ha-tagged sqstm1  (Addgene inc)

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: Palmostatin B alters autophagy in HeLa cells, assessed through LC3B and SQSTM1 immunoblotting. HeLa cells were treated with 0.1% DMSO or 100 μM palmostatin B (PalmB) for 18 h. Autophagy was induced by 4 h EBSS treatment or induced and inhibited by 4 h EBSS/2 h treatment with 500 nM BafA1. (A) Representative images of immunoblotting conducted for autophagy markers SQSTM1 and LC3‐II with and without Palm B treatment and autophagy induction. (B) Quantification of immunoblots for SQSTM1 normalized to ACTB/β‐actin. Densitometry was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA. (C) Quantification of immunoblots for LC3‐II normalized to ACTB. Quantification was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA).

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques: Western Blot, Software

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: SQSTM1 is palmitoylated and degraded by the lysosome. HeLa cells were induced to undergo autophagy through serum deprivation (−FBS) in the presence or absence of 400 nM BafA1. (A) Palmitoylation of SQSTM1 and Ras was detected by the acyl‐RAC assay. Composite gel, all samples were run on the same gel. (B) During autophagy SQSTM1 palmitoylation increased while (C) total SQSTM1 levels decreased (degraded by the lysosome). BafA1 significantly increased both (B) SQSTM1 palmitoylation and (C) total protein. (D) S‐acylated Ras and (E) protein levels were not significantly affected by either serum deprivation or BafA1 (two‐way ANOVA, n = 3, SEM, * p < 0.05).

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques:

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: SQSTM1 is palmitoylated at the di‐cysteine C289, 290 motif, which is required for localization to the lysosome. (A) IP‐ABE was performed from transfected HeLa cells expressing the indicated forms of SQSTM1‐HA in the presence or absence of BafA1 and palmitoylation was detected by streptavidin (STR). Composite gel, all samples were run on the same gel. (B) Only mutation of the C289, 290 di‐cysteine motif significantly reduced S‐acylation of SQSTM1‐HA, regardless of the addition of BafA1 ( n = 4–7, *< 0.05, ***< 0.0001, two‐way ANOVA). (C) HeLa cells expressing RFP‐LC3 (autophagosome marker) and the indicated SQSTM1‐HA were stained with Lysotracker Far Red for 1‐h prior to fixation. Cells were either grown in complete media (fed) as a control or subjected to growth in serum‐deprived media (fasted) for 4 h to induce autophagy and visualized by confocal microscopy ( n = 3). Scale bar = 10 μm. Co‐localization was measured as Pearson's colocalization coefficient (PCC) for (D) SQSTM1‐HA and RFP‐LC3, (E) SQSTM1‐HA and lysosomes, and (F) RFP‐LC3 and lysosomes. Statistical significance was determined by two‐way ANOVA with Bonferroni's post hoc comparison; * p < 0.05. Data are represented as mean ± SEM.

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques: Transfection, Expressing, Mutagenesis, Marker, Staining, Control, Confocal Microscopy, Comparison

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: SQSTM1 palmitoylation is significantly reduced in HD. (A, B) SQSTM1 palmitoylation is significantly reduced in the cortex from HD patient brains. (A) Palmitoylation of SQSTM1 was detected in patient brains by acyl‐resin assisted capture (ARAC). Tissues used were from patients of similar age at death, CAG size, and post‐mortem interval. Male and female samples were included in controls and HD samples. Two‐tailed unpaired t ‐test, SEM ( n = 5,4 for Ctrl, HD). Composite gel, all samples were run on the same gel. (B) Total SQSTM1 levels were examined in HD patients. (C, D) Significantly reduced SQSTM1 palmitoylation in the brains of HD mice is restored by fasting. Eight‐month‐old YAC128 mice were fasted for 24 h, as previously described. (C) Palmitoylation of SQSTM1 was detected using IP‐ABE (SQSTM1 is immunoprecipitated prior to ABE—top) and quantified (bottom). The arrow on the blot indicates the antibody heavy chain band. Composite gel, all samples were run on the same gel. (D) Total SQSTM1 levels were immunodetected (top) and quantified (bottom). Statistical significance was determined by two‐way ANOVA with Bonferroni's post hoc comparison; * p < 0.05, *** p < 0.0001. Data are represented as SEM.

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques: Two Tailed Test, Immunoprecipitation, Comparison

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: Palmostatin B increases localization of HTT to the autophagosomes. HeLa cells were treated with 0.1% DMSO or 10 μM Palmostatin B (PalmB) for 16–18 h. (A) Representative images of immunofluorescence conducted on the treated cells for endogenous SQSTM1 and endogenous HTT. Lysosomes were stained with 50 nM LysoT4racker Far Red (Thermofisher Scientific, L12492 ); 1 h incubation at 37°C prior to fixation. Scale bar = 50 μm. Co‐localization was measured as Pearson's colocalization coefficient (PCC) for (B) SQSTM1 and HTT, (C) SQSTM1 and lysosomes, and (D) HTT and lysosomes. Statistical significance was determined by the two‐tailed unpaired t ‐test; * p < 0.05, *** p < 0.001. Data are represented as mean ± SEM ( n = 3).

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques: Immunofluorescence, Staining, Incubation, Two Tailed Test

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