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$HSPB1 interacts <t>with</t> <t>p62/SQSTM1</t> and supports synergistically its unconventional secretion. ( A ) HeLa cells were transiently transfected with V5-HSPB1-V5 and HA-p62/SQSTM1. Twenty four hours after transfection, cell lysates were prepared and immunoprecipitation was performed using either anti-V5 or anti-HA antibody. HSPB1 and p62/SQSTM1 interaction was detected in both orientations. In addition, an unconjugated control IgG antibody was used to verify the specificity of the interaction (lane 4 of the top and bottom panels). ( B – C ) HeLa cells were transiently transfected with GFP-HSPB1 and HA-p62/SQSTM1. After 24 h, cells were semi-permeabilized by digitonin treatment, fixed, subjected to immunofluorescence and analysed by confocal microscopy. Magnifications (10×) of the merged channels are shown in the insets. The co-localization analysis was performed using an ImageJ plug-in (JaCoP). The graph shows Mander’s coefficient (fraction of HSPB1-positive structures overlapping with p62-positive structures and reverse, grey bars). Scale bars: 10 μm. ( D ) HeLa cells were placed in DMEM 1% FBS for 8 h, then secreted fraction and cell lysate were collected. Secreted fraction was centrifuged at 100 000 g for 2 h. Pellet (P100) was resuspended in lysis buffer and processed for western blot analysis using a p62/SQSTM1 antibody. P62/SQSTM1 is present in the P100 fraction and in the cell lysate but not in the S100. ( E – F ) HeLa cells were transfected with HSPB1-V5, P62-HA or both. After 24 h, the cells were placed in 1% FBS for 8 h to allow protein secretion. Both cell lysate and secreted fraction were collected and analysed. p62/SQSTM1 secretion is increased by HSPB1 over-expression as shown also in the quantification (F). ( G – H ) HeLa cells were co-transfected with V5-HSPB1 and HA-p62/SQSTM1. Twenty four hours after transfection, cells were washed three times and placed in serum-free DMEM (NO FBS) for the indicated times (from 0 to 24 h). Secreted fractions were collected from 0.5 up to 24 h. Both HSPB1 and p62/SQSTM1 secretion increases over time, but the amount of p62/SQSTM1 is decreased after 24 h. ( I – J ) Schematic diagram depicting the experimental protocol deployed to assess the dynamic variations of HSPB1 and p62/SQSTM1 secretion upon serum deprivation. HeLa cells were serum starved or left in complete medium. Then cells were placed in 1% FBS or NO FBS for 8 h to allow protein secretion and 1 h before collection 1% FBS was added to the rescue condition. Cell lysates were collected and analysed using the indicated antibodies. AKT phosphorylation is reduced by serum deprivation and is increased again after re-addition of FBS. ( K – L ) HeLa cells were transiently transfected with V5-HSPB1 and HA-p62/SQSTM1. After 24 h, cells were serum starved or left in complete medium. Then, cells were placed in 1% FBS for 8 h to allow protein secretion, and 1 h before collection, FBS was added back to the rescue condition. p62/SQSTM1 secretion is increased after serum starvation and the secretion is reversible. The graphs in (F), (H) and (L) report the quantitative analysis of HSPB1 and p62/SQSTM1 protein secretion relative to loading control from at least three independent experiments. The y -axis values are shown as the OUT/IN relative ratio and the error bars denote standard deviations. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.001; NS: non-significant).$
P62 Sqstm1 Construct, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein"

Article Title: The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddad047

$HSPB1 interacts with p62/SQSTM1 and supports synergistically its unconventional secretion. ( A ) HeLa cells were transiently transfected with V5-HSPB1-V5 and HA-p62/SQSTM1. Twenty four hours after transfection, cell lysates were prepared and immunoprecipitation was performed using either anti-V5 or anti-HA antibody. HSPB1 and p62/SQSTM1 interaction was detected in both orientations. In addition, an unconjugated control IgG antibody was used to verify the specificity of the interaction (lane 4 of the top and bottom panels). ( B – C ) HeLa cells were transiently transfected with GFP-HSPB1 and HA-p62/SQSTM1. After 24 h, cells were semi-permeabilized by digitonin treatment, fixed, subjected to immunofluorescence and analysed by confocal microscopy. Magnifications (10×) of the merged channels are shown in the insets. The co-localization analysis was performed using an ImageJ plug-in (JaCoP). The graph shows Mander’s coefficient (fraction of HSPB1-positive structures overlapping with p62-positive structures and reverse, grey bars). Scale bars: 10 μm. ( D ) HeLa cells were placed in DMEM 1% FBS for 8 h, then secreted fraction and cell lysate were collected. Secreted fraction was centrifuged at 100 000 g for 2 h. Pellet (P100) was resuspended in lysis buffer and processed for western blot analysis using a p62/SQSTM1 antibody. P62/SQSTM1 is present in the P100 fraction and in the cell lysate but not in the S100. ( E – F ) HeLa cells were transfected with HSPB1-V5, P62-HA or both. After 24 h, the cells were placed in 1% FBS for 8 h to allow protein secretion. Both cell lysate and secreted fraction were collected and analysed. p62/SQSTM1 secretion is increased by HSPB1 over-expression as shown also in the quantification (F). ( G – H ) HeLa cells were co-transfected with V5-HSPB1 and HA-p62/SQSTM1. Twenty four hours after transfection, cells were washed three times and placed in serum-free DMEM (NO FBS) for the indicated times (from 0 to 24 h). Secreted fractions were collected from 0.5 up to 24 h. Both HSPB1 and p62/SQSTM1 secretion increases over time, but the amount of p62/SQSTM1 is decreased after 24 h. ( I – J ) Schematic diagram depicting the experimental protocol deployed to assess the dynamic variations of HSPB1 and p62/SQSTM1 secretion upon serum deprivation. HeLa cells were serum starved or left in complete medium. Then cells were placed in 1% FBS or NO FBS for 8 h to allow protein secretion and 1 h before collection 1% FBS was added to the rescue condition. Cell lysates were collected and analysed using the indicated antibodies. AKT phosphorylation is reduced by serum deprivation and is increased again after re-addition of FBS. ( K – L ) HeLa cells were transiently transfected with V5-HSPB1 and HA-p62/SQSTM1. After 24 h, cells were serum starved or left in complete medium. Then, cells were placed in 1% FBS for 8 h to allow protein secretion, and 1 h before collection, FBS was added back to the rescue condition. p62/SQSTM1 secretion is increased after serum starvation and the secretion is reversible. The graphs in (F), (H) and (L) report the quantitative analysis of HSPB1 and p62/SQSTM1 protein secretion relative to loading control from at least three independent experiments. The y -axis values are shown as the OUT/IN relative ratio and the error bars denote standard deviations. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.001; NS: non-significant).$
Figure Legend Snippet: HSPB1 interacts with p62/SQSTM1 and supports synergistically its unconventional secretion. ( A ) HeLa cells were transiently transfected with V5-HSPB1-V5 and HA-p62/SQSTM1. Twenty four hours after transfection, cell lysates were prepared and immunoprecipitation was performed using either anti-V5 or anti-HA antibody. HSPB1 and p62/SQSTM1 interaction was detected in both orientations. In addition, an unconjugated control IgG antibody was used to verify the specificity of the interaction (lane 4 of the top and bottom panels). ( B – C ) HeLa cells were transiently transfected with GFP-HSPB1 and HA-p62/SQSTM1. After 24 h, cells were semi-permeabilized by digitonin treatment, fixed, subjected to immunofluorescence and analysed by confocal microscopy. Magnifications (10×) of the merged channels are shown in the insets. The co-localization analysis was performed using an ImageJ plug-in (JaCoP). The graph shows Mander’s coefficient (fraction of HSPB1-positive structures overlapping with p62-positive structures and reverse, grey bars). Scale bars: 10 μm. ( D ) HeLa cells were placed in DMEM 1% FBS for 8 h, then secreted fraction and cell lysate were collected. Secreted fraction was centrifuged at 100 000 g for 2 h. Pellet (P100) was resuspended in lysis buffer and processed for western blot analysis using a p62/SQSTM1 antibody. P62/SQSTM1 is present in the P100 fraction and in the cell lysate but not in the S100. ( E – F ) HeLa cells were transfected with HSPB1-V5, P62-HA or both. After 24 h, the cells were placed in 1% FBS for 8 h to allow protein secretion. Both cell lysate and secreted fraction were collected and analysed. p62/SQSTM1 secretion is increased by HSPB1 over-expression as shown also in the quantification (F). ( G – H ) HeLa cells were co-transfected with V5-HSPB1 and HA-p62/SQSTM1. Twenty four hours after transfection, cells were washed three times and placed in serum-free DMEM (NO FBS) for the indicated times (from 0 to 24 h). Secreted fractions were collected from 0.5 up to 24 h. Both HSPB1 and p62/SQSTM1 secretion increases over time, but the amount of p62/SQSTM1 is decreased after 24 h. ( I – J ) Schematic diagram depicting the experimental protocol deployed to assess the dynamic variations of HSPB1 and p62/SQSTM1 secretion upon serum deprivation. HeLa cells were serum starved or left in complete medium. Then cells were placed in 1% FBS or NO FBS for 8 h to allow protein secretion and 1 h before collection 1% FBS was added to the rescue condition. Cell lysates were collected and analysed using the indicated antibodies. AKT phosphorylation is reduced by serum deprivation and is increased again after re-addition of FBS. ( K – L ) HeLa cells were transiently transfected with V5-HSPB1 and HA-p62/SQSTM1. After 24 h, cells were serum starved or left in complete medium. Then, cells were placed in 1% FBS for 8 h to allow protein secretion, and 1 h before collection, FBS was added back to the rescue condition. p62/SQSTM1 secretion is increased after serum starvation and the secretion is reversible. The graphs in (F), (H) and (L) report the quantitative analysis of HSPB1 and p62/SQSTM1 protein secretion relative to loading control from at least three independent experiments. The y -axis values are shown as the OUT/IN relative ratio and the error bars denote standard deviations. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.001; NS: non-significant).

Techniques Used: Transfection, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Lysis, Western Blot, Over Expression

$Effect of HSPB1 mutants on the unconventional secretion of p62/SQSTM1. ( A – B ) HeLa cells were transiently transfected with 3A-HSPB1 and 3D-HSPB1 phosphorylation mutants. After 24 h, cells were placed in DMEM 1% FBS for 8 h and secreted fractions were collected, pre-cleared and centrifuged at 100 000× g for 2 h. Pellet (P100) fractions were lysed and processed for western blot analysis using an anti-FLAG antibody. The graph in (B) reports the quantitative analysis of 3A-HSPB1 and 3D-HSPB1 protein levels contained in the P100 fractions, relative to the inputs, from at least three independent experiments. ( C – D ) HeLa cells were transiently transfected with HA-p62/SQSTM1 and either 3A-HSPB1 or 3D-HSPB1 expression constructs. After 24 h, cell extracts were collected and 3A-HSPB1, 3D-HDSPB1 (E) or p62/SQSTM1 (F) was immunoprecipitated using the indicated antibodies. Co-immunoprecipitation was then evaluated with specific antibodies, as indicated. 3A-HSPB1 and 3D-HSPB1 are both able to co-immunoprecipitate with p62/SQSTM1, but the phospho-mimetic mutant (3D) shows a higher affinity for p62/SQSTM1 compared with the 3A. ( E – F ) HeLa cells were co-transfected with p62-HA and either 3A-HSPB1 or 3D-HSPB1. Twenty four hours after transfection, cells were serum starved overnight or left in complete medium. Then, cells were washed and placed in 1% FBS or serum-free DMEM (NO FBS) to allow protein secretion, and 1 h before collection, FBS was added to the rescue condition. Both cell lysates and secreted fractions were collected. 3D-HSPB1 strongly increases p62/SQSTM1 secretion both in steady state and in serum starvation. The graph in (F) and the inset report the quantitative analysis and the best-fit functional curve of HSPB1 and p62/SQSTM1 protein secretion, respectively, relative to loading control from three independent experiments. The y -axis values in the graph are shown as the OUT/IN relative ratio and the error bars denote standard deviations. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * P < 0.05; * * P < 0.01; NS: non-significant).$
Figure Legend Snippet: Effect of HSPB1 mutants on the unconventional secretion of p62/SQSTM1. ( A – B ) HeLa cells were transiently transfected with 3A-HSPB1 and 3D-HSPB1 phosphorylation mutants. After 24 h, cells were placed in DMEM 1% FBS for 8 h and secreted fractions were collected, pre-cleared and centrifuged at 100 000× g for 2 h. Pellet (P100) fractions were lysed and processed for western blot analysis using an anti-FLAG antibody. The graph in (B) reports the quantitative analysis of 3A-HSPB1 and 3D-HSPB1 protein levels contained in the P100 fractions, relative to the inputs, from at least three independent experiments. ( C – D ) HeLa cells were transiently transfected with HA-p62/SQSTM1 and either 3A-HSPB1 or 3D-HSPB1 expression constructs. After 24 h, cell extracts were collected and 3A-HSPB1, 3D-HDSPB1 (E) or p62/SQSTM1 (F) was immunoprecipitated using the indicated antibodies. Co-immunoprecipitation was then evaluated with specific antibodies, as indicated. 3A-HSPB1 and 3D-HSPB1 are both able to co-immunoprecipitate with p62/SQSTM1, but the phospho-mimetic mutant (3D) shows a higher affinity for p62/SQSTM1 compared with the 3A. ( E – F ) HeLa cells were co-transfected with p62-HA and either 3A-HSPB1 or 3D-HSPB1. Twenty four hours after transfection, cells were serum starved overnight or left in complete medium. Then, cells were washed and placed in 1% FBS or serum-free DMEM (NO FBS) to allow protein secretion, and 1 h before collection, FBS was added to the rescue condition. Both cell lysates and secreted fractions were collected. 3D-HSPB1 strongly increases p62/SQSTM1 secretion both in steady state and in serum starvation. The graph in (F) and the inset report the quantitative analysis and the best-fit functional curve of HSPB1 and p62/SQSTM1 protein secretion, respectively, relative to loading control from three independent experiments. The y -axis values in the graph are shown as the OUT/IN relative ratio and the error bars denote standard deviations. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * P < 0.05; * * P < 0.01; NS: non-significant).

Techniques Used: Transfection, Western Blot, Expressing, Construct, Immunoprecipitation, Mutagenesis, Functional Assay

$Serum starvation acts upstream of HSPB1-p62/SQSTM1 and triggers the secretion of mutant huntingtin. ( A – B ) HeLa cells were transiently transfected with mutant HTT. After 24 h, cells were serum starved or left in complete medium. Secreted fractions were collected and analysed by western blot. Serum starvation increases mut HTT secretion. ( C – D ) HeLa cells were co-transfected with V5-HSPB1 and mut HTT. After 24 h, cells were either kept in full medium or serum starved overnight. Cell lysates were collected and HSPB1 was immunoprecipitated using an anti-V5 antibody. The graph in (D) reports the quantitative analysis of mut HTT co-immunoprecipitated by HSPB1. Serum starvation does not affect the HSPB1/mut HTT interaction. ( E ) HeLa cells were transiently transfected with mut HTT for 24 h. Cells were left in complete medium or serum starved overnight in the presence or absence of 10-mm Z-VAD, and then secretion assays were performed in the same conditions. Secreted fractions were collected and analysed by western blot. Z-VAD treatment inhibit the proteolytic cleavage of mut HTT, without interfering with its secretion. ( F – G ) HeLa cells were transiently transfected with mut HTT. After 24 h, cells were treated or not with 10 μm LY-294002 overnight. Then cells were placed in 1% FBS (replenishing the LY-294002) for 8 h and both cell lysates and secreted fraction were collected and analysed. LY-294002 is able to mimic serum starvation and increases mut HTT secretion. ( H – I ) HeLa cells were transiently transfected with mut HTT with or without HA-AKT. Cells were kept in complete medium or serum starved overnight. Then, cells were placed in 1% FBS to allow protein secretion and 1 h before collection FBS was added to the rescue condition. The over-expression of AKT reduces mut HTT secretion both at steady state and in response to serum starvation/rescue conditions. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.01; NS: non-significant).$
Figure Legend Snippet: Serum starvation acts upstream of HSPB1-p62/SQSTM1 and triggers the secretion of mutant huntingtin. ( A – B ) HeLa cells were transiently transfected with mutant HTT. After 24 h, cells were serum starved or left in complete medium. Secreted fractions were collected and analysed by western blot. Serum starvation increases mut HTT secretion. ( C – D ) HeLa cells were co-transfected with V5-HSPB1 and mut HTT. After 24 h, cells were either kept in full medium or serum starved overnight. Cell lysates were collected and HSPB1 was immunoprecipitated using an anti-V5 antibody. The graph in (D) reports the quantitative analysis of mut HTT co-immunoprecipitated by HSPB1. Serum starvation does not affect the HSPB1/mut HTT interaction. ( E ) HeLa cells were transiently transfected with mut HTT for 24 h. Cells were left in complete medium or serum starved overnight in the presence or absence of 10-mm Z-VAD, and then secretion assays were performed in the same conditions. Secreted fractions were collected and analysed by western blot. Z-VAD treatment inhibit the proteolytic cleavage of mut HTT, without interfering with its secretion. ( F – G ) HeLa cells were transiently transfected with mut HTT. After 24 h, cells were treated or not with 10 μm LY-294002 overnight. Then cells were placed in 1% FBS (replenishing the LY-294002) for 8 h and both cell lysates and secreted fraction were collected and analysed. LY-294002 is able to mimic serum starvation and increases mut HTT secretion. ( H – I ) HeLa cells were transiently transfected with mut HTT with or without HA-AKT. Cells were kept in complete medium or serum starved overnight. Then, cells were placed in 1% FBS to allow protein secretion and 1 h before collection FBS was added to the rescue condition. The over-expression of AKT reduces mut HTT secretion both at steady state and in response to serum starvation/rescue conditions. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.01; NS: non-significant).

Techniques Used: Mutagenesis, Transfection, Western Blot, Immunoprecipitation, Over Expression

$The formation of the HSPB1-p62/SQSTM1-MUT HTT ternary complex is instrumental for the unconventional secretion of mutant huntingtin. ( A ) HeLa cells were transfected with mut HTT, HA-p62/SQSTM1 with or without V5-HSPB1. Twenty four hours after transfection, cells were placed in DMEM 1% FBS and cell lysate and secreted fraction were collected. Mutant HTT was immunoprecipitated using anti-FLAG antibody and Co-IP assessed with the indicated antibodies. Mutant HTT is able to interact with HSPB1 both intracellularly and in the secreted fractions, whereas p62/SQSTM1 interacts with mut HTT only intracellularly. ( B ) HeLa cells were transiently transfected with mut HTT and p62/SQSTM1. Twenty four hours after transfection, cells were kept in full medium or serum starved overnight, cell lysates were prepared and mut HTT immunoprecipitated with an anti-FLAG antibody. Serum starvation does reduce the interaction between mut HTT and p62/SQSTM1. ( C – D ) HeLa cells were silenced for scramble CTRL, HSPB1 or p62/SQSTM1 with 100-n m siRNA mix. HSPB1 and p62/SQSTM1 depletion were confirmed using specific antibodies. After 72 h, cells were re-transfected with the same mix, supplemented with 2 μg of FLAG-tagged mut HTT over-expression construct. After 48 h, cell lysates were prepared and mut HTT was immunoprecipitated using an anti-FLAG antibody and co-immunoprecipitation analysis performed with the indicated antibodies. ( E – F ) HeLa cells were silenced for Ctrl, HSPB1 or p62/SQSTM1 and transfected with mutant HTT as detailed in (C–D). Cells were semi-permeabilized with digitonin immediately before fixation. Indirect immunofluorescence was performed and analysed by confocal microscopy. Co-localization analysis was performed by using ImageJ software. Thirty cells per experimental condition were analysed and mean values with standard deviations are reported in the graph. Scale bar: 10 μ m . ( G ) Schematic representation of the hierarchy dictating the assembly and function of the HSPB1-p62-mut HTT ternary complex. ( H – I ) HeLa cells were silenced for scramble control, HSPB1 or p62/SQSTM1 with 100-n m siRNA and transfected with 2 μg of FLAG mut HTT, as detailed above. Cell lysate and secreted fraction were collected and analysed by western blot. The P -values for the co-localization and densitometric analyses were determined by Student’s t- test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.01; * * * P < 0.001).$
Figure Legend Snippet: The formation of the HSPB1-p62/SQSTM1-MUT HTT ternary complex is instrumental for the unconventional secretion of mutant huntingtin. ( A ) HeLa cells were transfected with mut HTT, HA-p62/SQSTM1 with or without V5-HSPB1. Twenty four hours after transfection, cells were placed in DMEM 1% FBS and cell lysate and secreted fraction were collected. Mutant HTT was immunoprecipitated using anti-FLAG antibody and Co-IP assessed with the indicated antibodies. Mutant HTT is able to interact with HSPB1 both intracellularly and in the secreted fractions, whereas p62/SQSTM1 interacts with mut HTT only intracellularly. ( B ) HeLa cells were transiently transfected with mut HTT and p62/SQSTM1. Twenty four hours after transfection, cells were kept in full medium or serum starved overnight, cell lysates were prepared and mut HTT immunoprecipitated with an anti-FLAG antibody. Serum starvation does reduce the interaction between mut HTT and p62/SQSTM1. ( C – D ) HeLa cells were silenced for scramble CTRL, HSPB1 or p62/SQSTM1 with 100-n m siRNA mix. HSPB1 and p62/SQSTM1 depletion were confirmed using specific antibodies. After 72 h, cells were re-transfected with the same mix, supplemented with 2 μg of FLAG-tagged mut HTT over-expression construct. After 48 h, cell lysates were prepared and mut HTT was immunoprecipitated using an anti-FLAG antibody and co-immunoprecipitation analysis performed with the indicated antibodies. ( E – F ) HeLa cells were silenced for Ctrl, HSPB1 or p62/SQSTM1 and transfected with mutant HTT as detailed in (C–D). Cells were semi-permeabilized with digitonin immediately before fixation. Indirect immunofluorescence was performed and analysed by confocal microscopy. Co-localization analysis was performed by using ImageJ software. Thirty cells per experimental condition were analysed and mean values with standard deviations are reported in the graph. Scale bar: 10 μ m . ( G ) Schematic representation of the hierarchy dictating the assembly and function of the HSPB1-p62-mut HTT ternary complex. ( H – I ) HeLa cells were silenced for scramble control, HSPB1 or p62/SQSTM1 with 100-n m siRNA and transfected with 2 μg of FLAG mut HTT, as detailed above. Cell lysate and secreted fraction were collected and analysed by western blot. The P -values for the co-localization and densitometric analyses were determined by Student’s t- test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.01; * * * P < 0.001).

Techniques Used: Mutagenesis, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Construct, Immunofluorescence, Confocal Microscopy, Software, Western Blot

Hemagglutination Assay:

Article Title: The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein
Article Snippet: The EGFP-CD63 was purchased from ADDGENE (catalogue #62964) ( ). .. The HA-tagged p62/SQSTM1 construct has been previously described ( ) and was purchased from ADDGENE (catalogue: #28027). ..

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Journal: Human Molecular Genetics

Article Title: The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein

doi: 10.1093/hmg/ddad047

Figure Lengend Snippet: HSPB1 interacts with p62/SQSTM1 and supports synergistically its unconventional secretion. ( A ) HeLa cells were transiently transfected with V5-HSPB1-V5 and HA-p62/SQSTM1. Twenty four hours after transfection, cell lysates were prepared and immunoprecipitation was performed using either anti-V5 or anti-HA antibody. HSPB1 and p62/SQSTM1 interaction was detected in both orientations. In addition, an unconjugated control IgG antibody was used to verify the specificity of the interaction (lane 4 of the top and bottom panels). ( B – C ) HeLa cells were transiently transfected with GFP-HSPB1 and HA-p62/SQSTM1. After 24 h, cells were semi-permeabilized by digitonin treatment, fixed, subjected to immunofluorescence and analysed by confocal microscopy. Magnifications (10×) of the merged channels are shown in the insets. The co-localization analysis was performed using an ImageJ plug-in (JaCoP). The graph shows Mander’s coefficient (fraction of HSPB1-positive structures overlapping with p62-positive structures and reverse, grey bars). Scale bars: 10 μm. ( D ) HeLa cells were placed in DMEM 1% FBS for 8 h, then secreted fraction and cell lysate were collected. Secreted fraction was centrifuged at 100 000 g for 2 h. Pellet (P100) was resuspended in lysis buffer and processed for western blot analysis using a p62/SQSTM1 antibody. P62/SQSTM1 is present in the P100 fraction and in the cell lysate but not in the S100. ( E – F ) HeLa cells were transfected with HSPB1-V5, P62-HA or both. After 24 h, the cells were placed in 1% FBS for 8 h to allow protein secretion. Both cell lysate and secreted fraction were collected and analysed. p62/SQSTM1 secretion is increased by HSPB1 over-expression as shown also in the quantification (F). ( G – H ) HeLa cells were co-transfected with V5-HSPB1 and HA-p62/SQSTM1. Twenty four hours after transfection, cells were washed three times and placed in serum-free DMEM (NO FBS) for the indicated times (from 0 to 24 h). Secreted fractions were collected from 0.5 up to 24 h. Both HSPB1 and p62/SQSTM1 secretion increases over time, but the amount of p62/SQSTM1 is decreased after 24 h. ( I – J ) Schematic diagram depicting the experimental protocol deployed to assess the dynamic variations of HSPB1 and p62/SQSTM1 secretion upon serum deprivation. HeLa cells were serum starved or left in complete medium. Then cells were placed in 1% FBS or NO FBS for 8 h to allow protein secretion and 1 h before collection 1% FBS was added to the rescue condition. Cell lysates were collected and analysed using the indicated antibodies. AKT phosphorylation is reduced by serum deprivation and is increased again after re-addition of FBS. ( K – L ) HeLa cells were transiently transfected with V5-HSPB1 and HA-p62/SQSTM1. After 24 h, cells were serum starved or left in complete medium. Then, cells were placed in 1% FBS for 8 h to allow protein secretion, and 1 h before collection, FBS was added back to the rescue condition. p62/SQSTM1 secretion is increased after serum starvation and the secretion is reversible. The graphs in (F), (H) and (L) report the quantitative analysis of HSPB1 and p62/SQSTM1 protein secretion relative to loading control from at least three independent experiments. The y -axis values are shown as the OUT/IN relative ratio and the error bars denote standard deviations. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.001; NS: non-significant).

Article Snippet: The HA-tagged p62/SQSTM1 construct has been previously described ( ) and was purchased from ADDGENE (catalogue: #28027).

Techniques: Transfection, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Lysis, Western Blot, Over Expression

Journal: Human Molecular Genetics

Article Title: The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein

doi: 10.1093/hmg/ddad047

Figure Lengend Snippet: Effect of HSPB1 mutants on the unconventional secretion of p62/SQSTM1. ( A – B ) HeLa cells were transiently transfected with 3A-HSPB1 and 3D-HSPB1 phosphorylation mutants. After 24 h, cells were placed in DMEM 1% FBS for 8 h and secreted fractions were collected, pre-cleared and centrifuged at 100 000× g for 2 h. Pellet (P100) fractions were lysed and processed for western blot analysis using an anti-FLAG antibody. The graph in (B) reports the quantitative analysis of 3A-HSPB1 and 3D-HSPB1 protein levels contained in the P100 fractions, relative to the inputs, from at least three independent experiments. ( C – D ) HeLa cells were transiently transfected with HA-p62/SQSTM1 and either 3A-HSPB1 or 3D-HSPB1 expression constructs. After 24 h, cell extracts were collected and 3A-HSPB1, 3D-HDSPB1 (E) or p62/SQSTM1 (F) was immunoprecipitated using the indicated antibodies. Co-immunoprecipitation was then evaluated with specific antibodies, as indicated. 3A-HSPB1 and 3D-HSPB1 are both able to co-immunoprecipitate with p62/SQSTM1, but the phospho-mimetic mutant (3D) shows a higher affinity for p62/SQSTM1 compared with the 3A. ( E – F ) HeLa cells were co-transfected with p62-HA and either 3A-HSPB1 or 3D-HSPB1. Twenty four hours after transfection, cells were serum starved overnight or left in complete medium. Then, cells were washed and placed in 1% FBS or serum-free DMEM (NO FBS) to allow protein secretion, and 1 h before collection, FBS was added to the rescue condition. Both cell lysates and secreted fractions were collected. 3D-HSPB1 strongly increases p62/SQSTM1 secretion both in steady state and in serum starvation. The graph in (F) and the inset report the quantitative analysis and the best-fit functional curve of HSPB1 and p62/SQSTM1 protein secretion, respectively, relative to loading control from three independent experiments. The y -axis values in the graph are shown as the OUT/IN relative ratio and the error bars denote standard deviations. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * P < 0.05; * * P < 0.01; NS: non-significant).

Article Snippet: The HA-tagged p62/SQSTM1 construct has been previously described ( ) and was purchased from ADDGENE (catalogue: #28027).

Techniques: Transfection, Western Blot, Expressing, Construct, Immunoprecipitation, Mutagenesis, Functional Assay

Journal: Human Molecular Genetics

Article Title: The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein

doi: 10.1093/hmg/ddad047

Figure Lengend Snippet: Serum starvation acts upstream of HSPB1-p62/SQSTM1 and triggers the secretion of mutant huntingtin. ( A – B ) HeLa cells were transiently transfected with mutant HTT. After 24 h, cells were serum starved or left in complete medium. Secreted fractions were collected and analysed by western blot. Serum starvation increases mut HTT secretion. ( C – D ) HeLa cells were co-transfected with V5-HSPB1 and mut HTT. After 24 h, cells were either kept in full medium or serum starved overnight. Cell lysates were collected and HSPB1 was immunoprecipitated using an anti-V5 antibody. The graph in (D) reports the quantitative analysis of mut HTT co-immunoprecipitated by HSPB1. Serum starvation does not affect the HSPB1/mut HTT interaction. ( E ) HeLa cells were transiently transfected with mut HTT for 24 h. Cells were left in complete medium or serum starved overnight in the presence or absence of 10-mm Z-VAD, and then secretion assays were performed in the same conditions. Secreted fractions were collected and analysed by western blot. Z-VAD treatment inhibit the proteolytic cleavage of mut HTT, without interfering with its secretion. ( F – G ) HeLa cells were transiently transfected with mut HTT. After 24 h, cells were treated or not with 10 μm LY-294002 overnight. Then cells were placed in 1% FBS (replenishing the LY-294002) for 8 h and both cell lysates and secreted fraction were collected and analysed. LY-294002 is able to mimic serum starvation and increases mut HTT secretion. ( H – I ) HeLa cells were transiently transfected with mut HTT with or without HA-AKT. Cells were kept in complete medium or serum starved overnight. Then, cells were placed in 1% FBS to allow protein secretion and 1 h before collection FBS was added to the rescue condition. The over-expression of AKT reduces mut HTT secretion both at steady state and in response to serum starvation/rescue conditions. The P -values for the densitometric analyses were determined by factorial ANOVA or Student’s t -test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.01; NS: non-significant).

Article Snippet: The HA-tagged p62/SQSTM1 construct has been previously described ( ) and was purchased from ADDGENE (catalogue: #28027).

Techniques: Mutagenesis, Transfection, Western Blot, Immunoprecipitation, Over Expression

Journal: Human Molecular Genetics

Article Title: The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein

doi: 10.1093/hmg/ddad047

Figure Lengend Snippet: The formation of the HSPB1-p62/SQSTM1-MUT HTT ternary complex is instrumental for the unconventional secretion of mutant huntingtin. ( A ) HeLa cells were transfected with mut HTT, HA-p62/SQSTM1 with or without V5-HSPB1. Twenty four hours after transfection, cells were placed in DMEM 1% FBS and cell lysate and secreted fraction were collected. Mutant HTT was immunoprecipitated using anti-FLAG antibody and Co-IP assessed with the indicated antibodies. Mutant HTT is able to interact with HSPB1 both intracellularly and in the secreted fractions, whereas p62/SQSTM1 interacts with mut HTT only intracellularly. ( B ) HeLa cells were transiently transfected with mut HTT and p62/SQSTM1. Twenty four hours after transfection, cells were kept in full medium or serum starved overnight, cell lysates were prepared and mut HTT immunoprecipitated with an anti-FLAG antibody. Serum starvation does reduce the interaction between mut HTT and p62/SQSTM1. ( C – D ) HeLa cells were silenced for scramble CTRL, HSPB1 or p62/SQSTM1 with 100-n m siRNA mix. HSPB1 and p62/SQSTM1 depletion were confirmed using specific antibodies. After 72 h, cells were re-transfected with the same mix, supplemented with 2 μg of FLAG-tagged mut HTT over-expression construct. After 48 h, cell lysates were prepared and mut HTT was immunoprecipitated using an anti-FLAG antibody and co-immunoprecipitation analysis performed with the indicated antibodies. ( E – F ) HeLa cells were silenced for Ctrl, HSPB1 or p62/SQSTM1 and transfected with mutant HTT as detailed in (C–D). Cells were semi-permeabilized with digitonin immediately before fixation. Indirect immunofluorescence was performed and analysed by confocal microscopy. Co-localization analysis was performed by using ImageJ software. Thirty cells per experimental condition were analysed and mean values with standard deviations are reported in the graph. Scale bar: 10 μ m . ( G ) Schematic representation of the hierarchy dictating the assembly and function of the HSPB1-p62-mut HTT ternary complex. ( H – I ) HeLa cells were silenced for scramble control, HSPB1 or p62/SQSTM1 with 100-n m siRNA and transfected with 2 μg of FLAG mut HTT, as detailed above. Cell lysate and secreted fraction were collected and analysed by western blot. The P -values for the co-localization and densitometric analyses were determined by Student’s t- test using STATVIEW v4.53 (Abacus Concepts) ( n = 3, * * P < 0.01; * * * P < 0.001).

Article Snippet: The HA-tagged p62/SQSTM1 construct has been previously described ( ) and was purchased from ADDGENE (catalogue: #28027).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Construct, Immunofluorescence, Confocal Microscopy, Software, Western Blot

Western blot showing the relative expression levels of the Venus tagged SQSTM1 and LC3 constructs compared to endogenous SQSTM1 and LC3. HeLa cells transfected with the indicated Venus-tagged SQSTM1 or LC3 constructs in duplicate were lysed and resolved by SDS-PAGE followed by western blotting with (A) anti-SQSTM1 or (B) anti-LC3 antibodies. Mm, molecular mass markers in kilodaltons.

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Western blot showing the relative expression levels of the Venus tagged SQSTM1 and LC3 constructs compared to endogenous SQSTM1 and LC3. HeLa cells transfected with the indicated Venus-tagged SQSTM1 or LC3 constructs in duplicate were lysed and resolved by SDS-PAGE followed by western blotting with (A) anti-SQSTM1 or (B) anti-LC3 antibodies. Mm, molecular mass markers in kilodaltons.

Article Snippet: 12 Mouse wild-type SQSTM1, SQSTM1 K7A,D69A , and SQSTM1 D337-339A constructs were purchased from Addgene (38277, 38281, and 38280, deposited by Noboru Mizushima).

Techniques: Western Blot, Expressing, Construct, Transfection, SDS Page

The nucleocytoplasmic ratio of soluble Venus-LC3 is decreased in the presence of overexpressed Cerulean-SQSTM1. Representative confocal images of the localization of Venus, Venus-LC3, Venus-LC3G120A, Venus-LC3F52A,L53A, and Venus-LC3R70A when transiently co-expressed with (A) Cerulean or (B) Cerulean-SQSTM1 in HeLa cells. Scale bar: 10 μm. (C) An automated image analysis routine was used to quantify the N/C ratio of the soluble (puncta independent) Venus fluorescence (YFP channel) in the cells co-expressing the indicated Venus-tagged constructs in combination with either Cerulean (light gray bars) or Cerulean-SQSTM1 (dark gray bars). Bars are the mean ± 95% confidence interval for N = 20 cells from 2 independent experiments. Statistical comparisons are between the grouped bars. ns, p > 0.05; ***, p ≤ 0.001.

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: The nucleocytoplasmic ratio of soluble Venus-LC3 is decreased in the presence of overexpressed Cerulean-SQSTM1. Representative confocal images of the localization of Venus, Venus-LC3, Venus-LC3G120A, Venus-LC3F52A,L53A, and Venus-LC3R70A when transiently co-expressed with (A) Cerulean or (B) Cerulean-SQSTM1 in HeLa cells. Scale bar: 10 μm. (C) An automated image analysis routine was used to quantify the N/C ratio of the soluble (puncta independent) Venus fluorescence (YFP channel) in the cells co-expressing the indicated Venus-tagged constructs in combination with either Cerulean (light gray bars) or Cerulean-SQSTM1 (dark gray bars). Bars are the mean ± 95% confidence interval for N = 20 cells from 2 independent experiments. Statistical comparisons are between the grouped bars. ns, p > 0.05; ***, p ≤ 0.001.

Article Snippet: 12 Mouse wild-type SQSTM1, SQSTM1 K7A,D69A , and SQSTM1 D337-339A constructs were purchased from Addgene (38277, 38281, and 38280, deposited by Noboru Mizushima).

Techniques: Fluorescence, Expressing, Construct

FRET reports on the close physical proximity of both soluble and puncta-associated Cerulean-SQSTM1 and Venus-LC3. FRET analysis was performed on cells co-expressing Cerulean-SQSTM1 with the indicated Venus-LC3 constructs and semi-automatically analyzed across multiple cells. The data were separately analyzed for (A) soluble SQSTM1 and (B) puncta-associated pools of SQSTM1. FRET was also measured between Cerulean-SQSTM1 and empty Venus as a negative control. Bars are the mean ± 95% confidence interval for N = 9–30 cells from 3 independent experiments. Statistical comparisons are with the negative control. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0 .001.

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: FRET reports on the close physical proximity of both soluble and puncta-associated Cerulean-SQSTM1 and Venus-LC3. FRET analysis was performed on cells co-expressing Cerulean-SQSTM1 with the indicated Venus-LC3 constructs and semi-automatically analyzed across multiple cells. The data were separately analyzed for (A) soluble SQSTM1 and (B) puncta-associated pools of SQSTM1. FRET was also measured between Cerulean-SQSTM1 and empty Venus as a negative control. Bars are the mean ± 95% confidence interval for N = 9–30 cells from 3 independent experiments. Statistical comparisons are with the negative control. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0 .001.

Article Snippet: 12 Mouse wild-type SQSTM1, SQSTM1 K7A,D69A , and SQSTM1 D337-339A constructs were purchased from Addgene (38277, 38281, and 38280, deposited by Noboru Mizushima).

Techniques: Expressing, Construct, Negative Control

Venus-LC3's rate of diffusion decreases when co-expressed with Cerulean-SQSTM1. A quantitative FRAP assay was carried out on cells co-expressing the indicated Venus- and Cerulean-tagged constructs using a rectangular imaging ROI centered on the cytoplasm, and small circular bleach region (rn = 1 μm) placed in a region devoid of puncta. (A) Examples of the early time-points from normalized FRAP recoveries are shown for Venus-LC3 co-expressed with Cerulean (light gray circles) or with Cerulean-SQSTM1 (dark gray circles). The solid lines are fits to a single component diffusion model. Residuals for the fits are indicated in the lower panel. (B) D values for the indicated Venus-LC3 constructs in cells co-expressing either Cerulean (light gray bars) or Cerulean-SQSTM1 (dark gray bars). Empty Venus was included as a control. Bars show the mean ± 95% confidence intervals for N = 19–64 cells from 2–4 independent experiments. Statistical comparisons are between the grouped bars. ns, p > 0.05; ***, p ≤ 0.001.

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Venus-LC3's rate of diffusion decreases when co-expressed with Cerulean-SQSTM1. A quantitative FRAP assay was carried out on cells co-expressing the indicated Venus- and Cerulean-tagged constructs using a rectangular imaging ROI centered on the cytoplasm, and small circular bleach region (rn = 1 μm) placed in a region devoid of puncta. (A) Examples of the early time-points from normalized FRAP recoveries are shown for Venus-LC3 co-expressed with Cerulean (light gray circles) or with Cerulean-SQSTM1 (dark gray circles). The solid lines are fits to a single component diffusion model. Residuals for the fits are indicated in the lower panel. (B) D values for the indicated Venus-LC3 constructs in cells co-expressing either Cerulean (light gray bars) or Cerulean-SQSTM1 (dark gray bars). Empty Venus was included as a control. Bars show the mean ± 95% confidence intervals for N = 19–64 cells from 2–4 independent experiments. Statistical comparisons are between the grouped bars. ns, p > 0.05; ***, p ≤ 0.001.

Article Snippet: 12 Mouse wild-type SQSTM1, SQSTM1 K7A,D69A , and SQSTM1 D337-339A constructs were purchased from Addgene (38277, 38281, and 38280, deposited by Noboru Mizushima).

Techniques: Diffusion-based Assay, FRAP Assay, Expressing, Construct, Imaging, Control

$Apparent molecular mass and mobile fractions for Venus, Venus-LC3, and Venus-LC3 mutants co-expressing either Cerulean or Cerulean-SQSTM1 based on the FRAP diffusion measurements in live HeLa cells under basal conditions.$

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Apparent molecular mass and mobile fractions for Venus, Venus-LC3, and Venus-LC3 mutants co-expressing either Cerulean or Cerulean-SQSTM1 based on the FRAP diffusion measurements in live HeLa cells under basal conditions.

Article Snippet: 12 Mouse wild-type SQSTM1, SQSTM1 K7A,D69A , and SQSTM1 D337-339A constructs were purchased from Addgene (38277, 38281, and 38280, deposited by Noboru Mizushima).

Techniques: Diffusion-based Assay

Soluble Venus-LC3 interacts with overexpressed Cerulean-SQSTM1 even when SQSTM1's PB1 domain is disrupted. (A) Representative confocal images of the localization of Venus-LC3 in HeLa cells co-expressing either Cerulean, as a negative control, or the indicated Cerulean-SQSTM1 constructs. Scale bar: 10 μm. (B) Quantification of the N/C ratios for soluble Venus-LC3 (YFP channel) in the presence of the indicated constructs. Bars show the mean ± 95% confidence intervals for N = 20–31 cells from 2–3 independent experiments. (C) Quantification of FRET between soluble Venus-LC3 and the indicated Cerulean-SQSTM1 constructs. FRET between Venus-LC3 and Cerulean was assessed as a negative control. Bars show the mean ± 95% confidence intervals for N = 30 cells from 3 independent experiments. (D) D for Venus-LC3 in cells co-expressing the indicated Cerulean-SQSTM1 constructs or empty Cerulean. Bars show the mean ± 95% confidence interval for N=30–35 cells from 3 independent experiments. Statistics are for comparisons with Venus-LC3 co-expressing wild-type SQSTM1. ns, p > 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Soluble Venus-LC3 interacts with overexpressed Cerulean-SQSTM1 even when SQSTM1's PB1 domain is disrupted. (A) Representative confocal images of the localization of Venus-LC3 in HeLa cells co-expressing either Cerulean, as a negative control, or the indicated Cerulean-SQSTM1 constructs. Scale bar: 10 μm. (B) Quantification of the N/C ratios for soluble Venus-LC3 (YFP channel) in the presence of the indicated constructs. Bars show the mean ± 95% confidence intervals for N = 20–31 cells from 2–3 independent experiments. (C) Quantification of FRET between soluble Venus-LC3 and the indicated Cerulean-SQSTM1 constructs. FRET between Venus-LC3 and Cerulean was assessed as a negative control. Bars show the mean ± 95% confidence intervals for N = 30 cells from 3 independent experiments. (D) D for Venus-LC3 in cells co-expressing the indicated Cerulean-SQSTM1 constructs or empty Cerulean. Bars show the mean ± 95% confidence interval for N=30–35 cells from 3 independent experiments. Statistics are for comparisons with Venus-LC3 co-expressing wild-type SQSTM1. ns, p > 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

Article Snippet: 12 Mouse wild-type SQSTM1, SQSTM1 K7A,D69A , and SQSTM1 D337-339A constructs were purchased from Addgene (38277, 38281, and 38280, deposited by Noboru Mizushima).

Techniques: Expressing, Negative Control, Construct

Nucleocytoplasmic distribution of Venus-SQSTM1 constructs. Representative images of the subcellular distribution of the indicated SQSTM1 and SQSTM1 isoform 2 constructs (A) following 2 h in the presence of vehicle or (B) following 2 h of LMB treatment. Scale bar: 10 μm. Data are representative of 3 independent experiments. (C) A semi-automated image analysis routine was used to quantify the N/C ratio of the soluble (puncta independent) Venus fluorescence (YFP channel) in the cells expressing the indicated Venus-tagged SQSTM1 constructs and nuclei labeled with DRAQ5 in the absence of LMB (light gray bars) or presence of LMB (dark gray bars). Bars are the mean ±95% confidence interval for N = 20 cells from 2 independent experiments. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01.

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Nucleocytoplasmic distribution of Venus-SQSTM1 constructs. Representative images of the subcellular distribution of the indicated SQSTM1 and SQSTM1 isoform 2 constructs (A) following 2 h in the presence of vehicle or (B) following 2 h of LMB treatment. Scale bar: 10 μm. Data are representative of 3 independent experiments. (C) A semi-automated image analysis routine was used to quantify the N/C ratio of the soluble (puncta independent) Venus fluorescence (YFP channel) in the cells expressing the indicated Venus-tagged SQSTM1 constructs and nuclei labeled with DRAQ5 in the absence of LMB (light gray bars) or presence of LMB (dark gray bars). Bars are the mean ±95% confidence interval for N = 20 cells from 2 independent experiments. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: 12 Mouse wild-type SQSTM1, SQSTM1 K7A,D69A , and SQSTM1 D337-339A constructs were purchased from Addgene (38277, 38281, and 38280, deposited by Noboru Mizushima).

Techniques: Construct, Fluorescence, Expressing, Labeling

FRET and FRAP analysis of soluble SQSTM1 homo-oligomers. Analysis of FRET between Cerulean- and Venus-tagged versions of the indicated (A) SQSTM1 constructs, or (B) SQSTM1iso. FRET was separately calculated for the soluble pools of protein (light gray bars) and puncta-associated pools (dark gray bars). FRET was also measured in cells co-expressing Cerulean-SQSTM1 and empty Venus as negative controls. Bars show the mean ± 95% confidence intervals for N = 27–30 cells from 3 independent experiments in (A), or N = 20 cells from 2 independent experiments in (B). Statistics are for the comparisons with the negative controls. FRAP analysis was used to quantify the diffusion coefficients of the indicated (C) Venus-tagged SQSTM1 constructs or (D) Venus-SQSTM1iso2. Diffusion coefficients for empty Venus were measured as internal controls. Bars show the mean ± 95% confidence intervals for N = 10–20 cells from 2 independent experiments in (A), and N = 31–35 cells from 3 independent experiments in (B). Statistics are for the comparisons with wild-type SQSTM1. ns, p > 0.05; ***, p ≤ 0.001.

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: FRET and FRAP analysis of soluble SQSTM1 homo-oligomers. Analysis of FRET between Cerulean- and Venus-tagged versions of the indicated (A) SQSTM1 constructs, or (B) SQSTM1iso. FRET was separately calculated for the soluble pools of protein (light gray bars) and puncta-associated pools (dark gray bars). FRET was also measured in cells co-expressing Cerulean-SQSTM1 and empty Venus as negative controls. Bars show the mean ± 95% confidence intervals for N = 27–30 cells from 3 independent experiments in (A), or N = 20 cells from 2 independent experiments in (B). Statistics are for the comparisons with the negative controls. FRAP analysis was used to quantify the diffusion coefficients of the indicated (C) Venus-tagged SQSTM1 constructs or (D) Venus-SQSTM1iso2. Diffusion coefficients for empty Venus were measured as internal controls. Bars show the mean ± 95% confidence intervals for N = 10–20 cells from 2 independent experiments in (A), and N = 31–35 cells from 3 independent experiments in (B). Statistics are for the comparisons with wild-type SQSTM1. ns, p > 0.05; ***, p ≤ 0.001.

Article Snippet: 12 Mouse wild-type SQSTM1, SQSTM1 K7A,D69A , and SQSTM1 D337-339A constructs were purchased from Addgene (38277, 38281, and 38280, deposited by Noboru Mizushima).

Techniques: Construct, Expressing, Diffusion-based Assay

Review

p62 sqstm1 construct (Addgene inc)

Structured Review

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1) Product Images from "The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein"

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