Structured Review

Fisher Scientific leupeptin
In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of <t>leupeptin</t> (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P
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1) Product Images from "Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms"

Article Title: Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00819

In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of leupeptin (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P
Figure Legend Snippet: In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of leupeptin (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P

Techniques Used: In Vivo, In Vitro, Activity Assay, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Protease Inhibitor

2) Product Images from "Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation"

Article Title: Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation

Journal: Autophagy

doi: 10.1080/15548627.2017.1293767

CIP2A is a CMA substrate. (A) Immunoblot for CIP2A and ACTB protein in total protein lysates of NIH-3T3 cells, control (ctrl) or LAMP2A knocked down (LAMP2A [-]), transduced with a plasmid encoding MYC or the empty vector. (B) Densitometric quantification for CIP2A in blots as the one shown in (A). Values are relative to ctrl empty vector cells and normalized to Ponceau S staining (N = 6). ((C)and D) NIH-3T3 MYC-transduced cells, ctrl or LAMP2A (-) 12 h after incubation without additions (None) or in the presence of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin, N/L) or a proteasome inhibitor (MG132). (C) Immunoblot for the indicated proteins. (D) Densitometry analysis of CIP2A in blots as the one shown in C. Values are relative to None condition and normalized to Ponceau S staining (N = 6). In both graphs values are presented as mean ± SEM. Two-way ANOVA and the Bonferroni post-hoc test were used and differences were considered significant for * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ns represents nonsignificant changes.
Figure Legend Snippet: CIP2A is a CMA substrate. (A) Immunoblot for CIP2A and ACTB protein in total protein lysates of NIH-3T3 cells, control (ctrl) or LAMP2A knocked down (LAMP2A [-]), transduced with a plasmid encoding MYC or the empty vector. (B) Densitometric quantification for CIP2A in blots as the one shown in (A). Values are relative to ctrl empty vector cells and normalized to Ponceau S staining (N = 6). ((C)and D) NIH-3T3 MYC-transduced cells, ctrl or LAMP2A (-) 12 h after incubation without additions (None) or in the presence of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin, N/L) or a proteasome inhibitor (MG132). (C) Immunoblot for the indicated proteins. (D) Densitometry analysis of CIP2A in blots as the one shown in C. Values are relative to None condition and normalized to Ponceau S staining (N = 6). In both graphs values are presented as mean ± SEM. Two-way ANOVA and the Bonferroni post-hoc test were used and differences were considered significant for * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ns represents nonsignificant changes.

Techniques Used: Transduction, Plasmid Preparation, Staining, Incubation

MYC is a proteasome substrate and does not undergo degradation by CMA. (A) Immunoblot for the indicated proteins in total cell extracts of NIH-3T3 cells at time zero (Time 0) and 12 h after incubation without additions (None) or in the presence of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin, N/L) or a proteasome inhibitor (MG132) in media supplemented (Serum +) or not (Serum -) with serum. (B) Densitometry analysis of MYC levels in blots as the representative one shown in A. Values are expressed relative to untreated (None) serum + conditions and normalized to Ponceau S staining (N = 6). (C) Immunoblot for the indicated proteins in total cell extracts from MYC-transduced cells, control (ctrl) or LAMP2A knockdown (LAMP2A [-]) treated for 12 h with N/L and MG132 in serum-supplemented media. (D) Densitometry analysis of MYC in blots as the representative one shown in (C). Values are expressed relative to untreated (None) ctrl MYC-transduced cells and normalized to Ponceau S staining (N = 4). (E) Immunoblot for MYC and LAMP1 of homogenates (HOM.) and lysosome-enriched fractions (Lys enr.) isolated from control and LAMP2A (-) cells, transduced with MYC. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is shown as an example of a well-characterized CMA substrate. Right: Densitometry analysis of MYC levels in blots as the one shown here. Values are expressed relative to control homogenates after normalization by Ponceau S staining (N = 2). All values are presented as mean ± SEM. Two-way ANOVA and the Bonferroni post-hoc test were used and differences were considered significant for * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ns represents nonsignificant changes.
Figure Legend Snippet: MYC is a proteasome substrate and does not undergo degradation by CMA. (A) Immunoblot for the indicated proteins in total cell extracts of NIH-3T3 cells at time zero (Time 0) and 12 h after incubation without additions (None) or in the presence of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin, N/L) or a proteasome inhibitor (MG132) in media supplemented (Serum +) or not (Serum -) with serum. (B) Densitometry analysis of MYC levels in blots as the representative one shown in A. Values are expressed relative to untreated (None) serum + conditions and normalized to Ponceau S staining (N = 6). (C) Immunoblot for the indicated proteins in total cell extracts from MYC-transduced cells, control (ctrl) or LAMP2A knockdown (LAMP2A [-]) treated for 12 h with N/L and MG132 in serum-supplemented media. (D) Densitometry analysis of MYC in blots as the representative one shown in (C). Values are expressed relative to untreated (None) ctrl MYC-transduced cells and normalized to Ponceau S staining (N = 4). (E) Immunoblot for MYC and LAMP1 of homogenates (HOM.) and lysosome-enriched fractions (Lys enr.) isolated from control and LAMP2A (-) cells, transduced with MYC. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is shown as an example of a well-characterized CMA substrate. Right: Densitometry analysis of MYC levels in blots as the one shown here. Values are expressed relative to control homogenates after normalization by Ponceau S staining (N = 2). All values are presented as mean ± SEM. Two-way ANOVA and the Bonferroni post-hoc test were used and differences were considered significant for * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ns represents nonsignificant changes.

Techniques Used: Incubation, Staining, Isolation, Transduction

3) Product Images from "Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis"

Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

Journal: Nature cell biology

doi: 10.1038/ncb3166

Cells expressing CMA-mutant PLIN2 accumulate LD. ( a ) Immunoblot for indicated proteins of total cell lysates from untransfected cells (−) or cells transfected with wild-type (WT) or CMA-mutant (MT) PLIN2-GFP. o: overexpressed protein; e: endogenous protein. Representative blots of n=3 independent experiments. ( b ) Cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( c ) Cells expressing WT or MT PLIN2-GFP treated with OL with or without lysosomal inhibitors ammonium chloride and leupeptin (NL). Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( d ) Immunostaining for LAMP1 in cells treated as in (c). Top: Merged images. Bottom: Colocalized pixels in white. Insets: higher magnification. Graph: percentage colocalization of PLIN2-GFP with LAMP1. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Binding of WT and MT PLIN2-GFP from cellular extracts to GST-hsc70 immobilized on agarose beads. Representative blots of n=3 independent experiments. (f , g ) Immunostaining for hsc70 in OL-treated cells expressing WT or MT PLIN2-GFP. Colocalized pixels are in white. Insets: higher magnification. 3D-reconstruction shown in Supplementary Figure 4a . Graph: percentage colocalization of PLIN2-GFP with hsc70. n=5 independent experiments with 40 cells per condition in each experiment. ( h – j ) Live - cell imaging of cells cotransfected with WT or MT PLIN2-GFP and LAMP1-RFP and treated with OL. Sequential frames at 60s intervals are shown. Arrows: LD. Graphs: LD (i) kiss-run events/cell and (j) count/cell, tracking velocity, displacement and displacement rate. Small and large LD clusters defined as
Figure Legend Snippet: Cells expressing CMA-mutant PLIN2 accumulate LD. ( a ) Immunoblot for indicated proteins of total cell lysates from untransfected cells (−) or cells transfected with wild-type (WT) or CMA-mutant (MT) PLIN2-GFP. o: overexpressed protein; e: endogenous protein. Representative blots of n=3 independent experiments. ( b ) Cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( c ) Cells expressing WT or MT PLIN2-GFP treated with OL with or without lysosomal inhibitors ammonium chloride and leupeptin (NL). Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( d ) Immunostaining for LAMP1 in cells treated as in (c). Top: Merged images. Bottom: Colocalized pixels in white. Insets: higher magnification. Graph: percentage colocalization of PLIN2-GFP with LAMP1. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Binding of WT and MT PLIN2-GFP from cellular extracts to GST-hsc70 immobilized on agarose beads. Representative blots of n=3 independent experiments. (f , g ) Immunostaining for hsc70 in OL-treated cells expressing WT or MT PLIN2-GFP. Colocalized pixels are in white. Insets: higher magnification. 3D-reconstruction shown in Supplementary Figure 4a . Graph: percentage colocalization of PLIN2-GFP with hsc70. n=5 independent experiments with 40 cells per condition in each experiment. ( h – j ) Live - cell imaging of cells cotransfected with WT or MT PLIN2-GFP and LAMP1-RFP and treated with OL. Sequential frames at 60s intervals are shown. Arrows: LD. Graphs: LD (i) kiss-run events/cell and (j) count/cell, tracking velocity, displacement and displacement rate. Small and large LD clusters defined as

Techniques Used: Expressing, Mutagenesis, Transfection, Immunostaining, Binding Assay, Live Cell Imaging, Cell Tracking Assay

Failure to remove PLINs by CMA blocks macrolipophagy. ( a ) BODIPY493/503 staining in CTR and L2A(−) cells untreated or treated with OL, or treated with 3-methyladenine (3MA) or lysosomal inhibitors ammonium chloride and leupeptin (NL). Full fields shown in Supplementary Figure 5c . Graph: average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( b , c ) Cells stained as in (a) treated or not with rapamycin (Rapa). Insets: higher magnification. Graph: Average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( d ) Immunoblot for indicated proteins in homogenates (HOM) and lipid droplets (LD) isolated from livers for fed (F) and starved (S) rats. Representative blots of n=5 independent experiments. ( e , f ) Costaining for indicated ATG proteins and BODIPY493/503 in CTR and L2A(−) cells treated with OL. Insets: Higher magnification of the boxed areas with colocalized pixels in white. Graph: percentage colocalization with BODIPY. n=6 independent experiments with 40 cells per condition in each experiment. ( g ) Costaining for Rab7 in the same cells as in (e). Graph: percentage colocalization with BODIPY. n=4 independent experiments with 40 cells per condition in each experiment. ( h ) Immunoblot for indicated proteins of HOM and LD isolated from starved wild-type (+) or L2A knockout (−) mice livers. ( i ) Immunostaining for LC3 in cells expressing WT or MT PLIN2-GFP treated with OL. Inset: Higher magnification of the boxed area with colocalized pixels in white. 3D-reconstruction shown in Supplementary Figure 7c . Graph: percentage colocalization of LC3 with PLIN2-GFP. n=4 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ** P
Figure Legend Snippet: Failure to remove PLINs by CMA blocks macrolipophagy. ( a ) BODIPY493/503 staining in CTR and L2A(−) cells untreated or treated with OL, or treated with 3-methyladenine (3MA) or lysosomal inhibitors ammonium chloride and leupeptin (NL). Full fields shown in Supplementary Figure 5c . Graph: average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( b , c ) Cells stained as in (a) treated or not with rapamycin (Rapa). Insets: higher magnification. Graph: Average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( d ) Immunoblot for indicated proteins in homogenates (HOM) and lipid droplets (LD) isolated from livers for fed (F) and starved (S) rats. Representative blots of n=5 independent experiments. ( e , f ) Costaining for indicated ATG proteins and BODIPY493/503 in CTR and L2A(−) cells treated with OL. Insets: Higher magnification of the boxed areas with colocalized pixels in white. Graph: percentage colocalization with BODIPY. n=6 independent experiments with 40 cells per condition in each experiment. ( g ) Costaining for Rab7 in the same cells as in (e). Graph: percentage colocalization with BODIPY. n=4 independent experiments with 40 cells per condition in each experiment. ( h ) Immunoblot for indicated proteins of HOM and LD isolated from starved wild-type (+) or L2A knockout (−) mice livers. ( i ) Immunostaining for LC3 in cells expressing WT or MT PLIN2-GFP treated with OL. Inset: Higher magnification of the boxed area with colocalized pixels in white. 3D-reconstruction shown in Supplementary Figure 7c . Graph: percentage colocalization of LC3 with PLIN2-GFP. n=4 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ** P

Techniques Used: Staining, Isolation, Knock-Out, Mouse Assay, Immunostaining, Expressing

PLIN2 and PLIN3 are CMA substrates. ( a ) Immunoblot for indicated proteins of total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: PLIN2 levels relative to untreated CTR cells. n=7 independent experiments. ( b ) Immunostaining for PLIN2 in CTR and L2A(−) cells treated as in (a). Graph: average puncta/cell. n=5 independent experiments with 40 cells per condition in each experiment. ( c ) Coimmunostaining for PLIN2 and LAMP1 in CTR and L2A(−) cells treated or not with OL followed by treatment with lysosomal inhibitors, ammonium chloride and leupeptin (NL). Top: Colocalized pixels in white. Bottom: Merged image of the boxed area at higher magnification. ( d ) Graph: percentage colocalization of PLIN2 with LAMP1 from (c). n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins of homogenates (HOM), lysosomes with high (CMA+) and low (CMA−) activity isolated from fed or starved (Stv) rat livers. Representative blots from 5 independent experiments. ( f ) Immunoblot for indicated proteins of isolated lysosome-enriched fractions from OL-treated control and L2A(−) cells. ( g ) Immunoblot for indicated proteins of HOM, CMA+ and CMA− lysosomes isolated from fed or starved (Stv) livers of rat untreated or treated with leupeptin (Leup). Graph: PLIN2 and PLIN3 levels in leupeptin-treated CMA+ lysosomes relative to untreated. Representative blots from 3 independent experiments (other two shown in Supplementary Figure 2h ). Values are mean ± SEM. Differences are significant for * P
Figure Legend Snippet: PLIN2 and PLIN3 are CMA substrates. ( a ) Immunoblot for indicated proteins of total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: PLIN2 levels relative to untreated CTR cells. n=7 independent experiments. ( b ) Immunostaining for PLIN2 in CTR and L2A(−) cells treated as in (a). Graph: average puncta/cell. n=5 independent experiments with 40 cells per condition in each experiment. ( c ) Coimmunostaining for PLIN2 and LAMP1 in CTR and L2A(−) cells treated or not with OL followed by treatment with lysosomal inhibitors, ammonium chloride and leupeptin (NL). Top: Colocalized pixels in white. Bottom: Merged image of the boxed area at higher magnification. ( d ) Graph: percentage colocalization of PLIN2 with LAMP1 from (c). n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins of homogenates (HOM), lysosomes with high (CMA+) and low (CMA−) activity isolated from fed or starved (Stv) rat livers. Representative blots from 5 independent experiments. ( f ) Immunoblot for indicated proteins of isolated lysosome-enriched fractions from OL-treated control and L2A(−) cells. ( g ) Immunoblot for indicated proteins of HOM, CMA+ and CMA− lysosomes isolated from fed or starved (Stv) livers of rat untreated or treated with leupeptin (Leup). Graph: PLIN2 and PLIN3 levels in leupeptin-treated CMA+ lysosomes relative to untreated. Representative blots from 3 independent experiments (other two shown in Supplementary Figure 2h ). Values are mean ± SEM. Differences are significant for * P

Techniques Used: Incubation, Immunostaining, Activity Assay, Isolation

Failure to remove PLINs by CMA blocks lipolysis. ( a ) Beta-oxidation rates in CTR and L2A(−) cells treated with OL followed by lipase inhibitor diethylumbelliferyl phosphate (Deup) and/or lysosomal inhibitors ammonium chloride and leupeptin (NL). n=3 independent experiments. ( b , c ) Costaining for DPH and ATGL in CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) after OL treatment. Colocalized pixels are in white. Right: Higher magnification of boxed area. Graph: percentage colocalization of DPH with ATGL. n=6 independent experiments with 40 cells per condition in each experiment. ( d ) Costaining for BODIPY493/503 and ATGL in CTR cells incubated in OL > S+ treated or not with atypical retinoic acid derivative (aRAd). Middle: Higher magnification area. Bottom: Colocalized pixels. Graph: percentage colocalization of BODIPY with ATGL. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: ATGL levels relative to untreated CTR cells. n=3 independent experiments. ( f , g ) Coimmunoprecipitation (IP) of ATGL in OL > S+ (f) or OL-treated (g) CTR (+) and L2A(−) cells. Cells in (f) are expressing G0S2-GFP. 1/10 input shown. Representative blots from n=3 (f) and 5 (g) independent experiments. ( h, i ) Immunoblot for indicated proteins of homogenates (HOM) and lipid droplets (LD) isolated from fed (F) or starved (S) rat livers (h), or starved wild-type (+) or L2A knockout (−) mice livers (i). Representative blots of n=4 (i; 3 additional blots shown in Supplementary Figure 5c ) and 7(h) independent experiments. ( j ) Immunostaining for ATGL in OL-treated cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graph: percentage colocalization of ATGL with PLIN2-GFP. n=5 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for * P
Figure Legend Snippet: Failure to remove PLINs by CMA blocks lipolysis. ( a ) Beta-oxidation rates in CTR and L2A(−) cells treated with OL followed by lipase inhibitor diethylumbelliferyl phosphate (Deup) and/or lysosomal inhibitors ammonium chloride and leupeptin (NL). n=3 independent experiments. ( b , c ) Costaining for DPH and ATGL in CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) after OL treatment. Colocalized pixels are in white. Right: Higher magnification of boxed area. Graph: percentage colocalization of DPH with ATGL. n=6 independent experiments with 40 cells per condition in each experiment. ( d ) Costaining for BODIPY493/503 and ATGL in CTR cells incubated in OL > S+ treated or not with atypical retinoic acid derivative (aRAd). Middle: Higher magnification area. Bottom: Colocalized pixels. Graph: percentage colocalization of BODIPY with ATGL. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: ATGL levels relative to untreated CTR cells. n=3 independent experiments. ( f , g ) Coimmunoprecipitation (IP) of ATGL in OL > S+ (f) or OL-treated (g) CTR (+) and L2A(−) cells. Cells in (f) are expressing G0S2-GFP. 1/10 input shown. Representative blots from n=3 (f) and 5 (g) independent experiments. ( h, i ) Immunoblot for indicated proteins of homogenates (HOM) and lipid droplets (LD) isolated from fed (F) or starved (S) rat livers (h), or starved wild-type (+) or L2A knockout (−) mice livers (i). Representative blots of n=4 (i; 3 additional blots shown in Supplementary Figure 5c ) and 7(h) independent experiments. ( j ) Immunostaining for ATGL in OL-treated cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graph: percentage colocalization of ATGL with PLIN2-GFP. n=5 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for * P

Techniques Used: Incubation, Expressing, Isolation, Knock-Out, Mouse Assay, Immunostaining

4) Product Images from "Common γ-chain cytokine signaling is required for macroautophagy induction during CD4+ T-cell activation"

Article Title: Common γ-chain cytokine signaling is required for macroautophagy induction during CD4+ T-cell activation

Journal: Autophagy

doi: 10.1080/15548627.2015.1089374

Autophagy is induced by TCR activation. Freshly isolated naïve mouse CD4 + T cells ( A ) or in vitro differentiated effector Th1 ( B ) and Th2 ( C ) cells were incubated in either media alone or stimulated with plate bound anti-CD3 and soluble anti-CD28 antibodies for 12 to 24 h. To measure autophagy flux, ammonium chloride and leupeptin (NL) or vinblastine 100 µM (Vb) were added for the last 3 h of culture. Accumulation of LC3-II in the presence of inhibitors was measured by immunoblot on whole cell lysates. Bar graph represent mean+SEM of autophagy flux, measured as the difference between the intensity of the LC3-II band in cells cultured in the presence or absence of NL, from 6 (naïve) 12 (T H 1) or 10 (T H 2) independent experiments after 20 to 24 h of activation (paired 2-tailed Student t test. **, P
Figure Legend Snippet: Autophagy is induced by TCR activation. Freshly isolated naïve mouse CD4 + T cells ( A ) or in vitro differentiated effector Th1 ( B ) and Th2 ( C ) cells were incubated in either media alone or stimulated with plate bound anti-CD3 and soluble anti-CD28 antibodies for 12 to 24 h. To measure autophagy flux, ammonium chloride and leupeptin (NL) or vinblastine 100 µM (Vb) were added for the last 3 h of culture. Accumulation of LC3-II in the presence of inhibitors was measured by immunoblot on whole cell lysates. Bar graph represent mean+SEM of autophagy flux, measured as the difference between the intensity of the LC3-II band in cells cultured in the presence or absence of NL, from 6 (naïve) 12 (T H 1) or 10 (T H 2) independent experiments after 20 to 24 h of activation (paired 2-tailed Student t test. **, P

Techniques Used: Activation Assay, Isolation, In Vitro, Incubation, Cell Culture

5) Product Images from "NRBF2 regulates macroautophagy as a component of Vps34 Complex I"

Article Title: NRBF2 regulates macroautophagy as a component of Vps34 Complex I

Journal: The Biochemical journal

doi: 10.1042/BJ20140515

NRBF2 knockdown inhibits autophagy induction ( A ) Control or NRBF2-knockdown GFP–LC3 cells were incubated for 16 h in the absence or presence of 10%FBS. For the last 4 h, cells were incubated without or with the lysosomal inhibitors NH 4 Cl (20 mM) and leupeptin (200 µM). The cells were lysed and blotted for p62 and LC3. ( B ) LC3-II levels in each condition were quantified. The data are normalized for the levels of LC3-II in control serum-starved cells, and are means ± S.E.M. from three separate experiments. ( C ) p62 levels in each condition were quantified as above. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KnDn, knockdown; WT, wild-type.
Figure Legend Snippet: NRBF2 knockdown inhibits autophagy induction ( A ) Control or NRBF2-knockdown GFP–LC3 cells were incubated for 16 h in the absence or presence of 10%FBS. For the last 4 h, cells were incubated without or with the lysosomal inhibitors NH 4 Cl (20 mM) and leupeptin (200 µM). The cells were lysed and blotted for p62 and LC3. ( B ) LC3-II levels in each condition were quantified. The data are normalized for the levels of LC3-II in control serum-starved cells, and are means ± S.E.M. from three separate experiments. ( C ) p62 levels in each condition were quantified as above. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KnDn, knockdown; WT, wild-type.

Techniques Used: Incubation

6) Product Images from "Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms"

Article Title: Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00819

In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of leupeptin (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P
Figure Legend Snippet: In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of leupeptin (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P

Techniques Used: In Vivo, In Vitro, Activity Assay, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Protease Inhibitor

Related Articles

Gas Chromatography:

Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis
Article Snippet: .. Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise. .. For imaging experiments, cells were treated with etomoxir (10 μM; Sigma-Aldrich) for 6 h.

Western Blot:

Article Title: NRBF2 regulates macroautophagy as a component of Vps34 Complex I
Article Snippet: .. The lysosome inhibitors NH4 Cl and leupeptin (Fisher Scientific) were used together at 20 mM and 200 µM respectively for 4 h. The lysosomal inhibitor concanamycin A (Sigma–Aldrich) was used at 1 µM for 30 min. Primary antibodies used for immunoprecipitation and Western blotting were as follows: anti-NRBF2 (Cell Signaling Technology), anti-Vps34 for immunoprecipitation [ ], anti-Vps34 for Western blotting [ ], anti-Vps15 [ ], anti-Beclin-1 (BD Biosciences), anti- Atg14L (MBL International), anti-UVRAG (Cell Signaling Technology), anti-LC3 (Cell Signaling Technology), anti-p62 (MBL International), anti-V5 for immunoprecipitation (Thermo Scientific); anti-V5 for Western blotting (Life Technologies), anti-FLAG (Sigma–Aldrich); anti-actin (Sigma–Aldrich), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; MBL International) and anti-(rabbit IgG) (Jackson ImmunoResearch). .. Anti-HA (haemagglutinin) and anti-Myc antibodies were produced in-house.

Labeling:

Article Title: Pro-metastatic NEDD9 regulates individual cell migration via caveolin-1-dependent trafficking of integrins
Article Snippet: .. Cells, pretreated with 10uM MG132, 50uM of Leupeptin (Fisher Scientific) and 0.3mMprimaquine (Sigma) where indicated, were labeled for 30min on ice with Sulfo-NHS-S-S-biotin (Thermo Fisher Sci.) in PBS; followed by incubation at 37°C to allow internalization and recycling of the labeled surface molecules for times as indicated on the figures. .. At each time point, the residual surface biotin was removed by 10 min incubation with 200 mM MESNA (Sigma) three times.

Mouse Assay:

Article Title: Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms
Article Snippet: .. We injected intraperitoneally a single dose of leupeptin 20 mg/kg b.w. (BP2662, Fisher Scientific, Waltham, MA, United States) or saline into 6 h starved mice during dark cycle, and harvested tissues (aorta and liver) 2 h later. .. Atherosclerotic Lesion Analysis To analyze plaque area and composition, the upper aortic root was embedded in Tissue-Tek OCT Compound (Sakura Finetek Europe, Alphen aan den Rijn, Netherlands) and cryosectioned.

Immunoprecipitation:

Article Title: NRBF2 regulates macroautophagy as a component of Vps34 Complex I
Article Snippet: .. The lysosome inhibitors NH4 Cl and leupeptin (Fisher Scientific) were used together at 20 mM and 200 µM respectively for 4 h. The lysosomal inhibitor concanamycin A (Sigma–Aldrich) was used at 1 µM for 30 min. Primary antibodies used for immunoprecipitation and Western blotting were as follows: anti-NRBF2 (Cell Signaling Technology), anti-Vps34 for immunoprecipitation [ ], anti-Vps34 for Western blotting [ ], anti-Vps15 [ ], anti-Beclin-1 (BD Biosciences), anti- Atg14L (MBL International), anti-UVRAG (Cell Signaling Technology), anti-LC3 (Cell Signaling Technology), anti-p62 (MBL International), anti-V5 for immunoprecipitation (Thermo Scientific); anti-V5 for Western blotting (Life Technologies), anti-FLAG (Sigma–Aldrich); anti-actin (Sigma–Aldrich), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; MBL International) and anti-(rabbit IgG) (Jackson ImmunoResearch). .. Anti-HA (haemagglutinin) and anti-Myc antibodies were produced in-house.

Incubation:

Article Title: Pro-metastatic NEDD9 regulates individual cell migration via caveolin-1-dependent trafficking of integrins
Article Snippet: .. Cells, pretreated with 10uM MG132, 50uM of Leupeptin (Fisher Scientific) and 0.3mMprimaquine (Sigma) where indicated, were labeled for 30min on ice with Sulfo-NHS-S-S-biotin (Thermo Fisher Sci.) in PBS; followed by incubation at 37°C to allow internalization and recycling of the labeled surface molecules for times as indicated on the figures. .. At each time point, the residual surface biotin was removed by 10 min incubation with 200 mM MESNA (Sigma) three times.

Blocking Assay:

Article Title: Common γ-chain cytokine signaling is required for macroautophagy induction during CD4+ T-cell activation
Article Snippet: .. For any experimental condition, to measure autophagy flux lysosomal turnover of LC3-II was quantified on immunoblot by comparing the levels of LC3-II in untreated T cells and in T cells that were treated with 20 mM NH4 Cl and 100 µM leupeptin (Fisher scientific, BP2662) or 100 µM vinblastine (LC labs, V-7300) to block lysosomal proteases or autophagosome-lysosome fusion, respectively, for the last 3 h of culture in each specific condition analyzed. ..

Lysis:

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: .. Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4. .. GST and the GST fusion proteins were purified using 5-ml GSTrapFF columns (GE Healthcare) on an AKTA purifier UPC 10 fast-performance liquid chromatography (FPLC) system (GE).

Sonication:

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: .. Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4. .. GST and the GST fusion proteins were purified using 5-ml GSTrapFF columns (GE Healthcare) on an AKTA purifier UPC 10 fast-performance liquid chromatography (FPLC) system (GE).

Injection:

Article Title: Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms
Article Snippet: .. We injected intraperitoneally a single dose of leupeptin 20 mg/kg b.w. (BP2662, Fisher Scientific, Waltham, MA, United States) or saline into 6 h starved mice during dark cycle, and harvested tissues (aorta and liver) 2 h later. .. Atherosclerotic Lesion Analysis To analyze plaque area and composition, the upper aortic root was embedded in Tissue-Tek OCT Compound (Sakura Finetek Europe, Alphen aan den Rijn, Netherlands) and cryosectioned.

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    Fisher Scientific leupeptin
    In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of <t>leupeptin</t> (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P
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    In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of leupeptin (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms

    doi: 10.3389/fphar.2018.00819

    Figure Lengend Snippet: In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of leupeptin (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P

    Article Snippet: We injected intraperitoneally a single dose of leupeptin 20 mg/kg b.w. (BP2662, Fisher Scientific, Waltham, MA, United States) or saline into 6 h starved mice during dark cycle, and harvested tissues (aorta and liver) 2 h later.

    Techniques: In Vivo, In Vitro, Activity Assay, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Protease Inhibitor

    CIP2A is a CMA substrate. (A) Immunoblot for CIP2A and ACTB protein in total protein lysates of NIH-3T3 cells, control (ctrl) or LAMP2A knocked down (LAMP2A [-]), transduced with a plasmid encoding MYC or the empty vector. (B) Densitometric quantification for CIP2A in blots as the one shown in (A). Values are relative to ctrl empty vector cells and normalized to Ponceau S staining (N = 6). ((C)and D) NIH-3T3 MYC-transduced cells, ctrl or LAMP2A (-) 12 h after incubation without additions (None) or in the presence of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin, N/L) or a proteasome inhibitor (MG132). (C) Immunoblot for the indicated proteins. (D) Densitometry analysis of CIP2A in blots as the one shown in C. Values are relative to None condition and normalized to Ponceau S staining (N = 6). In both graphs values are presented as mean ± SEM. Two-way ANOVA and the Bonferroni post-hoc test were used and differences were considered significant for * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ns represents nonsignificant changes.

    Journal: Autophagy

    Article Title: Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation

    doi: 10.1080/15548627.2017.1293767

    Figure Lengend Snippet: CIP2A is a CMA substrate. (A) Immunoblot for CIP2A and ACTB protein in total protein lysates of NIH-3T3 cells, control (ctrl) or LAMP2A knocked down (LAMP2A [-]), transduced with a plasmid encoding MYC or the empty vector. (B) Densitometric quantification for CIP2A in blots as the one shown in (A). Values are relative to ctrl empty vector cells and normalized to Ponceau S staining (N = 6). ((C)and D) NIH-3T3 MYC-transduced cells, ctrl or LAMP2A (-) 12 h after incubation without additions (None) or in the presence of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin, N/L) or a proteasome inhibitor (MG132). (C) Immunoblot for the indicated proteins. (D) Densitometry analysis of CIP2A in blots as the one shown in C. Values are relative to None condition and normalized to Ponceau S staining (N = 6). In both graphs values are presented as mean ± SEM. Two-way ANOVA and the Bonferroni post-hoc test were used and differences were considered significant for * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ns represents nonsignificant changes.

    Article Snippet: For degradation assays, cells where treated with lysosomal proteolysis inhibitors ammonium chloride (20 mM; Sigma-Aldrich, A9434) and leupeptin (100 µM; Fisher Scientific, BP26621) or the proteasome inhibitor MG132 (10 µM; Sigma-Aldrich, M7449) for 12 h in the presence or absence of serum, as indicated.

    Techniques: Transduction, Plasmid Preparation, Staining, Incubation

    MYC is a proteasome substrate and does not undergo degradation by CMA. (A) Immunoblot for the indicated proteins in total cell extracts of NIH-3T3 cells at time zero (Time 0) and 12 h after incubation without additions (None) or in the presence of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin, N/L) or a proteasome inhibitor (MG132) in media supplemented (Serum +) or not (Serum -) with serum. (B) Densitometry analysis of MYC levels in blots as the representative one shown in A. Values are expressed relative to untreated (None) serum + conditions and normalized to Ponceau S staining (N = 6). (C) Immunoblot for the indicated proteins in total cell extracts from MYC-transduced cells, control (ctrl) or LAMP2A knockdown (LAMP2A [-]) treated for 12 h with N/L and MG132 in serum-supplemented media. (D) Densitometry analysis of MYC in blots as the representative one shown in (C). Values are expressed relative to untreated (None) ctrl MYC-transduced cells and normalized to Ponceau S staining (N = 4). (E) Immunoblot for MYC and LAMP1 of homogenates (HOM.) and lysosome-enriched fractions (Lys enr.) isolated from control and LAMP2A (-) cells, transduced with MYC. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is shown as an example of a well-characterized CMA substrate. Right: Densitometry analysis of MYC levels in blots as the one shown here. Values are expressed relative to control homogenates after normalization by Ponceau S staining (N = 2). All values are presented as mean ± SEM. Two-way ANOVA and the Bonferroni post-hoc test were used and differences were considered significant for * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ns represents nonsignificant changes.

    Journal: Autophagy

    Article Title: Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation

    doi: 10.1080/15548627.2017.1293767

    Figure Lengend Snippet: MYC is a proteasome substrate and does not undergo degradation by CMA. (A) Immunoblot for the indicated proteins in total cell extracts of NIH-3T3 cells at time zero (Time 0) and 12 h after incubation without additions (None) or in the presence of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin, N/L) or a proteasome inhibitor (MG132) in media supplemented (Serum +) or not (Serum -) with serum. (B) Densitometry analysis of MYC levels in blots as the representative one shown in A. Values are expressed relative to untreated (None) serum + conditions and normalized to Ponceau S staining (N = 6). (C) Immunoblot for the indicated proteins in total cell extracts from MYC-transduced cells, control (ctrl) or LAMP2A knockdown (LAMP2A [-]) treated for 12 h with N/L and MG132 in serum-supplemented media. (D) Densitometry analysis of MYC in blots as the representative one shown in (C). Values are expressed relative to untreated (None) ctrl MYC-transduced cells and normalized to Ponceau S staining (N = 4). (E) Immunoblot for MYC and LAMP1 of homogenates (HOM.) and lysosome-enriched fractions (Lys enr.) isolated from control and LAMP2A (-) cells, transduced with MYC. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is shown as an example of a well-characterized CMA substrate. Right: Densitometry analysis of MYC levels in blots as the one shown here. Values are expressed relative to control homogenates after normalization by Ponceau S staining (N = 2). All values are presented as mean ± SEM. Two-way ANOVA and the Bonferroni post-hoc test were used and differences were considered significant for * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. ns represents nonsignificant changes.

    Article Snippet: For degradation assays, cells where treated with lysosomal proteolysis inhibitors ammonium chloride (20 mM; Sigma-Aldrich, A9434) and leupeptin (100 µM; Fisher Scientific, BP26621) or the proteasome inhibitor MG132 (10 µM; Sigma-Aldrich, M7449) for 12 h in the presence or absence of serum, as indicated.

    Techniques: Incubation, Staining, Isolation, Transduction

    Cells expressing CMA-mutant PLIN2 accumulate LD. ( a ) Immunoblot for indicated proteins of total cell lysates from untransfected cells (−) or cells transfected with wild-type (WT) or CMA-mutant (MT) PLIN2-GFP. o: overexpressed protein; e: endogenous protein. Representative blots of n=3 independent experiments. ( b ) Cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( c ) Cells expressing WT or MT PLIN2-GFP treated with OL with or without lysosomal inhibitors ammonium chloride and leupeptin (NL). Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( d ) Immunostaining for LAMP1 in cells treated as in (c). Top: Merged images. Bottom: Colocalized pixels in white. Insets: higher magnification. Graph: percentage colocalization of PLIN2-GFP with LAMP1. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Binding of WT and MT PLIN2-GFP from cellular extracts to GST-hsc70 immobilized on agarose beads. Representative blots of n=3 independent experiments. (f , g ) Immunostaining for hsc70 in OL-treated cells expressing WT or MT PLIN2-GFP. Colocalized pixels are in white. Insets: higher magnification. 3D-reconstruction shown in Supplementary Figure 4a . Graph: percentage colocalization of PLIN2-GFP with hsc70. n=5 independent experiments with 40 cells per condition in each experiment. ( h – j ) Live - cell imaging of cells cotransfected with WT or MT PLIN2-GFP and LAMP1-RFP and treated with OL. Sequential frames at 60s intervals are shown. Arrows: LD. Graphs: LD (i) kiss-run events/cell and (j) count/cell, tracking velocity, displacement and displacement rate. Small and large LD clusters defined as

    Journal: Nature cell biology

    Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    doi: 10.1038/ncb3166

    Figure Lengend Snippet: Cells expressing CMA-mutant PLIN2 accumulate LD. ( a ) Immunoblot for indicated proteins of total cell lysates from untransfected cells (−) or cells transfected with wild-type (WT) or CMA-mutant (MT) PLIN2-GFP. o: overexpressed protein; e: endogenous protein. Representative blots of n=3 independent experiments. ( b ) Cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( c ) Cells expressing WT or MT PLIN2-GFP treated with OL with or without lysosomal inhibitors ammonium chloride and leupeptin (NL). Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( d ) Immunostaining for LAMP1 in cells treated as in (c). Top: Merged images. Bottom: Colocalized pixels in white. Insets: higher magnification. Graph: percentage colocalization of PLIN2-GFP with LAMP1. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Binding of WT and MT PLIN2-GFP from cellular extracts to GST-hsc70 immobilized on agarose beads. Representative blots of n=3 independent experiments. (f , g ) Immunostaining for hsc70 in OL-treated cells expressing WT or MT PLIN2-GFP. Colocalized pixels are in white. Insets: higher magnification. 3D-reconstruction shown in Supplementary Figure 4a . Graph: percentage colocalization of PLIN2-GFP with hsc70. n=5 independent experiments with 40 cells per condition in each experiment. ( h – j ) Live - cell imaging of cells cotransfected with WT or MT PLIN2-GFP and LAMP1-RFP and treated with OL. Sequential frames at 60s intervals are shown. Arrows: LD. Graphs: LD (i) kiss-run events/cell and (j) count/cell, tracking velocity, displacement and displacement rate. Small and large LD clusters defined as

    Article Snippet: Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise.

    Techniques: Expressing, Mutagenesis, Transfection, Immunostaining, Binding Assay, Live Cell Imaging, Cell Tracking Assay

    Failure to remove PLINs by CMA blocks macrolipophagy. ( a ) BODIPY493/503 staining in CTR and L2A(−) cells untreated or treated with OL, or treated with 3-methyladenine (3MA) or lysosomal inhibitors ammonium chloride and leupeptin (NL). Full fields shown in Supplementary Figure 5c . Graph: average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( b , c ) Cells stained as in (a) treated or not with rapamycin (Rapa). Insets: higher magnification. Graph: Average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( d ) Immunoblot for indicated proteins in homogenates (HOM) and lipid droplets (LD) isolated from livers for fed (F) and starved (S) rats. Representative blots of n=5 independent experiments. ( e , f ) Costaining for indicated ATG proteins and BODIPY493/503 in CTR and L2A(−) cells treated with OL. Insets: Higher magnification of the boxed areas with colocalized pixels in white. Graph: percentage colocalization with BODIPY. n=6 independent experiments with 40 cells per condition in each experiment. ( g ) Costaining for Rab7 in the same cells as in (e). Graph: percentage colocalization with BODIPY. n=4 independent experiments with 40 cells per condition in each experiment. ( h ) Immunoblot for indicated proteins of HOM and LD isolated from starved wild-type (+) or L2A knockout (−) mice livers. ( i ) Immunostaining for LC3 in cells expressing WT or MT PLIN2-GFP treated with OL. Inset: Higher magnification of the boxed area with colocalized pixels in white. 3D-reconstruction shown in Supplementary Figure 7c . Graph: percentage colocalization of LC3 with PLIN2-GFP. n=4 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ** P

    Journal: Nature cell biology

    Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    doi: 10.1038/ncb3166

    Figure Lengend Snippet: Failure to remove PLINs by CMA blocks macrolipophagy. ( a ) BODIPY493/503 staining in CTR and L2A(−) cells untreated or treated with OL, or treated with 3-methyladenine (3MA) or lysosomal inhibitors ammonium chloride and leupeptin (NL). Full fields shown in Supplementary Figure 5c . Graph: average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( b , c ) Cells stained as in (a) treated or not with rapamycin (Rapa). Insets: higher magnification. Graph: Average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( d ) Immunoblot for indicated proteins in homogenates (HOM) and lipid droplets (LD) isolated from livers for fed (F) and starved (S) rats. Representative blots of n=5 independent experiments. ( e , f ) Costaining for indicated ATG proteins and BODIPY493/503 in CTR and L2A(−) cells treated with OL. Insets: Higher magnification of the boxed areas with colocalized pixels in white. Graph: percentage colocalization with BODIPY. n=6 independent experiments with 40 cells per condition in each experiment. ( g ) Costaining for Rab7 in the same cells as in (e). Graph: percentage colocalization with BODIPY. n=4 independent experiments with 40 cells per condition in each experiment. ( h ) Immunoblot for indicated proteins of HOM and LD isolated from starved wild-type (+) or L2A knockout (−) mice livers. ( i ) Immunostaining for LC3 in cells expressing WT or MT PLIN2-GFP treated with OL. Inset: Higher magnification of the boxed area with colocalized pixels in white. 3D-reconstruction shown in Supplementary Figure 7c . Graph: percentage colocalization of LC3 with PLIN2-GFP. n=4 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ** P

    Article Snippet: Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise.

    Techniques: Staining, Isolation, Knock-Out, Mouse Assay, Immunostaining, Expressing

    PLIN2 and PLIN3 are CMA substrates. ( a ) Immunoblot for indicated proteins of total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: PLIN2 levels relative to untreated CTR cells. n=7 independent experiments. ( b ) Immunostaining for PLIN2 in CTR and L2A(−) cells treated as in (a). Graph: average puncta/cell. n=5 independent experiments with 40 cells per condition in each experiment. ( c ) Coimmunostaining for PLIN2 and LAMP1 in CTR and L2A(−) cells treated or not with OL followed by treatment with lysosomal inhibitors, ammonium chloride and leupeptin (NL). Top: Colocalized pixels in white. Bottom: Merged image of the boxed area at higher magnification. ( d ) Graph: percentage colocalization of PLIN2 with LAMP1 from (c). n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins of homogenates (HOM), lysosomes with high (CMA+) and low (CMA−) activity isolated from fed or starved (Stv) rat livers. Representative blots from 5 independent experiments. ( f ) Immunoblot for indicated proteins of isolated lysosome-enriched fractions from OL-treated control and L2A(−) cells. ( g ) Immunoblot for indicated proteins of HOM, CMA+ and CMA− lysosomes isolated from fed or starved (Stv) livers of rat untreated or treated with leupeptin (Leup). Graph: PLIN2 and PLIN3 levels in leupeptin-treated CMA+ lysosomes relative to untreated. Representative blots from 3 independent experiments (other two shown in Supplementary Figure 2h ). Values are mean ± SEM. Differences are significant for * P

    Journal: Nature cell biology

    Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    doi: 10.1038/ncb3166

    Figure Lengend Snippet: PLIN2 and PLIN3 are CMA substrates. ( a ) Immunoblot for indicated proteins of total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: PLIN2 levels relative to untreated CTR cells. n=7 independent experiments. ( b ) Immunostaining for PLIN2 in CTR and L2A(−) cells treated as in (a). Graph: average puncta/cell. n=5 independent experiments with 40 cells per condition in each experiment. ( c ) Coimmunostaining for PLIN2 and LAMP1 in CTR and L2A(−) cells treated or not with OL followed by treatment with lysosomal inhibitors, ammonium chloride and leupeptin (NL). Top: Colocalized pixels in white. Bottom: Merged image of the boxed area at higher magnification. ( d ) Graph: percentage colocalization of PLIN2 with LAMP1 from (c). n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins of homogenates (HOM), lysosomes with high (CMA+) and low (CMA−) activity isolated from fed or starved (Stv) rat livers. Representative blots from 5 independent experiments. ( f ) Immunoblot for indicated proteins of isolated lysosome-enriched fractions from OL-treated control and L2A(−) cells. ( g ) Immunoblot for indicated proteins of HOM, CMA+ and CMA− lysosomes isolated from fed or starved (Stv) livers of rat untreated or treated with leupeptin (Leup). Graph: PLIN2 and PLIN3 levels in leupeptin-treated CMA+ lysosomes relative to untreated. Representative blots from 3 independent experiments (other two shown in Supplementary Figure 2h ). Values are mean ± SEM. Differences are significant for * P

    Article Snippet: Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise.

    Techniques: Incubation, Immunostaining, Activity Assay, Isolation

    Failure to remove PLINs by CMA blocks lipolysis. ( a ) Beta-oxidation rates in CTR and L2A(−) cells treated with OL followed by lipase inhibitor diethylumbelliferyl phosphate (Deup) and/or lysosomal inhibitors ammonium chloride and leupeptin (NL). n=3 independent experiments. ( b , c ) Costaining for DPH and ATGL in CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) after OL treatment. Colocalized pixels are in white. Right: Higher magnification of boxed area. Graph: percentage colocalization of DPH with ATGL. n=6 independent experiments with 40 cells per condition in each experiment. ( d ) Costaining for BODIPY493/503 and ATGL in CTR cells incubated in OL > S+ treated or not with atypical retinoic acid derivative (aRAd). Middle: Higher magnification area. Bottom: Colocalized pixels. Graph: percentage colocalization of BODIPY with ATGL. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: ATGL levels relative to untreated CTR cells. n=3 independent experiments. ( f , g ) Coimmunoprecipitation (IP) of ATGL in OL > S+ (f) or OL-treated (g) CTR (+) and L2A(−) cells. Cells in (f) are expressing G0S2-GFP. 1/10 input shown. Representative blots from n=3 (f) and 5 (g) independent experiments. ( h, i ) Immunoblot for indicated proteins of homogenates (HOM) and lipid droplets (LD) isolated from fed (F) or starved (S) rat livers (h), or starved wild-type (+) or L2A knockout (−) mice livers (i). Representative blots of n=4 (i; 3 additional blots shown in Supplementary Figure 5c ) and 7(h) independent experiments. ( j ) Immunostaining for ATGL in OL-treated cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graph: percentage colocalization of ATGL with PLIN2-GFP. n=5 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for * P

    Journal: Nature cell biology

    Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    doi: 10.1038/ncb3166

    Figure Lengend Snippet: Failure to remove PLINs by CMA blocks lipolysis. ( a ) Beta-oxidation rates in CTR and L2A(−) cells treated with OL followed by lipase inhibitor diethylumbelliferyl phosphate (Deup) and/or lysosomal inhibitors ammonium chloride and leupeptin (NL). n=3 independent experiments. ( b , c ) Costaining for DPH and ATGL in CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) after OL treatment. Colocalized pixels are in white. Right: Higher magnification of boxed area. Graph: percentage colocalization of DPH with ATGL. n=6 independent experiments with 40 cells per condition in each experiment. ( d ) Costaining for BODIPY493/503 and ATGL in CTR cells incubated in OL > S+ treated or not with atypical retinoic acid derivative (aRAd). Middle: Higher magnification area. Bottom: Colocalized pixels. Graph: percentage colocalization of BODIPY with ATGL. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: ATGL levels relative to untreated CTR cells. n=3 independent experiments. ( f , g ) Coimmunoprecipitation (IP) of ATGL in OL > S+ (f) or OL-treated (g) CTR (+) and L2A(−) cells. Cells in (f) are expressing G0S2-GFP. 1/10 input shown. Representative blots from n=3 (f) and 5 (g) independent experiments. ( h, i ) Immunoblot for indicated proteins of homogenates (HOM) and lipid droplets (LD) isolated from fed (F) or starved (S) rat livers (h), or starved wild-type (+) or L2A knockout (−) mice livers (i). Representative blots of n=4 (i; 3 additional blots shown in Supplementary Figure 5c ) and 7(h) independent experiments. ( j ) Immunostaining for ATGL in OL-treated cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graph: percentage colocalization of ATGL with PLIN2-GFP. n=5 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for * P

    Article Snippet: Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise.

    Techniques: Incubation, Expressing, Isolation, Knock-Out, Mouse Assay, Immunostaining

    Autophagy is induced by TCR activation. Freshly isolated naïve mouse CD4 + T cells ( A ) or in vitro differentiated effector Th1 ( B ) and Th2 ( C ) cells were incubated in either media alone or stimulated with plate bound anti-CD3 and soluble anti-CD28 antibodies for 12 to 24 h. To measure autophagy flux, ammonium chloride and leupeptin (NL) or vinblastine 100 µM (Vb) were added for the last 3 h of culture. Accumulation of LC3-II in the presence of inhibitors was measured by immunoblot on whole cell lysates. Bar graph represent mean+SEM of autophagy flux, measured as the difference between the intensity of the LC3-II band in cells cultured in the presence or absence of NL, from 6 (naïve) 12 (T H 1) or 10 (T H 2) independent experiments after 20 to 24 h of activation (paired 2-tailed Student t test. **, P

    Journal: Autophagy

    Article Title: Common γ-chain cytokine signaling is required for macroautophagy induction during CD4+ T-cell activation

    doi: 10.1080/15548627.2015.1089374

    Figure Lengend Snippet: Autophagy is induced by TCR activation. Freshly isolated naïve mouse CD4 + T cells ( A ) or in vitro differentiated effector Th1 ( B ) and Th2 ( C ) cells were incubated in either media alone or stimulated with plate bound anti-CD3 and soluble anti-CD28 antibodies for 12 to 24 h. To measure autophagy flux, ammonium chloride and leupeptin (NL) or vinblastine 100 µM (Vb) were added for the last 3 h of culture. Accumulation of LC3-II in the presence of inhibitors was measured by immunoblot on whole cell lysates. Bar graph represent mean+SEM of autophagy flux, measured as the difference between the intensity of the LC3-II band in cells cultured in the presence or absence of NL, from 6 (naïve) 12 (T H 1) or 10 (T H 2) independent experiments after 20 to 24 h of activation (paired 2-tailed Student t test. **, P

    Article Snippet: For any experimental condition, to measure autophagy flux lysosomal turnover of LC3-II was quantified on immunoblot by comparing the levels of LC3-II in untreated T cells and in T cells that were treated with 20 mM NH4 Cl and 100 µM leupeptin (Fisher scientific, BP2662) or 100 µM vinblastine (LC labs, V-7300) to block lysosomal proteases or autophagosome-lysosome fusion, respectively, for the last 3 h of culture in each specific condition analyzed.

    Techniques: Activation Assay, Isolation, In Vitro, Incubation, Cell Culture