ATP production , and non - mitochondrial respiration . OCR data shown are representative of four independent experiments and expressed as mean ± SD ( n = 4 MHCIIlow ; n = 7 MHCIIhi ; n = 5 CD11c + ) , with statistical significance determined using one - way ANOVA test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : no significant difference ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mito Stress assay of sorted ATM subsets (red, MHCIIlow; green, MHCIIhi; and blue, CD11c+) from 16-week-HFD obese mice showing overall oxygen consumption rate (OCR), maximal respiration, spare capacity respiration, basal respiration, ATP production, and non-mitochondrial respiration. OCR data shown are representative of four independent experiments and expressed as mean ± SD (n= 4 MHCIIlow; n= 7 MHCIIhi; n=5 CD11c+), with statistical significance determined using one-way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001; ns: no significant difference).


Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169 - DTR ND - ( A ) or HFD - treated ( 16 weeks ) ( B ) mice . A representative analysis of three independent experiments is shown .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169-DTR ND- (A) or HFD-treated (16 weeks) (B) mice. A representative analysis of three independent experiments is shown.


eWAT was collected from lean ( C ) and obese ( HFD 16 weeks ) ( D ) WT or CD169 - DTR mice after 12 days DT - mediated ATM depletion . Representative pictures of dissected eWAT are shown on the left , the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right , with statistical significance calculated using the Student ' s t - test ( * * P < 0 . 01 and * * * * P < 0 . 0001 ) . The zoomed - in area shows clear , oily lipid accumulation in the absence of ATMs ( lower left part ) .

B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O

eWAT was collected from lean (C) and obese (HFD 16 weeks) (D) WT or CD169-DTR mice after 12 days DT-mediated ATM depletion. Representative pictures of dissected eWAT are shown on the left, the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right, with statistical significance calculated using the Student's t-test (**P < 0.01 and ****P < 0.0001). The zoomed-in area shows clear, oily lipid accumulation in the absence of ATMs (lower left part).


Adipocyte hypertrophy is visible in the absence of ATMs . Representative images of hematoxylin and eosin staining ( H & E ) ( E ) and corresponding adipocyte diameters ( F ) of lean and obese ( HFD 16 weeks ) eWAT from WT or CD169 - DTR mice treated for 7 or 12 days with DT . Scale bar , 50 μm . Each group comprised 3 - 5 mice . Statistical significance was determined using one - way ANOVA test . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; ns : no significant difference . The box and whisker plots show the median value and 10 - 90 percentiles of adipocyte diameters .

B-DISEASE I-DISEASE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE B-EXP_ASSAY O

Adipocyte hypertrophy is visible in the absence of ATMs. Representative images of hematoxylin and eosin staining (H&E) (E) and corresponding adipocyte diameters (F) of lean and obese (HFD 16 weeks) eWAT from WT or CD169-DTR mice treated for 7 or 12 days with DT. Scale bar, 50 μm. Each group comprised 3-5 mice. Statistical significance was determined using one-way ANOVA test. **P < 0.01; ****P < 0.0001; ns: no significant difference. The box and whisker plots show the median value and 10-90 percentiles of adipocyte diameters.